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. 2012 Feb;219(3):823-34.
doi: 10.1007/s00213-011-2409-y. Epub 2011 Jul 16.

Gβ5-RGS complexes are gatekeepers of hyperactivity involved in control of multiple neurotransmitter systems

Keqiang Xie  1 , Shencheng Ge, Victoria E Collins, Christy L Haynes, Kenneth J Renner, Robert L Meisel, Rafael Lujan, Kirill A Martemyanov
Affiliations

Gβ5-RGS complexes are gatekeepers of hyperactivity involved in control of multiple neurotransmitter systems

Keqiang Xie et al. Psychopharmacology (Berl). 2012 Feb.

Abstract

Rationale and objectives: Our knowledge about genes involved in the control of basal motor activity that may contribute to the pathology of the hyperactivity disorders, e.g., attention deficit hyperactivity disorder (ADHD), is limited. Disruption of monoamine neurotransmitter signaling through G protein-coupled receptors (GPCR) is considered to be a major contributing factor to the etiology of the ADHD. Genetic association evidence and functional data suggest that regulators of G protein signaling proteins of the R7 family (R7 RGS) that form obligatory complexes with type 5 G protein beta subunit (Gβ5) and negatively regulate signaling downstream from monoamine GPCRs may play a role in controlling hyperactivity.

Methods: To test this hypothesis, we conducted behavioral, pharmacological, and neurochemical studies using a genetic mouse model that lacked Gβ5, a subunit essential for the expression of the entire R7 RGS family.

Results: Elimination of Gβ5-RGS complexes led to a striking level of hyperactivity that far exceeds activity levels previously observed in animal models. This hyperactivity was accompanied by motor learning deficits and paradoxical behavioral sensitization to a novel environment. Neurochemical studies indicated that Gβ5-RGS-deficient mice had higher sensitivity of inhibitory GPCR signaling and deficits in basal levels, release, and reuptake of dopamine. Surprisingly, pharmacological treatment with monoamine reuptake inhibitors failed to alter hyperactivity. In contrast, blockade of NMDA receptors reversed the expression of hyperactivity in Gβ5-RGS-deficient mice.

Conclusions: These findings establish that Gβ5-RGS complexes are critical regulators of monoamine-NMDA receptor signaling cross-talk and link these complexes to disorders that manifest as hyperactivity, impaired learning, and motor dysfunctions.

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Figures

Figure 1
Figure 1. Gβ5−/− mice display dramatic hyperactivity, impaired habituation to novel environment and marked motor learning deficits
Behavior of Gβ5−/−mice and their wild-type littermates (Gβ5+/+) in the open field chamber (A-F). A, Gβ5−/−mice display paradoxical habituation to the novel environment and severe hyperactivity. ANOVA analysis revealed a significant habituation effect for both wild type (F(10,35) = 129.9, p < 0.001) and Gβ5−/− (F(10,35) = 83.8, p < 0.001). However, the direction of the effect was opposite: Gβ5+/+ mice showed progressive decrease in activity during habituation, whereas the activity of Gβ5−/− mice increased. This difference in the habituation behavior was found significant by two-way ANOVA analysis (genotype x trial interaction, F(35, 720) = 3.14, p < 0.001). Right panel shows habituation behavior of wild-type subjects on magnified scale. B-F, Quantification of typical mouse behavioral traits in the open field. Statistical significance of differences was analyzed using pair-wise comparison by Student’s t-test; **p < 0.01 (n=11/group). G, Gβ5−/−mice exhibit severe deficits in motor learning behavior in rotarod test. A two-way ANOVA analysis revealed significant effect of trial for both groups of mice (F(10,187) = 7.38, p < 0.001), genotype (F(1,187) = 120.25, p < 0.001) and interaction between trial and genotype (F(10,187) = 2.248, p < 0.05). Pairwise comparions by post hoc Tukey’s test revealed that Gβ5−/−mice had a deficit in rotarod performance from the third trial (**, p < 0.01).
Figure 2
Figure 2. Subcellular localization of Gβ5 in the striatum and hippocampus
Electron micrographs show immunolabeling for Gβ5 in different neuronal compartments of WT mice, as detected using a pre-embedding immunogold method. In striatum (A-C), and hippocampus (D-F) immunoparticles for Gβ5 were detected postsynaptically along the somatic plasma membrane (arrows), and intracellular sites (crossed arrows) of dendritic shafts (Den) and dendritic spines (s. In addition, immunoparticles for Gβ5 were detected at presynaptic sites, along the extrasynaptic plasma membrane (arrowheads) of axon terminals (at) establishing excitatory synapses with dendritic shafts or dendritic spines (s). Immunoreactivity for Gβ5 was completely absent in Gβ5−/−samples (C,F). Scale bar: 0.2 μm.
Figure 3
Figure 3. Dynamics of striatal dopamine transmission in Gβ5−/−mice
A, Measurements of basal extracellular levels of dopamine in the striatum of freely moving mice by microdialysis. B, Measurements of fast changes in dopamine release and re-uptake in coronal slices containing the striatum by cyclic voltammetry. Dopamine release was evoked by a single electrical pulse as indicated by arrow. Extracellular DA was sampled every 100 ms. Asterisk denotes statistically significant difference (*p < 0.05) as revealed by Student’s t-test. C and D, Quantification of kinetic parameters of dopamine concentration changes suggesting slower dopamine release and clearance in response to electrical stimulation in Gβ5−/−striatal slices. Clearance parameters were obtained by fitting recordings to a single exponential decay and analyzed by Student’s t-test (*p < 0.05, **p < 0.01 for pair-wise comparisons between wild-type (Gβ5+/+) and Gβ5−/−mice.
Figure 4
Figure 4. Enhanced GPCR signaling in Gβ5−/−mice
A, Gβ5−/−mice exhibit greater morphine-induced analgesia but normal nociceptive thresholds in the hot plate test (n=7 9) when compared with their wild-type littermates. A two-way ANOVA (doses by genotype) revealed a significant effect of dose (F(4,58) = 92.278, p < 0.001) indicating analgesic effect of morphine in both genotypes and significant difference between genotypes (F(1,58) = 61.339, p < 0.001). Gβ5−/−had higher sensitivity to morphine effects as indicated by the interaction between dose and genotype (F(4,58) = 13.804, p < 0.001). Pairwise post hoc Tukey’s test confirmed a significance of morphine effects (*p < 0.05; **p < 0.01, ***p < 0.001 morphine vs saline control) and differences between genotypes (###, p < 0.001). B, Elevated behavioral sensitivity of Gβ5−/−mice to motor depressant effects of the D2 receptor agonist quinpirole. Locomotor activity of wild-type Gβ5+/+ (n=6) and Gβ5−/−(n= 6) mice was measured in the open field chamber for 3 hr following i.p. drug administration and then normalized to the activity of saline-treated controls of the same genotype. ANOVA showed quinpirole had significant inhibitory effect on activity of both Gβ5+/+ (F(4,25)=13.137, p < 0.001) and Gβ5−/−mice (F(4,25)=20.462, p < 0.001). Post hoc Tukey’s test confirmed higher doses of quinpirole inhibited mice activity (*p < 0.05; **p < 0.01, ***p < 0.001 vs saline control). ANOVA analysis followed by Tukuey test showed significant difference at dose of 0.1 mg/kg between genotypes ( #p < 0.05, Gβ5−/−mice vs Gβ5+/+). C, Elevated behavioral sensitivity of Gβ5−/−mice to motor depressant effects of the mGluR2/3 receptor agonist LY379268. Locomotor activity of wild-type Gβ5+/+ (n=6) and Gβ5−/−(n= 6) mice was measured in the open field chamber for 3 hr following i.p. drug administration and then normalized to the activity of saline-treated controls of the same genotype. ANOVA showed LY379268 had significant inhibitory effect on activity of Gβ5−/−mice (F(4,25)=33.596, p < 0.001). Pairwise post hoc Tukey’s test confirmed higher doses of LY379268 inhibited activity of Gβ5−/−mice (***p < 0.001 vs saline control). ANOVA analysis followed by Tukey’s test showed significant difference at dose of 1.5 mg/kg between genotypes ( #p < 0.05, Gβ5−/−mice vs wild-type). Error bars represent SEM values.
Figure 5
Figure 5. Gβ5−/− mice are resistant to the effects of monoamine reuptake inhibitors
Mice (n=6/group) were injected i.p. with desipramine (10 mg/kg), atomoxine (10 mg/kg), citalopram (10 mg/kg) or amphetamine (1 mg/kg). Immediately after injection, mice were placed in an open field chamber and their activity was monitored for 2 hrs. Total distances run by animals after drug injection were normalized to those after the injection of saline. Statistically significant differences have been analyzed by Student’s t-test comparing drug-treated subjects and saline injected controls. (*p < 0.05, **p < 0.01 vs saline control). All error bars represent SEMs.
Figure 6
Figure 6. Paradoxical effects of NMDA receptor blockade in mice lacking Gβ5
Gβ5−/−mice or their wild-type littermates (n=5) were injected either with saline or various doses of MK-801 and their ambulatory activities were monitored in the open field chamber. The same subjects were used to test the different MK-801doses with 2 days allowed between treatments. ANOVA revealed a significant effect of MK-801 on activity of both Gβ5+/+ (F(4,29) = 6.896, p < 0.001) and Gβ5−/− (F(4,29) = 34.921, p < 0.001) mice. Pairwise post hoc Tukey’s test revealed significant differences between treatments and saline control when indicated (*p < 0.05; **p < 0.01). Error bars represent SEMs.
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