Title : Coexpressed D 1 - and D 2 - like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans

Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1-like and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interact to modulate the activity of the neuron in which they are expressed. D1-like and D2-like dopamine receptors are also coexpressed in the cholinergic ventral cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in dopamine response. These mutants were also defective in behaviors mediated by endogenous dopamine signaling including basal slowing and swimming-induced paralysis (SWIP). We used transgene rescue experiments to show that defects in these dopamine-specific behaviors were caused by abnormal signaling in the cholinergic motor neurons. To investigate the interaction between the D1-like and D2-like receptors specifically in these cholinergic motor neurons, we measured the sensitivity of dopamine signaling mutants and transgenic animals to the acetylcholinesterase inhibitor aldicarb. We found that D2 signaling inhibited acetylcholine release from the cholinergic motor neurons while D1 signaling stimulated release from these same cells. Thus, coexpressed D1 and D2-like dopamine receptors act antagonistically in vivo to modulate acetylcholine release from the cholinergic motor neurons of C. elegans. MATERIALS AND METHODS Nematode culture: Worm strains were maintained at 20oC under standard conditions and double and triple mutants were generated using standard methods (Brenner 1974). Mutants analyzed in behavioral assays shown were as follows: ace2(g72) I, goa-1(sa734) I, egl-30(tg26) I, eat-16(ad702) I, cat-2(e1112) II, glr-1(nd38) III, glr-1(n2461), dat-1(ok157) III, dgk-1(sy428) X, dop-3(vs106) X, dop-1(vs100) X, ace1(nd35) X. DA resistance screen: Synchronized populations of fourth larval stage (L4) N2 animals were mutagenized with 30μM ethyl methanesulfonate for four hr and cultured on NGM plates for 24 hr before F1 embryos were harvested by bleach treatment of gravid adults. Synchronized L4 F1 progeny were cloned to individual wells of untreated flat-bottomed 96 well plates, each well containing 50μL of OD550=10 OP50 culture suspended in S complete media. Worm cultures were grown at 20oC for three days in a humidified container and were then washed three times with 100μL of water and tested for resistance to a 40mM DA solution. A culture was scored positive for DA resistance if ~25% of the animals in an individual well remained thrashing in DA solution for more than four min. Resistant animals were immediately rescued from resistant cultures and cloned individually into liquid media and their broods were again tested for DA resistance. A strain was considered homozygous >75% of the progeny were resistant to 40mM liquid DA after four min. This screen had advantages over our previous genetic screen (Chase et al. 2004) that allowed us to identify additional components of DA signaling. In our previous screen, F2 progeny of mutagenized animals were placed onto agar plates containing 40mM DA. After 20 min, rare animals were selected that were capable of spontaneous movement. The F2 animals transferred to DA plates in this screen originated from a population of F1 animals and so we refer to this screen as “non-clonal” to distinguish it from our new “clonal” screen in which each group of animals tested for DA resistance are from a single F1 parent. The “non-clonal” screen had two shortcomings: 1) partially resistant mutants (like dop-3) could not be recovered; and 2) many primary isolates did not retest (false positives). In the current screen, F1 progeny of mutagenized animals were grown individually in liquid culture microtiter plates. The F1 animals are heterozygous for induced mutations and give rise to F2 broods containing both mutant heterozygotes and homozygotes. Because the DA resistance test was performed on a population of F2 progeny from a single F1 parent in the new “clonal” screen, ~25% of the animals in a positive culture would be resistant to DA. This decreased the number of false positives that were isolated. Wells in which just a few animals were moving were considered not to be DAresistant. Secondly, because the animals were tested for DA resistance in liquid assay rather than on agar plates, animals could be scored for resistance immediately upon exposure to DA and thus even partially resistant mutants could be isolated. Mapping mutations: Mutations were mapped as described (Wicks et al. 2001) by mating to the polymorphic mapping strain CB4856, identifying cross-progeny, and rehomozygosing each mutation in the F2 generation. Rehomozygosed mutants were identified by placing F2 animals on agar plates containing 40mM DA and selecting animals that were resistant to paralysis after four min. Mutations that mapped near previously identified genes involved in DA signaling (goa-1, eat-16, and dop-3) were tested by standard complementation analysis using appropriate null strains. Transgenic animals: For rescue of DOP-3 in the cholinergic motor neurons of dat1; dop-3 mutants, 50ng/μL of pCL31 (acr-2::GFP) and 25ng/μL of pCL34 (acr-2::DOP-3) plasmids were co-injected with 15ng/μL of pJK4 (myo-2::GFP). For rescue of DOP-3 in the GABAergic motor neurons of dat-1; dop-3 mutants, 50ng/μL of pCL32 (unc47::GFP) and 25ng/μL of pCL35 (unc-47::DOP-3) were co-injected with 15ng/μL of pJK4. For rescue of DOP-3 in the cholinergic motor neurons of dop-3 ace-1 mutants, 50ng/μL of pCL31 and 25ng/μL of pCL34 were co-injected with 15ng/μL of pJK4. For rescue of DOP-1 in the cholinergic motor neurons of dop-3 dop-1 ace-1 mutants, 50ng/μL of pCL31 and 25ng/μL of pCL33 (acr-2::DOP-1) were co-injected with 15ng/μL of pJK4. Five independent transgenic lines were established for each experimental group, and 50 L4 animals from each line were selected that displayed the most complete expression of GFP in the cholinergic or GABAergic motor neurons and assayed for the appropriate behavior. Five control lines carrying the empty vector for each experimental condition were generated and assayed in parallel. All transgenic lines were generated using standard methods (Mello et al. 1991), and all constructs were derived from the pPD49.26 vector (Addgene) using standard subcloning procedures. acr-2 is a cholinergic neuron specific promoter (Nurrish et al. 1999), and unc-47 is a GABAergic neuron specific promoter (Eastman et al. 1999). For the double transgenic animals shown in Figure 3C an acr-2::GFP construct (pCL31) was coinjected with pL15EK (both at 50ng/ul) into the strain MT8189, GFP-positive animals were identified and mated with unc-47::mCherry expressing males (strain IZ501(UfIs34), a generous gift from M. Francis). Of the 302 neurons found in the C. elegans hermaphrodite, the acr-2 promoter is active in exactly 59 neurons, 39 of which are cholinergic motor neurons in the ventral cord that express DOP-1 and DOP-3, and innervate body wall muscles to control locomotion. The promoter is also active in six RMD motor neurons and in six IL1 sensory neurons that innervate head muscles to control foraging but not body movement, and in the two PVQ interneurons. The cell bodies of these neurons can be seen in Figure 3A. The unc-47 promoter is active in exactly 26 neurons, 19 of which are GABAergic motor neurons in the ventral cord that express DOP-3 and innervate body wall muscles to control locomotion. The promoter is also active in four RME neurons that innervate head muscles to control foraging but not body movement, in the AVL and DVB neurons that innervate enteric muscles to control defecation, and in the RIS interneuron, ablation of which has no effect on locomotion (McIntire et al. 1993). Behavioral assays: For DA dose-response assays ~25 young adults for each strain were incubated undisturbed for 10 min on plates containing the indicated concentration of DA, and then scored for paralysis. Animals were considered paralyzed when they did not exhibit at least one spontaneous body bend in a five sec observation period. Assays were repeated in triplicate for a total of at least 75 animals per strain. For acute aldicarb exposure, 1mM aldicarb plates were made by adding a 0.5M stock solution to molten low-salt agar at 55oC to a final concentration of 1mM. Plates were stored inverted in the dark at room temperature for 24 hr, then stored at 4oC and used within one week. Plates were allowed to equilibrate at room temperature for 30 min prior to the assay. Approximately twenty-five young adult animals were picked away from food and placed in the center of a 1mM aldicarb plate and prodded every five min with a platinum worm pick and scored for paralysis. For all figures that include glr-1(nd38) mutants paralysis was defined as the inability to exhibit at least one body bend in a five sec period following prodding. For all other figures paralysis was defined as the inability to exhibit at least two body bends in a five sec period following prodding. Each assay was done in triplicate for a total of at least 75 animals per strain. Basal slowing assays were done as previously described (Chase et al. 2004). Briefly, the locomotion rates of staged young adult animals were quantified by counting the number of body bends completed in five consecutive 20 sec intervals in the presence or absence of HB101 bacteria. Plates with bacteria were prepared by spreading 35μl of HB101 bacteria (A600=0.70-0.75) across each plate and incubation overnight at 37oC. Data were collected for six animals per condition for a total of 30 measurements per condition. Percent slowing was calculated by dividing the difference between locomotion rates on and off food by the locomotion rate off food. Swimming induced paralysis assays were performed by picking 10 L4 animals away from food and then placing them in a 50μL water droplet on a Menzel Glaser 10-well diagnostic slide #X1XER308B# and scoring for movement after 10 min. Movement was scored as the presence of free alternating body bends characteristic of C. elegans swimming behavior (Pierce-Shimomura et al. 2008). In the case of locomotion-defective mutants, movement was scored as the continual exhibition of spontaneous body bends. This assay was repeated for a total of 50 animals per strain. Statistical analyses: Comparisons shown Figures 2B, 3B and 4 were done using two-tailed Students t-test. In all other figures error bars represent the means of three trials and SEM. In these other figures (figures 2A, 5AC, and 6A, B) we compared the curves of each mutant to the wildtype or other appropriate control (see below) using a two-way ANOVA with repeated measures followed by a Bonferromi multiple comparisons post hoc test. In Figure 2A the curves of all mutants were statistically different from WT at multiple DA concentrations: (ace-1(nd35) at 15, 20, 30, 40mM; p<0.0001) (dop-3(vs106), glr-1(nd35) and glr-1(A/T) at 15, 20,30,40, 60 and 80mM; p<0.0001). For Figure 5A the curves for dop-3(vs106) and ace-1(nd35) mutants were not statistically different from WT (p>0.05) but the curves for glr-1(nd38) and glr-1(A/T) animals were: (glr-1(nd35) at t=35-70 min; p<0.0001 and glr-1(A/T) at t= 40-70 min; p<0.0001). For figure 5B the curve for glr-1(nd38); dop-3(vs106) double mutants was statistically different from that for glr-1(nd38) animals at t= 30, 35, 40 and 45 min; p<0.0001). In Figure 5C the curve for dop-3(vs106) ace-1(nd35) double mutants was statistically different from that for ace-1(nd35) and dop-3(vs106) single mutants at t=25 min; p<0.001 and at t=30-60 min; p<0.0001). In Figure 6A we compared the curve for dop-3(vs106) ace-1(nd35) double mutants expressing the empty transgene to that for dop-3(vs106) ace-1(nd35) double mutants expressing the acr-2::DOP-3 rescuing transgene and found they were different (at t= 20 min. p<0.001 at t=25-65 min. p<0.0001). In Figure 6B we compared the curve for dop-1(vs100) dop-3(vs106) ace1(nd35) triple mutant expressing the empty transgene with the triple mutant expressing the acr-2::DOP-1 rescuing transgene and found they were different (at t= 20 min. p<0.001 at t=25-65 min. p<0.0001). INTRODUCTION Dopamine (DA) modulates neural activity in the mammalian brain by acting through two classes of G protein-coupled receptors, with D1 and D5 receptors in the D1-like class and D2, D3, and D4 receptors in the D2-like class. Pharmacological agents that distinguish between classes of receptor, but not between receptors within a class, have been used to show that signaling by D1and D2-like receptors can have synergistic or antagonistic effects on gene expression and behavior (Plaznik et al. 1989; Keefe and Gerfen 1995; Kelley et al. 1998; Gong et al., 1999; McNamara et al. 2003). The cellular and molecular mechanisms that underlie these effects have not been clearly established and are likely to be difficult to dissect as many neurons in the brain express more than one DA receptor and the G proteins and signaling pathways activated by DA receptors vary depending on the region of the brain and type of neurons in which they are expressed (Stoof and Kebabian 1981; Undie and Friedman 1990; Surmeier et al. 1992; Jin et al. 2001). Understanding how signaling pathways regulated by coexpressed DA receptors interact to modulate neural function is critical to understanding how abnormal DA signaling in the brain contributes to neurological disorders including schizophrenia and Parkinson’s disease. In C. elegans DA is made and released from eight mechanosensory neurons and acts extrasynaptically to control behavior (Sulston et al. 1975; Chase et al. 2004; Sanyal et al. 2004). DA acts through D1-like (DOP-1) and D2-like (DOP-3) DA receptors in C. elegans and these receptors are expressed on distinct neurons but are also coexpressed on some neurons (Suo et al. 2002; Chase et al. 2004). Orthologs of each of the major G proteins that couple to DA receptors in mammals, including Gαs, Gαi/o, and Gαq, are expressed throughout the C. elegans nervous system (Jansen et al. 1999). C. elegans movement is modulated by DA acting through DOP-1 and DOP-3 receptors such that signaling through DOP-3 and Gαo inhibits locomotion, and signaling through DOP-1, Gαq and PLCβ antagonizes DOP-3 signaling (Chase et al. 2004). While the G proteins and other downstream signaling components of these receptor signaling pathways in C. elegans are conserved and function downstream of DA receptors in mammals, how the receptor signaling pathways functionally interact (in either organism) to modulate neural function remains largely untested. To begin to understand how coexpressed D1-like and D2-like receptors act antagonistically in vivo we performed a genetic screen for DA signaling mutants in C. elegans. The genes we identified allowed us to show that signaling through coexpressed D1and D2-like receptors oppositely modulate acetylcholine release by acting through Gαq and Gαo signaling pathways, respectively, directly in the cholinergic