R7BP complexes with RGS9-2 and RGS7 in the striatum differentially control motor learning and locomotor responses to cocaine

Abstract

In the striatum, signaling through G protein-coupled dopamine receptors mediates motor and reward behavior, and underlies the effects of addictive drugs. The extent of receptor responses is determined by RGS9-2/Gbeta5 complexes, a striatally enriched regulator that limits the lifetime of activated G proteins. Recent studies suggest that the function of RGS9-2/Gbeta5 is controlled by the association with an additional subunit, R7BP, making elucidation of its contribution to striatal signaling essential for understanding molecular mechanisms of behaviors mediated by the striatum. In this study, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration, consistent with the essential role of R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not show higher sensitivity to locomotor-stimulating effects of cocaine. Using a striatum-specific knockdown approach, we show that the sensitivity of motor stimulation to cocaine is instead dependent on RGS7, whose complex formation with R7BP is dictated by RGS9-2 expression. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.

Figure 1

Baseline behavioral characteristics are unaltered on the genetic elimination of R7BP. R7BP knockout (R7BP^−/−) mice (n=9) and wild-type (R7BP^+/+, WT) litter mates (n=5) were placed in locomotor open field test chamber for 3 h and analyzed for the behavioral traits. (a) Distance traveled per 5-min interval was plotted over the 3-h evaluation period, or used to calculate cumulative total distance traveled (b). Quantification and one-way ANOVA reveals that travel velocity (c), stereotypy counts (d), vertical jumps (e), thigmotaxis (f), and central zone entries (g) between R7BP^−/− and R7BP^+/+ littermates were all statistically insignificant.

Figure 2

R7BP^−/− mice show deficits in motor coordination. (a) R7BP^−/− mice (n=14) and WT (n=8) were analyzed using an accelerated rotarod test over the course of three trials/day, for 3 days totaling nine trials. A two-way ANOVA (trial by genotype) revealed a significant effect of genotype (F_(1, 180)=23.96, p<0.001), and a significant effect of trial (F_(8, 180)=20.601, p<0.001). (b) Calculated mean latencies to fall off the rotarod across all trials were 178±8 (R7BP^+/+) and 130±6 (R7BP^−/−). (c) The mean latency to fall off the rotarod for the three trials for each day revealed a significant difference between genotypes on days 2 (p<0.01) and 3 (p<0.05).

Figure 3

Lentiviral expression of R7BP in dorsal striatum improves the performance of R7BP^−/− in rotarod task. (a) Strategy for lentivirus-mediated protein expression in vivo. Shown schematic illustrates location of lentiviral construct (R7BP or LacZ) unilateral injection sites at four depth points (3.1, 2.8, 2.5, and 2.2 mm) of 5-day-old R7BP^−/− mice. Each side of the brain received four 600-nl injections with either R7BP or control LacZ lentivirus within the striatum. (b) Verification of lentiviral-mediated R7BP expression in adult R7BP^−/− mice. Left, at postnatal day 25, brains were sectioned at 300-μm thickness, and LacZ/R7BP lentivirus injected striatum was isolated. Tissue sections were solubilized with 1% Triton X-100 containing buffer, lysates containing 10 μg of total protein were separated on 12.5% PAGE, and subjected to western blotting with the indicated antibodies. Shown is maximal expression of R7BP detected within the isolated sections. Maximal R7BP-mediated lentiviral expression in R7BP^−/− mice were estimated to be ∼30% of the corresponding levels in R7BP^+/+ mice using quantitative densitometry analysis with ImageJ software package. Right, western blot analysis of proteins immunoprecipitated from the total lysates by 2 μg of anti-R7BP antibodies. (c) Verification of R7BP expression around injection sites by immunostaining with anti-R7BP antibodies. R7BP^−/− mice were injected with 90 nl of R7BP expressing lentivirus at each injection site, and killed at 10 weeks. In total, 1–2 injection sites in a 40-μm brain slice were typically observed in one plane, with two injection sites shown. (d) Performance of R7BP^−/− mice bilaterally injected with either R7BP (n=9) or LacZ (n=5) expressing lentiviruses (90 nl per injections site) in rotarod test. ANOVA analysis revealed a significant increase in fall latencies on R7BP restoration into the striatum (F_(1, 125)=16.41, p<0.001) as well as a significant effect of trial (F_(8, 125)=9.62, p<0.001) (e). Mean latency to fall off the rotarod across all trials. Values are: 106±11 s for LacZ injected mice and 153±9 s for mice injected with R7BP expressing virus.

Figure 4

Locomotor stimulatory effects of cocaine administration are unaltered in R7BP^−/− mice. (a) Locomotor activities of R7BP^−/− (n=12) and their wild-type litermates (n=6) in response to increasing dozes of cocaine administration. Mice were injected with saline or cocaine (1, 5, 10, and 20 mg/kg) and their locomotor activity was measured for 1 h. A two-way ANOVA (dose by genotype) revealed a significant effect of dose (F_(4, 80)=23.128, p<0.001) but no effect of genotype (F_(1, 80)=3.248, p=0.075). (b) Locomotor sensitization to repeated cocaine administration. Mice of both genotypes (n=18 for R7BP^−/− and n=11 for R7BP^+/+) were given daily injections of saline for 3 days (S1–3), followed by 5 daily injections of 10 mg/kg cocaine (C1–C5). A two-way ANOVA (day by genotype) revealed a significant effect of trial number (F_(7, 211)=13.38, p<0.001) as both groups showed higher locomotor activity with each additional cocaine injection (ie, sensitization). However, there was no statistical difference between genotypes (F_(1, 211)=0.30, p=0.86).

Figure 5

Increased sensitivity of R7BP^−/− mice to locomotor-stimulating effects of morphine. Mice of both genotypes (n=9 for R7BP^−/− and n=5 for R7BP^+/+) were injected with increasing doses of morphine (1, 5, 10, and 20 mg/kg) and their locomotor activities were measured for 3 h after the administration. Total distance traveled in locomotor chambers was plotted as bar graphs. A two-way ANOVA (dose × genotype) revealed both a significant effect of dose (F_(4, 60)=71.99, p<0.001), and a significant effect of genotype (F_(1, 60)=14.37, p<0.001).

Figure 6

Knockdown of RGS7 in the striatum potentiates locomotor sensitization to cocaine. (a) Verification of lentivirus-mediated RGS7 knockdown in wild-type mice. Wild-type C57BL/6J mice at postnatal day 5 were unilaterally injected (600 nl per injection site) with either miR–CTR control knockdown lentivirus or miR–αRGS7 knockdown lentivirus into the striatum similarly as illustrated in Figure 3a. At postnatal day 25, brains were sectioned at 300-μm thickness, and miR–αRGS7/miR–CTR lentivirus injected striatum was isolated. Tissue sections were solubilized with 1% Triton X-100 containing IP buffer, 10 μg of total extracted protein was separated on 12.5% PAGE, and subjected to western blot analysis with the indicated antibodies. Shown is a representative maximal effect of RGS7 knockdown from one 300-μm section. Quantification of protein change shown below was performed from three maximal RGS7 knockdown sections per brain, with two mice analyzed. Image analysis was performed by densitometry quantification using ImageJ software package. Analysis of the data by Student's t-test, revealed a statistically significant difference between relative changes in RGS7 protein compared with β-actin (^***p<0.001), and no difference (p>>0.05) in relative changes between RGS9-2 and R7BP protein compared with β-actin. (b) Western blot analysis of proteins IP by 2 μg of anti-R7BP antibody from the total lysates shown in panel (a). Densitometry quantification of proteins present in immunoprecipitated fractions are presented below. (c) Knockdown of RGS7 potentiates sensitization to cocaine. Wild-type mice were bilaterally injected with either RGS7 knockdown (RGS7 KD) lentivirus (n=10) or control knockdown (Control KD) lentivirus (n=8) with 90 nl per injections site. At 8 weeks of age, animals were given daily injections of saline for 3 days (S1–3), followed by five daily injections of 10 mg/kg cocaine (C1–5). A two-way ANOVA (day by genotype) revealed a significant effect of day (F_(7, 128)=13.48, p<0.001) as both groups showed higher locomotor activity with each additional cocaine injection (ie, sensitization). Injection of RGS7 KD virus produced higher responses compared with the injection with control virus (F_(1, 125)=28.67, p<0.001) and a higher degree of sensitization as indicated by a statistically significant interaction between day and virus (F_(7, 128)=2.42, p=0.023). (d) RGS7 knockdown does not hamper motor learning performance as tested by rotarod test. A two-way ANOVA (trial by genotype) followed by Tukey's test revealed a significant improvement in rotarod performance with RGS7 knockdown virus within the first two trials (p<0.05), but no significant difference between the two viruses for the remaining trials.