Brain region specific actions of regulator of G protein signaling 4 oppose morphine reward and dependence but promote analgesia

Abstract

Background: Regulator of G protein signaling 4 (RGS4) is one of the smaller members of the RGS family of proteins, which are known to control signaling amplitude and duration via interactions with G protein alpha subunits or other signaling molecules. Earlier evidence suggests dynamic regulation of RGS4 levels in neuronal networks mediating actions of opiates and other drugs of abuse, but the consequences of RGS4 actions in vivo are largely unknown.

Methods: In this study, we use constitutive and nucleus accumbens-inducible RGS4 knockout mice as well as mice overexpressing RGS4 in the nucleus accumbens via viral mediated gene transfer, to examine the influence of RGS4 on behavioral responses to opiates. We also use electrophysiology and immunoprecipitation assays to further understand the mechanisms underlying the tissue-specific actions of RGS4.

Results: Inducible knockout or selective overexpression of RGS4 in the nucleus accumbens reveals that, in this brain region, RGS4 acts as a negative regulator of morphine reward, whereas in the locus coeruleus RGS4 opposes morphine physical dependence. In contrast, we show that RGS4 does not affect morphine analgesia or tolerance but is a positive modulator of certain opiate analgesics, such as methadone and fentanyl.

Conclusions: These findings provide fundamentally novel information concerning the role of RGS4 in the cellular mechanisms underlying the diverse actions of opiate drugs in the nervous system.

Figure 1. Selective deletion of RGS4 in the NAc increases sensitivity to morphine reward

Low (a, Zeiss 10x) and high (b, Leica confocal 40x) power magnifications of immunofluorescence for GFP in the NAc in AAV-CreGFP-injected floxed RGS4 mice. To verify recombination, the infected area was isolated using laser capture microdissection, and the extracted RNA was analyzed by qPCR to measure RGS4 and GFP expression. (c) shows RGS4 levels (normalized to GAPDH) in AAV-CreGFP and AAV-GFP infected brains (p<0.01, t-test). Mice lacking RGS4 in the NAc CPP at 3 mg/kg morphine, whereas their wildtype controls CPP at 5 mg/kg (d, n=5–6 per group). Conversely, overexpression of RGS4 in the NAc of C57Bl/6 mice, via infection with an HSV-RGS4 vector, prevents CPP to morphine (5 mg/kg s.c.) compared to control animals which were injected with an HSV-LacZ vector (e, n=7–8 per group). Selective deletion of RGS4 from the NAc increases sensitivity to the locomotor activating effects of repeated morphine exposure (10 mg/kg) (f, n=6 per group). For all behavioral studies, data are expressed as means ± S.E.M., p<0,01, two way ANOVA followed by Bonferroni test.

Figure 2. RGS4 knockout mice exhibit more severe opiate withdrawal

(a) RGS4 knockout (KO) mice show a greater degree of morphine physical dependence as compared to wildtype (WT) littermates; several signs of naloxone precipitated opiate withdrawal are more intense in mutant mice compared to WT littermates. Data are expressed as mean ± S.E.M. (n=8 per group). (b) In contrast, selective deletion of the RGS4 gene in the NAc (as described in Fig. 1) has little effect on opiate withdrawal; it leads to a decrease in ptosis only and a trend for increase in jumping behaviour which was not statistically significant. Data are expressed as mean ± S.E.M. (n=8 per group) *p<0.01 for genotype versus treatment, two way ANOVA followed by Bonferroni post hoc test.

Figure 3. RGS4 modulates LC firing via a cAMP-dependent mechanism

(a) No significant difference was observed in the sensitivity of DAMGO between LC neurons obtained from RGS4 wildtype (WT) and knockout (KO) mice (n=11–12 per data point; 4–5 mice each group). The sensitivity of LC neurons to forskolin (an activator of adenylyl cyclase) was determined in RGS4 KO mice and their wildtype littermates. (b) Effect of forskolin on LC firing in brain slices from drug-naïve RGS4 KO and WT mice. There is no difference in the effect of forskolin on LC firing rate between genotypes (n=13–16 per data point; 4–5 mice each group). (c) In contrast, after chronic morphine treatment, there was a significant difference in the sensitivity of LC neurons to forskolin: LC neurons became significantly more sensitive to forskolin in the KO group as compared to WT mice (n=12–13 per data point, 4–5 mice each group * p<0.05, two way ANOVA).

Figure 4. Agonist selective regulation of hot plate analgesia by RGS4

RGS4 knockout (KO) mice show normal responses to morphine in the 52°C hot plate assay (a). In addition, there is no genotype effect on the rate of analgesic tolerance to morphine (b, n=9–10 per group). Data are expressed as % of maximal possible effect (MPE=[Latency-baseline]/[cutoff-latency]). (c) In contrast to morphine, RGS4 KO mice are less sensitive to the analgesic actions of fentanyl and methadone (n=8–10 per group). (d) shows analgesic responses to fentanyl at different time points within 1.5 hr after morphine administration (n=7 per group) and (e) shows a dose response to fentanyl in the hot plate assay for RGS4 KO and wildtype (WT) mice (n=5–8 per group). Deletion of RGS4 from the NAc leads to a rightward shift in fentanyl dose response in the hot plate assay (f, 9–10 per group) but does not affect responses to morphine in this test (g, n=9–10 per group). For all behavioral experiments, data are expressed as mean ± S.E.M. *p<0.01 for genotype versus treatment, two way ANOVA followed by Bonferroni post hoc test.

Figure 5. RGS4 and MOR signal transduction in striatum

Quantitation of RGS4 and RGS2 levels in NAc 2 hrs after acute morphine or fentanyl administration by western blotting. Data are expressed as means ± S.E.M, n=3–4 per group, *, p<0.05 t-test.

Figure 6. Morphine and fentanyl promote the formation of distinct RGS4 complexes in striatum

(a) Mice received s.c. injections of saline, fentanyl (0.125 mg/kg), or morphine (15 mg/kg) and striata were extracted 30 min later. Striatal extracts were IP’d with an anti-MOR antibody and the immunoprecipitate was analyzed by western blot (WB) for Gαq. Fentanyl promotes the formation of complexes between MOR and Gαq. p<0.01 for fentanyl versus saline and morphine, one way ANOVA followed by Dunnett’s post hoc test. (b) Mice received acute injections of saline, fentanyl, or morphine as in (a) and striata were dissected 30 min later. Striatal extracts were IP’d with an anti-Gαq antibody and the immunoprecipitate was immunoblotted for RGS4. Fentanyl and morphine promote the formation of complexes between Gαq and RGS4. p<0.01 between treatment, one way ANOVA followed by Dunnett post hoc test. (c) Mice were treated as in (a) and striatal extracts were immunoprecipitated with an anti-MOR antibody and the immunoprecipitate was immunoblotted for RGS4. Fentanyl treatment decreases MOR-RGS4 complex levels in striatum. p<0.01 for fentanyl versus saline and morphine, one way ANOVA followed by Dunnett’s post hoc test.