Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans

Abstract

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- and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1- 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. 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- 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.

Figure 1

Mutations and signaling pathways that control locomotion in C. elegans. (A) Schematic of the opposing dopamine-signaling pathways that act to modulate locomotion behavior in C. elegans. Dopamine inhibits locomotion by binding to DOP-3 and activating Gαo signaling, while dopamine enhances locomotion by binding to DOP-1 and activating Gαq signaling. The G protein-signaling components identified in our genetic screen are indicated by shaded ovals. Names of C. elegans-signaling proteins are shown inside the ovals and names of their mammalian homologs are shown outside the ovals. (B) Alignment of the third transmembrane domain region (TMDIII) of AMPA glutamate receptors from C. elegans and mammals. The residues mutated in glr-1(nd38) and the Lurcher mouse are indicated. A solid background indicates identical residues; a shaded background indicates chemically similar residues. (C) Schematic of the neural circuitry in C. elegans that controls locomotion behavior. Many sensory neurons, including PLM, ASH, and AVM, innervate the command interneurons (AVD, AVA, AVE, AVB, and PVC), which integrate these signals and activate the cholinergic motor neurons to control forward and backward locomotion. GLR-1 expression is largely restricted to the command interneurons, the DOP-1 and DOP-3 receptors are coexpressed in the cholinergic motor neurons, and ACE-1 is expressed in the muscle cells. Arrows indicate chemical synapses between the indicated cell types and bars indicate gap junctions.

Figure 2

Analysis of dopamine-signaling defects in ace-1(nd35) and glr-1(nd38) mutants. (A) Dose-response curves measuring paralysis induced by exogenous dopamine. Shown is the percentage of animals moving 10 min after being placed on agar plates containing the indicated concentrations of dopamine. Each data point represents the mean ± SEM for three trials totaling at least 75 animals. (B) Quantitative analysis of basal slowing behavior. For each strain, locomotion rates in the absence of bacteria (open bars) and the presence of bacteria (solid bars) were calculated as the average of 30 observations. Error margins shown indicate 95% confidence intervals. Asterisks indicate values significantly different from the 46% slowing seen in the wild type (Student's t-test: *P < 0.01, **P < 0.0001). The percentage slowing in the presence of bacteria for each strain is shown at the right.

Figure 3

Quantitative analysis of swimming-induced paralysis. Swimming-induced paralysis is mediated by DOP-3 signaling in the ventral-cord motor neurons. (A) Photomicrograph of a double-transgenic animal in which green fluorescent protein (GFP) is expressed in the cholinergic motor neurons using the acr-2 promoter, and red fluorescent protein (mCherry) is expressed in the GABAergic motor neurons using the unc-47 promoter (bottom bracket). A few other neurons located in the head (top bracket) express GFP or mCherry, but these neurons do not innervate body-wall muscles to control locomotion. Scale bar, 20 μm. (B) SWIP behavior of control nontransgenic strains. Each measurement shown represents the mean of five trials of 10 L4 animals each for a total of 50 animals. Error bars represent 95% confidence intervals. (C) SWIP behavior of dat-1; dop-3 double mutants carrying transgenes. The promoters used for transgenic expression are indicated at the bottom. Shaded bars represent measurements from control strains carrying empty vector transgenes, which have promoters but no receptor sequences. Solid bars represent measurements from strains carrying transgenes from which the promoters express the DOP-3 receptor. For each transgene, measurements of 50 animals for each of five independent transgenic lines were averaged, and the means and 95% confidence intervals are shown. An asterisk indicates that receptor expression gave significant rescue compared to the control (unpaired Student's t-test: P < 0.0001). Both the acr-2 and the unc-47 promoters gave significant rescue, indicating that swimming-induced paralysis is caused, at least in part, by dopamine acting through DOP-3 receptors expressed in both the cholinergic and the GABAergic motor neurons.

Figure 4

Quantitative analysis of SWIP behavior of dopamine-signaling mutants. (A–D) Each measurement 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. (A) dat-1 mutants are paralyzed compared to wild-type animals. (B) Mutations in the DOP-1 and DOP-3 receptors have opposite effects on SWIP. (C) All dopamine-signaling mutants predicted to increase acetylcholine signaling in the neuromuscular junction suppress dat-1 swimming-induced paralysis. (D) glr-1(nd38) and ace-1(nd35) mutations suppress SWIP. Asterisks in C and D indicate a significant difference from dat-1 mutants (Student’s t-test: **P < 0.0001). Asterisks in B indicate significant difference between the dat-1; dop-1 dop-3 triple mutant and the double mutants indicated (Student's t-test: *P < 0.05, **P < 0.0001).

Figure 5

dop-3(vs106), ace-1(nd35), and glr-1(nd38) mutations all cause increased acetylcholine release into the neuromuscular junction. (A–C) Quantitative analysis of acetylcholine release into the neuromuscular junction. Shown is the percentage of animals paralyzed at the indicated times after being placed on agar plates containing 1 mm of aldicarb. Each data point represents the mean ± SEM for three trials totaling at least 75 animals.

Figure 6

DOP-1 and DOP-3 receptors act antagonistically in the cholinergic motor neurons to modulate acetylcholine release. (A and B) Quantitative analysis of acetylcholine release into the neuromuscular junction. Shown is the percentage of animals paralyzed at the indicated times after being placed on agar plates containing 1 mm of aldicarb. Each data point for nontransgenic animals represents the mean ± SEM for three trials totaling at least 75 animals. For transgenic animals, each data point represents the average of 250 animals (two trials of 25 animals per line and a total of five lines per transgene). (A) The DOP-3 receptor acts to inhibit acetylcholine release from the cholinergic motor neurons. (B) The DOP-1 receptor acts to enhance acetylcholine release from the cholinergic motor neurons.