D1 Dopamine Receptor Signaling Is Modulated by the R7 RGS Protein EAT-16 and the R7 Binding Protein RSBP-1 in Caenoerhabditis elegans Motor Neurons

Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1) required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior

Quantitative analysis of SWIP behavior in knockdown or null mutants of dopamine signaling genes.

<p>(A), SWIP behavior of XP292 animals fed dsRNA-expressing bacteria. The dsRNA fed to XP292 animals is indicated below each bar. XP292 animals fed bacteria containing empty vector pL4440 become paralyzed within 10 min of swimming while animals fed bacteria expressing dsRNA that targets <i>dop-3</i> continue to swim under these conditions. Students t test, asterisks indicate p<0.001. (B), SWIP behavior of animals with null mutations in genes identified in the dsRNAi screen. EAT-16, RSBP-1, UNC-43, FLP-1, AND GRK-1 are required for SWIP behavior caused by <i>dat-1</i> mutation. Each measurement shown in either panel represents the mean of five trials of 10 L4 animals each for a total of 50 animals per dsRNA fed or mutant strain. Error bars represent standard error of the mean. All strains were compared using one-way ANOVA with Bonferroni's post hoc test. Single asterisks indicate p<0.001. Double asterisks indicate p<0.01. Except where indicated by the connecting line, all statistical comparisons shown are to <i>dat-1</i> single mutants. We note that <i>grk-1</i> single mutants showed significant SWIP when compared to wild-type animals but that <i>grk-1</i> also suppressed <i>dat-1</i>-induced SWIP.</p

Quantitative analysis of dopamine response in <i>rsbp-1; dop-3</i> and <i>rsbp-1; dop-1</i> double mutants.

<p>Shown are the percentages of animals moving 20 min after being placed on agar plates containing 60 mM dopamine. Each data point represents the mean ± standard error of the mean (s.e.m.) for three trials totaling at least 75 animals. One-way ANOVA with Bonferroni post hoc test was used to compare all strains. Single asterisk indicates p<0.001, double asterisk indicates p<0.05. RSBP-1 acts with the R7 RGS protein EAT-16 to modulate signaling by the D1/DOP-1 receptor.</p

Rescue of <i>rsbp-1</i> function by transgenic expression using cell-specific promoters.

<p>Animals were tested for paralysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037831#pone-0037831-g002" target="_blank">Figure 2A</a>, except that a single concentration of DA was used (40 mM). For control, non-transgenic animals (white bars), each measurement shown represents the mean and 95% confidence interval of the mean for at least 75 animals. Gray and black bars represent measurements from <i>rsbp-1</i> mutants carrying transgenes. The promoters used for transgene expression are indicated at the bottom. Gray bars represent measurements form control strains carrying empty vector transgenes, which have promoters but no RSBP-1 sequences. We observed no significant differences in the response of these transgenic animals when compared to each other or to <i>rsbp-1</i> null mutants that lacked transgenes (p<0.001). Black bars represent measurements from strains carrying transgenes from which the promoters express RSBP-1. For each transgene, measurements of at least 75 animals for each of two or three lines were averaged, and the means and 95% confidence intervals are shown. Asterisks indicate that RSBP-1 expression gave significant rescue compared to control animals that contained promoter but not RSBP-1 coding sequence. Asterisk indicates p<0.001. The intermediate response of <i>myo-3p</i>::RSBP-1 animals was different from both <i>unc-47p</i>::RSBP-1 animals (p<0.001) and from all other rescue strains (p<0.001). All comparisons done using one-way ANOVA with Bonferroni post hoc test. The <i>unc-17</i> promoter gave nearly complete rescue of <i>rsbp-1</i> while the <i>unc-47</i> promoter had no significant effect (p = 0.32, student's t test) on behavior indicating that RSBP-1 acts in the cholinergic motor neurons and not the GABAergic neurons to mediate DA signaling.</p

Analysis of dopamine signaling defects in <i>rsbp-1</i> mutants.

<p>(A), Dose-response curves measuring locomotion behavior in response to exogenous dopamine. Shown are the percentages of animals moving 20 min after being placed on agar plates containing the indicated concentrations of dopamine. Each data point represents the mean ± standard error of the mean for three trials totaling at least 75 animals. (Two way ANOVA with Bonferroni post hoc test, *p<0.0001, ^§p<0.01, ^†p<0.05 when compared to the wildtype). (B), Quantitative analysis of basal slowing behavior. For each strain, locomotion rates in the absence of bacteria (white bars) and presence of bacteria (black bars) were calculated as the average of 30 observations. Error margins shown indicate 95% confidence intervals. Asterisks indicate values significantly different from the 45% slowing seen in the wildtype. (One-way ANOVA with Bonferroni post hoc test, asterisks indicate p<0.001). The percent slowing in the presence of bacteria for each strain is shown at the right. <i>rsbp-1</i> mutants are defective in endogenous dopamine signaling.</p

Fluorescence of animals expressing <i>rsbp-1</i> and <i>dop-1</i> promoter transgenes.

<p>(A), young adult transgenic animal showing expression of <i>rsbp-1p</i>::GFP in neurons of the head and retrovesicular ganglia (left bracket), pre-anal ganglia and tail neurons (right bracket), and vulval muscle cells (large arrows). Expression is also seen in the cell bodies and processes of ventral cord motor neurons. (small arrows indicate positions of the ventral cord neuron cell bodies). Faint green fluorescence can also be seen in body-wall and pharyngeal muscle cells. (B–D), high-power magnification images of the ventral cord area of a double transgenic animal expressing <i>rsbp-1p</i>::mCherry and <i>dop-1p</i>::GFP transgenes. In all images dorsal is up and anterior is left. (B), Nomarski image of double transgenic animal shown in panels C–E. (C), red fluorescence of the mCherry protein expressed from the extrachromosomal transgene <i>rsbp-1p</i>::mCherry. (D), green fluorescence of GFP protein expressed from the chromosomally integrated transgene <i>dop-1p</i>::GFP. (E), merged image showing coexpression of <i>rspb-1</i> and <i>dop-1</i> transgenes in cholinergic motor neurons. Asterisks indicate the positions of cell bodies of GABAergic motor neurons that express <i>rsbp-1p</i>::mCherry but not <i>dop-1p</i>::GFP. RSBP is expressed in both cholinergic and GABAergic motor neurons of the ventral cord and is thus coexpressed with both DOP-1 and DOP-3 receptors. Some cells of the ventral cord shown in panel E express the <i>dop-1p</i>::GFP but not the <i>rsbp-1p</i>::mCherry transgene. The relative position of these non-mCherry-expressing cells varies among transgenic animals suggesting that the lack of expression of the <i>rsbp-1p</i>::mCherry transgene in some cells that express the <i>dop-1p</i>::GFP transgene is due to random loss of the extrachromosomal transgene during cell division and not due to restricted expression of <i>rsbp-1</i>.</p

Genes identified in the dsRNAi screen.

*<p>FLP-1 encodes up to eight invertebrate-specific FMRFamide-related peptides.</p>**<p>EAT-16 is similar in both sequence and domain structure to all four human R7 RGS protein family members but is not clearly more related to one member than the others.</p

Quantitative behavioral analysis of wild-type and <i>rsbp-1</i>, <i>eat-16</i>, and <i>egl-10</i> mutant animals.

<p>(A), SWIP behavior of wild-type or mutant animals. Each measurement shown represents the mean of five trials of 10 L4 animals each for a total of 50 animals per strain. Error bars represent 95% confidence intervals. All strains were compared using one-way ANOVA with Bonferroni post hoc test. Only <i>dat-1</i> and <i>egl-10</i> single mutants, <i>dat-1; egl-10</i> double mutants, and <i>rsbp-1</i>; <i>dat-1</i>; <i>egl-10</i> triple mutants were statistically different from the wildtype (p<0.001). (B), Dose-response curves measuring paralysis induced by exogenous dopamine. Shown are the percentages of animals moving 20 min after being placed on agar plates containing the indicated concentrations of dopamine. Each data point represents the mean ± standard error of the mean (s.e.m.) for three trials totaling at least 75 animals. The responses of <i>eat-1</i> and <i>rsbp-1</i> mutants are not statistically different from each other at any concentration of dopamine. The response of <i>egl-10</i> mutants is significantly different from the wild-type at the indicated concentrations of dopamine (Two way ANOVA with Bonferroni post hoc test, *p<0.0001, ^§p<0.01).</p