Dopamine receptor DOP-1 engages a sleep pathway to modulate swimming in C. elegans.

Animals require robust yet flexible programs to support locomotion. Here we report a pathway that connects the D1-like dopamine receptor DOP-1 with a sleep mechanism to modulate swimming in C. elegans. We show that DOP-1 plays a negative role in sustaining swimming behavior. By contrast, a pathway through the D2-like dopamine receptor DOP-3 negatively regulates the initiation of swimming, but its impact fades quickly over a few minutes. We find that DOP-1 and the GPCR kinase (G-protein-coupled receptor kinase-2) function in the sleep interneuron RIS, where DOP-1 modulates the secretion of a sleep neuropeptide FLP-11. We further show that DOP-1 and FLP-11 act in the same pathway to modulate swimming. Together, these results delineate a functional connection between a dopamine receptor and a sleep program to regulate swimming in C. elegans. The temporal transition between DOP-3 and DOP-1 pathways highlights the dynamic nature of neuromodulation for rhythmic movements that persist over time.

Introduction

Animals use various locomotion strategies to benefit their survival. Even simple forms of locomotion like crawling, walking, or swimming require the nervous system to engage muscles in a timely and precise manner. Both invertebrate and vertebrate animals have developed neural circuits to produce stable motor outputs with rhythmic movements (Getting, 1989; Grillner, 2006; Marder and Bucher, 2001; Marder and Calabrese, 1996; Pearson, 2000; Ruder and Arber, 2019). To readily respond to challenges, these neural circuits must be robust yet flexible.

Decades of research show that sustained movements start with activities of central pattern generators (CPGs), components of the nervous system that are capable of producing oscillatory activity without receiving extrinsic information (Delcomyn, 1980; Grillner, 2006; Kiehn, 2006; Marder and Bucher, 2001; Marder et al., 2005). However, to smoothly perform behavioral tasks, a complex network of sensory inputs and feedback mechanisms need to be engaged to shape the activity of CPGs for motor coordination and rhythm modulation (Marder et al., 2015; Marder and Thirumalai, 2002; Miles and Sillar, 2011). The nematode C. elegans utilizes undulatory locomotion to crawl and swim (Cohen and Sanders, 2014; Pierce-Shimomura et al., 2008; Zhen and Samuel, 2015). Multiple CPG oscillators, made up of motor neurons in the C. elegans ventral nerve cord, generate intrinsic network oscillations for body movements (Fouad et al., 2018; Gao et al., 2018; Wen et al., 2018). While it is clear that signals from upstream interneurons and the electrical coupling within the ventral nerve cord are necessary for coordinating pattern generators to produce robust yet flexible oscillations (Kawano et al., 2011; Wen et al., 2012; Xu et al., 2018), the precise modulatory mechanisms that establish dynamic rhythmicity remain largely unknown.

Dopamine is a neurotransmitter of diverse functions including rhythm modulation in both vertebrate and invertebrate animals (Cermak et al., 2020; Harris-Warrick et al., 1998; Puhl and Mesce, 2008; Sharples et al., 2014; Svensson et al., 2003; Vidal-Gadea et al., 2011). In C. elegans, dopamine regulates behaviors such as habituation of mechanical stimuli (Ardiel et al., 2016; Rose and Rankin, 2001; Sanyal et al., 2004), slowing of locomotion in the presence of food (Sawin et al., 2000), the transition from swimming to crawling (Vidal-Gadea et al., 2011), spatial pattern selection (Han et al., 2017), as well as learning and decision-making (Calhoun et al., 2015; Rengarajan et al., 2019; Wang et al., 2014). The impact of dopamine on behaviors in C. elegans is determined by several factors. First, dopamine receptors are widely expressed in the nervous system (Bentley et al., 2016; Chase et al., 2004; Sugiura et al., 2005; Suo et al., 2002, 2003; Tsalik et al., 2003), resulting in many interaction nodes between dopamine and motor circuits. Second, different dopamine receptors in C. elegans, like those in vertebrates, have distinct functions. For instance, in worms, the D1-like dopamine receptor DOP-1 and the D2-like dopamine receptor DOP-3 have opposite effects on locomotion (Chase et al., 2004) and acetylcholine release (Allen et al., 2011), likely through cell-autonomous mechanisms in motor neurons. Third, the strength of dopamine signals through receptor-mediated responses is tuned by G proteins and their regulatory mechanisms (Jiang et al., 2001; Wang et al., 1995). In the case of basal slowing of locomotion on food, DOP-3 activates the Gαo pathway, while DOP-1 acts through the Gαq signaling pathway (Chase et al., 2004). Recently, Topalidou et al. identified a mechanism at the interface between locomotion and dopamine signaling in which the G-protein-coupled receptor kinase-2 (GRK-2) negatively modulates a DOP-3 pathway in premotor interneurons (Topalidou et al., 2017b). GRK-2 leads to inactivation of GOA-1 and activation of NALCN/NCA channels, which subsequently support rhythmic outputs of body bends during crawling and swimming.

Neuropeptides are also key regulators of locomotory activity. Their action to inhibit or activate locomotion is often linked to developmental clocks (Erion et al., 2016; Krupp et al., 2013; Selcho et al., 2017; Taghert and Nitabach, 2012; Xu et al., 2008). During development, C. elegans undergoes four larval molts with a cycle of approximately 8-10 hr (Moss, 2007). During each larval molt, the worms enter into a prolonged period (∼2 hr) of behavioral quiescence with inactivation of locomotion and feeding behaviors, with properties similar to the sleep-like state in mammals and Drosophila melanogaster (Hendricks et al., 2000; Huber et al., 2004; Nelson and Raizen, 2013; Raizen et al., 2008; Shaw et al., 2000). Neuropeptide pathways have been found to regulate this developmentally timed behavior quiescence in C. elegans (Choi et al., 2013; Turek et al., 2016; Van der Auwera et al., 2020). Among them, a FMRFamide-related neuropeptide FLP-11, stored in the sleep interneuron RIS, strongly inhibits locomotion at the onset of sleep (Turek et al., 2016).

In this study, we report a functional link between the dopamine receptor DOP-1 and the sleep peptide FLP-11 for sustaining swimming behavior. We find that DOP-1 and DOP-3 negatively regulate C. elegans swimming through two different circuits. While both pathways are responsible for the defective swimming pattern of grk-2 mutants, the impact of DOP-1 and DOP-3 on swimming is restricted to distinct temporal periods. Together, these results demonstrate a functional integration of a dopamine receptor and a sleep quiescence program in living animals, revealing the dynamic nature of modulation underlying the seemingly constant appearance of repetitive movements.

Results

A requirement for different signals during early and late stages of swimming

We quantified the swimming frequency by tracing changes of mid-point body angle over time. As previously reported (Topalidou et al., 2017b), grk-2 mutants exhibited severe defects in swimming behavior (Figure 1), supporting a positive role of GRK-2 in modulating swimming. The body bend frequency of grk-2 mutants was decreased to less than 0.5 Hz from approximately 2 Hz in wild type animals. The reduction in swimming frequency in grk-2 mutants was largely suppressed by a dop-3(vs106) null mutation (Figures 1A and 1C). These results are consistent with previous findings showing that DOP-3 is a negative regulator of swimming (Topalidou et al., 2017b). However, at 10 min, the body bend frequency of the grk-2; dop-3 double mutant was significantly decreased to a level (∼0.4 Hz) that is indistinguishable from the frequency of grk-2 single mutant swimming (Figures 1B and 1C). Indeed, more than 60% of the grk-2; dop-3 mutants completely stopped swimming by 10 min (Figure 1C right panel). These results showed that the dop-3 mutation suppresses the swimming defect of grk-2 mutants at 1 min but not at 10 min. The reduction of swimming frequency was unlikely due to osmolarity changes upon transferring worms from NGM plates to M9 buffer, as grk-2; dop-3 mutants showed similar decreases in swimming frequency in NGM salt solution (See also Figure S1A).

Figure 1

The grk-2; dop-3 double mutant worms failed to sustain swimming

Changes of mid-point body angles were plotted over a time window of 20 s during swimming. For clarity, positive angles are indicated in red and negative angles are indicated in blue.

(A) Worms were placed in M9 buffer for 1 min. Representative swimming traces are shown for wild-type (WT: N2; top panel), grk-2(gk268) single mutant (middle panel), and grk-2(gk268); dop-3(vs106) double mutant worms (lower panel).

(B) Worms were placed in M9 buffer for 10 min. Representative swimming traces show that grk-2; dop-3 double mutants stopped swimming at the 10 min time point.

(C) Quantification of data. Left: Body bend frequency was quantified and represented as mean ± SEM for WT, grk-2(gk268), dop-3(vs106), and grk-2(gk268); dop-3(vs106) mutants. The grk-2(gk268); dop-3(vs106) double mutants exhibited a swimming defect at the 10 min time point. ∗∗∗p < 0.001, (unpaired Student's t-test). Middle and right panels: Cumulative fraction plots of swimming frequency distribution at 1 min (middle) and 10 min (right) time points. Swimming of grk-2(gk268); dop-3(vs106) double mutants exhibited a shift from high frequency at 1 min to low frequency at 10 min. The frequency distribution curves of grk-2 single and grk-2; dop-3 double mutants were significantly different at 1 min, but they became identical at 10 min (Kolmogorov-Smirnov test). ∗∗∗p < 0.001, n.s., not significant. Statistical analysis and graphing were carried out using Prism 8 (GraphPad).

To determine whether the grk-2; dop-3 mutants had lost their physical ability to swim at 10 min, we poked these worms using a platinum wire. We found that more than 90% of the worms bended their body immediately after poking (See also Figure S1B). However, when examined at 30 s after poking, about 70% of the worms had stopped swimming (See also Figure S1B). These data indicate that the grk-2; dop-3 worms at 10 min were in a quiescent rather than a paralyzed state, as they could initiate but were unable to sustain the body bend activity. Interestingly, after entering the quiescent state, the grk-2; dop-3 mutants stopped pharyngeal pumping (See also Video S1), similar to worms undergoing lethargus on food (Raizen et al., 2008; Van Buskirk and Sternberg, 2007), but different from those experiencing exercise-induced quiescence (Schuch et al., 2020).

Video S1. grk-2; dop-3 mutant worms stopped pharyngeal pumping after entering swimming quiescence, related to figure 1

A 45-s video recording shows that pharyngeal pumping was completely stopped in grk-2; dop-3 double mutants that had entered swimming quiescence after 10-min swimming.

Together, these results show that the grk-2; dop-3 mutants enter a quiescent state at 10 min, indicating that the impact of the D2-like dopamine receptor DOP-3 on swimming fades quickly, and additional mechanisms are necessary for sustained swimming. In contrast to dop-3(vs106), a cat-2(e1112) mutation that disrupts tyrosine hydroxylase for dopamine synthesis suppressed swimming defects in grk-2 mutants at both 1 min and 10 min (See also Figure S1C), suggesting that swimming at 10 min is regulated by dopamine through receptors other than DOP-3.

DOP-1 and DOP-3 act in distinct phases of swimming

C. elegans locomotion is modulated by dopamine through the antagonistic action of the dopamine receptors DOP-1 and DOP-3 (Chase et al., 2004). To determine whether DOP-1 plays a role in delayed swimming quiescence, we introduced the dop-1(vs100) mutation into grk-2; dop-3 mutant worms, which can initiate but fail to sustain swimming. We found that grk-2; dop-1 dop-3 triple mutants remained active swimmers after 10 min (Figure 2A). Body bend frequency of grk-2; dop-1 dop-3 triple mutants was identical at 1 min and 10 min (Figure 2B), and the cumulative distribution of the swimming frequency was shifted to significantly higher values in the grk-2; dop-1 dop-3 triple mutants compared to the grk-2; dop-3 double mutants (Figure 2C). As a control, we investigated the role of DOP-4, the other D1-like dopamine receptor in C. elegans. We found that the dop-4(tm1392) mutant did not restore swimming of grk-2; dop-3 mutants at 10 min (See also Figure S2). To further confirm the role of dop-1, we examined the swimming of grk-2; dop-1 double mutants. We found that these mutant worms were more active in swimming at 10 min than they were at 1 min, indicating a late role of DOP-1 in inhibiting swimming (Figure 3A). Together, these data show that both DOP-1 and DOP-3 are negative regulators of swimming, but their roles are distributed to distinct phases of swimming: DOP-3 modulates early swimming while DOP-1 regulates the late phase of swimming.

Figure 2

DOP-1 and DOP-3 support distinct phases of swimming

(A) Representative swimming traces are shown for grk-2(gk268); dop-1(vs100) dop-3(vs106) triple mutants (A). Left panel, swimming at 1 min. Right panel, swimming at 10 min.

(B) Body bend frequency was represented as mean ± SEM. grk-2; dop-1 dop-3 triple mutants showed identical swimming frequencies at 1 min and 10 min ∗∗∗p < 0.001, (unpaired Student's t-test). n.s., not significant.

(C) Cumulative fraction plots of swimming frequency distribution show a significant difference between grk-2; dop-3 double and grk-2; dop-1 dop-3 triple mutants at 10 min (Kolmogorov-Smirnov test).

Figure 3

DOP-1, neuropeptide FLP-11 and APTF-1 modulate swimming at 10 min.

(A) Representative traces show the swimming behavior of grk-2(gk268); dop-1(vs100) double mutants at 1 min (left) and 10 min (middle). Body bend frequency was quantified and represented as mean ± SEM in the right panel. ∗∗∗ indicates p < 0.001 (unpaired Student's t-test).

(B) Representative traces show the swimming behavior of grk-2(gk268); flp-11(tm2706) double mutants at 1 min (left) and 10 min (middle). Body bend frequency was quantified and represented as mean ± SEM in the right panel. ∗∗∗ indicates p < 0.001 (unpaired Student's t-test).

(C) Representative traces show the swimming behavior of aptf-1(gk794); grk-2(gk268) double mutants at 1 min (left) and 10 min (middle). Body bend frequency was quantified and represented as mean ± SEM (right). Statistical analysis and graphing were carried out using Prism 8 (GraphPad).

The sleep neuropeptide FLP-11 regulates swimming at a late-phase

To gain insight into the mechanisms behind the late-phase quiescence, we looked into pathways that are capable of inducing locomotion quiescence. In particular, we investigated several genes encoding neuropeptides that reduce locomotion activity during sleep-like states. We created double mutants between grk-2 and individual mutations that disrupt flp-11, flp-18, flp-21, or pdf-1 (Bhardwaj et al., 2018; Choi et al., 2013; Turek et al., 2016). We found that grk-2; flp-11 double mutants exhibited strong recovery of swimming at 10 min (Figure 3B). By contrast, disruption of flp-18, pdf-1, or flp-21 did not restore swimming in grk-2 mutants (See also Figure S3). Finally, removal of the AP2 transcription factor APTF-1, which controls the expression of FLP-11 (Turek et al., 2013, 2016), induced a significant recovery of swimming in grk-2 mutants at 10 min, which is similar to the swimming recovery in grk-2; flp-11 double mutants (Figure 3C). Together, these findings indicate that the FLP-11 pathway plays a role in the late stage of swimming.

DOP-3 and FLP-11 pathways act independently

Because grk-2; flp-11 and grk-2; dop-3 worms swim at different time points, we hypothesized that the FLP-11 and DOP-3 pathways function in independently continuous swimming. In support of this idea, simultaneous removal of both flp-11 and dop-3 from grk-2 mutants led to recovered swimming at both 1 and 10 min with identical frequencies (Figure 4A; 1 min: 1.02 ± 0.04 Hz; and 10 min: 0.94 ± 0.04 Hz; n.s.), similar to grk-2; dop-1 dop-3 mutants. The additive impact of DOP-3 and FLP-11 on the sustainability of swimming supports the idea that these genetic pathways act in distinct phases of swimming. Similar to the dop-1 mutation (Figure 2B), deletion of flp-11 from grk-2; dop-3 double mutants also caused a minor reduction in the rate of body bends at 1 min (Figure 4A), suggesting that FLP-11 and DOP-1 have an additional role in supporting early swimming, the opposite of their role in regulating swimming at 10 min.

Figure 4

dop-1, but not dop-3, functions in the same genetic pathway with flp-11

(A) Representative traces show the swimming behavior of grk-2(gk268); flp-11(tm2706) dop-3(vs106) triple mutants at 1 min (left) and 10 min (middle). Body bend frequency was quantified and represented as mean ± SEM (right). ∗∗∗p < 0.001, (unpaired Student's t-test). n.s., not significant.

(B) The left panel shows a schematic diagram for the CRISPR-Cas9 and homologous recombination strategy used to disrupt the flp-11 gene in grk-2; dop-1 mutants. The genotype of the flp-11(pek248) mutant allele was confirmed by PCR amplification using oligos JBO-5540 and JBO-5541. Genotyping results are shown in the right panel.

(C) Body bend frequency was analyzed for grk-2(gk268); dop-1(vs100) double mutants and grk-2(gk268); dop-1(vs100) flp-11(pek248) triple mutants, and was represented as mean ± SEM. ∗∗∗p < 0.001, ∗∗p < 0.01, (unpaired Student's t-test). Statistical analysis and graphing were carried out using Prism 8 (GraphPad).

DOP-1 and FLP-11 act in the same genetic pathway

Next, we examined the genetic relationship between dop-1 and flp-11. Because flp-11 and dop-1 are located close to each other (∼0.7cM) on chromosome X, we employed a CRISPR-Cas9 and homologous recombination strategy to directly disrupt the flp-11 gene in grk-2; dop-1 mutants (Figure 4B). We obtained a flp-11(pek248) mutant allele that carries a deletion within the first intron and the second exon of flp-11 and a large insertion (1.9kb) of a hygromycin-resistance (HygR) cassette in the second exon (Figure 4B). Because these changes disrupt the gene structure of flp-11, we expect that the pek248 mutation is a null allele. Introduction of the flp-11(pek248) mutation into grk-2; dop-1 worms did not further enhance the phenotype of grk-2; dop-1 mutants (Figure 4C), indicating that flp-11 and dop-1 function in the same genetic pathway.

Premotor interneurons and the sleep neuron RIS regulate different phases of swimming

GRK-2 is expressed in many neurons including acetylcholine head neurons and body motor neurons (Topalidou et al., 2017b). Since expression of GRK-2 in premotor interneurons significantly restores locomotion activity during crawling (Topalidou et al., 2017b), we asked whether swimming is also controlled by GRK-2 in premotor interneurons. We introduced a single-copy transgene of nmr-1p::grk-2 into grk-2 mutants. These worms were fully active in early swimming (1 min) with a frequency of 1.7 ± 0.07 Hz (Figure 5), indicating a positive role of GRK-2 in premotor interneurons to initiate swimming. However, the swimming frequency of these worms was significantly reduced to 1.1 ± 0.11 Hz by 10 min, demonstrating that GRK-2 in premotor interneurons could not fully sustain swimming. Therefore, additional neurons must be involved to ensure the continuity of swimming.

Figure 5

GRK-2 acts in premotor interneurons to modulate early phase of swimming

Representative traces show the swimming behavior of transgenic grk-2(gk268) worms carrying a single-copy nmr-1p::grk-2 transgene at 1 min (left) and 10 min (middle), respectively. Body bend frequency was plotted as mean ± SEM (right). ∗∗∗p < 0.001, (unpaired Student's t-test). Statistical analysis and graphing were carried out using Prism 8 (GraphPad).

FLP-11 is secreted from the sleep neuron RIS to inhibit locomotion during development (Steuer Costa et al., 2019; Turek et al., 2016). Consistent with previous findings, we found that the flp-11 promoter (flp-11p) drove specific expression in the RIS neuron (See also Figure S4A). The tight pattern of flp-11 expression prompted us to determine whether the RIS sleep neuron is the functional site for GRK-2 and DOP-1.

First, we asked whether grk-2 is expressed in the RIS neuron. We generated transgenic worms carrying a nuclear green fluorescence reporter 2xNLS:mNeonGreen in RIS (flp-11p::2xnls::mNeongreen) and a cytoplasmic red fluorescence reporter TagRFP under the control of grk-2p (grk-2p::tagrfp). Using confocal fluorescence microscopy, we found that the RIS neuron had both nuclear green and cytoplasmic red fluorescence (Figure 6A), indicating that GRK-2 is expressed in RIS, supporting previous observations through transcriptome analysis (Konietzka et al., 2020).

Figure 6

The sleep interneuron RIS is the functional site for GRK-2 and DOP-1 to regulate late-stage swimming

(A) An image showing GRK-2 expression in the RIS neuron. Transgenic worms carrying grk-2p::tagrfp and flp-11p::2xnls::mNeonGreen were examined using an Olympus FV1000 scanning confocal microscope. The green fluorescent protein mNeonGreen was excited by a 488 nm laser, and the red fluorescent protein TagRFP was excited using a 559 nm laser. The 559 nm laser was also used to collect DIC (differential interference contrast) images. The fluorescent image was superimposed on the DIC image for clarity.

(B) Representative swimming traces for grk-2(gk268); dop-3(vs106) double mutants, in the presence (left) or absence (middle) of a single-copy transgene flp-11p::grk-2. Body bend frequency at 10 min was quantified and shown as mean ± SEM in the right panel. ∗∗∗p < 0.001, (unpaired Student's t-test).

(C) Representative swimming traces for grk-2(gk268); dop-1(vs100) dop-3(vs106) triple mutants, in the presence (left) or absence (middle) of a flp-11p::dop-1 transgene. Body bend frequency at 10 min was quantified and shown as mean ± SEM (right). ∗∗∗p < 0.001, (unpaired Student's t-test).

(D) The left panel shows a schematic diagram of the coelomocyte assay used to quantify FLP-11 secretion from RIS. Worms carrying an integrated transgene (pekIs116 [flp-11p::flp-11::yfp]) were examined using the Olympus FV1000 scanning confocal microscope. The yellow fluorescent protein mVenus was excited using the 488 nm laser. Representative images of anterior coelomocytes in wild type (wt), grk-2(gk268); dop-3(vs106) double, and grk-2(gk268); dop-1(vs100) dop-3(vs106) triple mutant worms are shown in the middle. Fluorescence intensity of YFP puncta in coelomocytes was quantified and is shown as mean ± SEM in the right panel. ∗∗∗p < 0.001, (one-way ANOVA with Dunnett's multiple comparisons test).

Next, to determine whether GRK-2 acts in RIS to sustain swimming, we expressed a flp-11p::grk-2 transgene in grk-2; dop-3 worms that are able to initiate swimming but fail to sustain it at late stages (Figure 1), and examined the ability of these transgenic worms to swim at the 10 min time point. In contrast to grk-2; dop-3 worms that fail to sustain swimming, expression of flp-11p::grk-2 fully restored the body-bend frequency of grk-2; dop-3 worms at 10 min (Figure 6B). These data show that GRK-2 in the RIS sleep neuron is sufficient to support late-stage swimming. As a control, GRK-2 expression in the premotor interneurons by nmr-1p did not show rescue activity at 10 min (See also Figures S4B, and S4C), confirming that the role of premotor neuron GRK-2 is mainly limited to the early phase swimming. Together, these results indicate that GRK-2 functions in RIS and premotor interneurons to support distinct temporal phases of swimming.

Finally, we examined the role of DOP-1 in RIS using cell-specific rescue experiments. DOP-1, like GRK-2, is expressed in many neurons including RIS (Konietzka et al., 2020; Tsalik et al., 2003). We found that expression of a flp-11p::dop-1 transgene in grk-2; dop-1 dop-3 mutants inhibited late-stage swimming (Figure 6C), indicating that DOP-1 in RIS is sufficient to negatively modulate late-stage swimming. In addition, the flp-11p::dop-1 transgene slightly increased the swimming frequency at 1 min (See also Figure S4D), suggesting that DOP-1 may have a minor role in RIS to promote early swimming. In summary, these cell-specific rescue experiments show that GRK-2 and DOP-1 act in RIS to modulate late-phase swimming.

DOP-1 regulates FLP-11 secretion from the RIS neuron

To understand the functional connection between DOP-1 and FLP-11 in RIS, we next quantified the levels of FLP-11 secretion using an established coelomocyte uptake assay (Ch'ng et al., 2008; Fares and Greenwald, 2001; Sieburth et al., 2007). FLP-11:YFP was expressed in RIS using the flp-11 promoter. We quantified YFP fluorescence intensity in coelomocytes as a readout of secreted FLP-11:YFP that had been taken up by the scavenger coelomocytes. Our results showed a low level of yellow fluorescence in coelomocytes in wild-type worms (Figure 6D), supporting the idea that there is little FLP-11 secretion in awake wild-type worms (Turek et al., 2016). By contrast, significantly increased levels of YFP signal were found in grk-2; dop-3 coelomocytes (Figure 6D), suggesting that FLP-11 secretion was elevated in these double mutants. Finally, we found that, in grk-2; dop-1 dop-3 triple mutants, coelomocyte YFP fluorescence was returned to a low level (Figure 6D), similar to that in wild-type animals. These data show that the secretion of the sleep peptide FLP-11 from the RIS neuron was promoted by the dopamine receptor DOP-1. Because RIS is a sleep neuron and its activity is regulated, we speculated that uncoupling FLP-11 secretion from RIS activity could cause irregularity of swimming activity. Consistent with this idea, we found that ectopic expression of FLP-11 in body wall muscles of wild-type worms slightly but significantly reduced swimming frequency at both 1 min and 10 min (See also Figure S4E), suggesting that miscontrolled FLP-11 secretion inhibits swimming.

Neuropeptide receptors couple FLP-11 to swimming quiescence

As a neuropeptide, FLP-11 requires G-protein-coupled receptors to carry out its function. Previous studies have identified FRPR-3, NPR-4, and NPR-22 as candidate receptors for FLP-11 (Chew et al., 2018; Cohen et al., 2009; Frooninckx et al., 2012; Mertens et al., 2004, 2006). To determine which neuropeptide receptor acts in the FLP-11 pathway to regulate late-phase swimming quiescence, we examined grk-2; frpr-3 double mutant worms, and found that removal of FRPR-3 led to a partial but significant increase in swimming frequency at 10 min (Figure 7A). Both npr-22 and npr-4 mutations, when introduced into grk-2; frpr-3 mutants, each further enhanced the restoration of swimming (Figure 7A). Finally, the grk-2; npr-22; frpr-3; npr-4 quadruple mutants showed restored swimming frequency that was indistinguishable from grk-2; flp-11 double mutants at 10 min (Figures 7A and 7B). These data show that FLP-11 engages several receptors, whose activation reduces swimming at a late phase.

Figure 7

Multiple receptors are responsible for coupling FLP-11 to swimming regulation

(A) Body bend frequency of mutant worms that carry grk-2(gk268) and mutations disrupting candidate FLP-11 receptors FRPR-3, NPR-22, and NPR-4 was determined and plotted as mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001, compared to grk-2 single mutants (one-way ANOVA with Dunnett's multiple comparisons test).

(B) Representative swimming traces of grk-2; npr-22; frpr-3; npr-4 mutants at 1 min (left) and 10 min (right).

(C) Representative swimming traces of transgenic grk-2; npr-22; frpr-3; npr-4 mutant worms that carry twk-47p::frpr-3 at 1 min (left) and 10 min (right).

(D) Body bend frequency at 10 min. Data are represented as mean ± SEM. ∗∗∗p < 0.001 (unpaired Student's t-test).

Expression of FRPR-3, NPR-4, and NPR-22 is fairly broad in C. elegans (Chew et al., 2018; Cohen et al., 2009; Turek et al., 2016). For example, FRPR-3 is expressed in approximately 30 neurons, and NPR-4 is expressed in about 10 neurons. This complex pattern of expression makes it difficult to pinpoint their site-of-action. To get a glimpse into where these neuropeptide receptors function, we investigated the interneuron AVK, which expresses FRPR-3, and receives direct synaptic input from RIS. Previous studies have shown that inhibition of AVK induces slowed swimming, as well as frequent and prolonged stops during crawling (Hums et al., 2016; Oranth et al., 2018). To determine the role of AVK FRPR-3 in swimming, we expressed FRPR-3 using the AVK specific promoter twk-47p (Lorenzo et al., 2020) in grk-2; npr-22; frpr-3; npr-4 mutants. We found that expression of FRPR-3 in AVK significantly reduced swimming frequency at 10 min (Figures 7C and 7D). These data indicate that expression of FRPR-3 in AVK interneurons is sufficient to induce quiescence during late-stage swimming, suggesting that AVK is one of the neurons that receive FLP-11 signals from RIS to regulate swimming.

Discussion

We showed that swimming of C. elegans is controlled by two distinct dopaminergic pathways. In particular, we found that the D1-like dopamine receptor DOP-1 negatively regulates the frequency of body bends through FLP-11, a neuropeptide required for behavior quiescence during development. We showed that the DOP-1/FLP-11 pathway acts together with the known DOP-3/NALCN regulatory pathway to ensure a continuous pattern of swimming (Pierce-Shimomura et al., 2008; Topalidou et al., 2017b). While both pathways regulate swimming, their action differs. Physiologically, the DOP-3/NALCN pathway functions early during an initial phase of swimming. By contrast, the DOP-1/FLP-11 regulation acts late to inhibit swimming. The DOP-1/FLP-11 and DOP-3/NALCN pathways also differ at the circuit level. The DOP-3/NALCN pathway functions in premotor interneurons (Topalidou et al., 2017b). However, DOP-1 and FLP-11 act in the RIS sleep interneuron. Together, these results show that orchestration of dopaminergic and neuropeptidergic signals sets the foundation for sustained swimming.

An integrated dopamine signaling network for distinct phases of rhythmic behavior

Swimming is a classic example of a rhythmic behavior that requires neurons to produce locomotory patterns. Unlike reflexes, body bends during swimming occur in continuous sequences in a repeated manner. In C. elegans, dopamine plays a key role in modulating several aspects of swimming. For instance, dopamine is necessary and sufficient for worms to switch locomotory patterns from swimming to crawling, and such transition of locomotory gaits requires D1-like dopamine receptors (DOP-1 and DOP-4), but not D2-like receptors (DOP-2 and DOP-3) (Vidal-Gadea et al., 2011). In addition, worms that lack the plasma membrane dopamine transporter DAT-1, when placed in water, swim briefly and then become paralyzed (termed swim-induced paralysis; SWIP) (Hardaway et al., 2012; McDonald et al., 2007). While SWIP and the swimming quiescence of grk-2; dop-3 double mutants appear to be phenotypically similar, the underlying mechanisms are different – SWIP depends solely on DOP-3 (McDonald et al., 2007), which is absent in the grk-2; dop-3 mutant worms. Instead, the swimming quiescence of grk-2; dop-3 mutants is mediated by DOP-1 and FLP-11, a neuropeptide secreted from the sleep neuron RIS. Unlike the swim-to-crawl transition, DOP-4 is not required for the swimming quiescence observed in our study. Together, C. elegans swimming is modulated by a network of distinct dopaminergic pathways.

While the frequency of movements remains largely constant over a long period of swimming, mechanisms for sustaining the rhythm appear to be dynamic. The impact of the DOP-3 pathway is at the initial phase of swimming, and it quickly fades with time. By contrast, the engagement of the DOP-1/FLP-11 pathway is slow, and it acts to modulate swimming rhythm at late time points (see Figure 8). The integration of these pathways may be more complex than simple arithmetic addition, as they act in distinct circuits (premotor vs. sleep neurons) and take effect at different time points, indicating the necessity for dynamic recruitment of circuits and fine-tuning of the strength of modulations. While our results established a genetic link between dopamine and sleep mechanisms, the underlying neural circuits and their regulatory dynamics require further investigation.

Figure 8

Model for dopamine modulation of swimming in C. elegans

The dopamine receptors DOP-1 and DOP-3 negatively regulate C. elegans swimming through two different circuits, and produce physiological impact on swimming at distinct temporal periods. The D1-like dopamine receptor DOP-1 negatively regulates the frequency of body bends through FLP-11, a neuropeptide released from the sleep neuron RIS. The AP2 transcription factor APTF-1 controls FLP-11 expression in RIS. The FLP-11 signal inhibits AVK activity via receptors including FRPR-3, and AVK positively regulates swimming (Hums et al., 2016; Oranth et al., 2018). The known DOP-3/NALCN pathway functions in premotor neurons to modulate an initial phase of swimming. DOP-3 negatively controls NALCN, and NALCN acts in premotor neurons to promote swimming. By contrast, the DOP-1/FLP-11 regulation acts in the sustained phase of swimming.

How does a system maintain stable output of rhythmic movements while the underlying circuits undergo changes? Possibly, regulatory enzymes in G protein signaling pathways (e.g., the GPCR kinase GRK-2) may function to fine-tune the weight of individual cellular signals (e.g., dopamine and neuropeptides) to stabilize the output of a system. In C. elegans, GRK-2 might function as a positive regulator of swimming by inactivating two dopamine receptors, DOP-1 in the RIS neuron and DOP-3 in premotor interneurons, which negatively regulate swimming via peptide signaling and channel modulation, respectively.

The delicate nature of balanced states of modulatory pathways can be easily disrupted by pathologic conditions and sometimes even by unwanted side effects from therapeutic treatments. For example, long-term usage of L-dopa and dopamine agonists in Parkinson's patients alters dopamine receptor function through mis-regulated G-protein pathways, which leads to movement defects with disrupted rhythm (Corvol et al., 2004; Heumann et al., 2014; Lanza et al., 2018; Nagatsua and Sawadab, 2009), suggesting a functional link between dopamine-dependent G-protein pathways and abnormal rhythm regulation. The diversity of dopamine receptors and their non-uniform distribution across the nervous system add additional layers of complexity, which may explain the breadth of the phenotypic spectrum seen in patients with dopaminergic dysfunction. In fact, the abnormality of rhythmic control in Parkinson's patients often goes beyond locomotion, such as walking and swimming, and reaches a number of non-motor activities including breathing and sleep (Connolly and Lang, 2014; Kalia and Lang, 2015; Olanow et al., 2009; Stefani and Hogl, 2020), revealing a complex but perhaps vulnerable network of dopaminergic regulation of rhythmic behaviors.

Recurrent mechanisms are engaged in biological events with different rhythms

Biological rhythms are diverse. They enable organisms to organize behavior and metabolism on different time scales for various biological purposes. For example, a circadian system typically oscillates once per 24 hr, while actions during running and swimming may take place more than once per second. It is not surprising that underlying mechanisms for slow and fast rhythmic events are distinct. However, previous studies have revealed some striking mechanistic similarities in rhythmic regulation on distinct time scales, i.e., recurrent mechanisms that impact neuronal excitability and control locomotory quiescence are often engaged in both fast and slow rhythmic activities. For example, dopamine has a conserved role in modulating sleep-wake states in worms, flies, and mice (Andretic et al., 2005; Dzirasa et al., 2006; Kume et al., 2005; Singh et al., 2014). Similarly, FLP-11 is a neuropeptide known to regulate slow developmental processes by inducing sleep-like behavior prior to each of the four molts in C. elegans (Turek et al., 2016). The periodicity of the molting cycle is controlled by lin-42, a gene homologous to the fly circadian gene per (Jeon et al., 1999; Monsalve et al., 2011). NALCN channels are also involved in sleep regulation during circadian cycles in flies, mice, and humans (Cochet-Bissuel et al., 2014; Flourakis et al., 2015; Funato et al., 2016; Lozic et al., 2016). Meanwhile, our findings here, as well as others, show that FLP-11 and NALCN channels also modulate locomotion and swimming behaviors, which may occur on a timescale of a second or less (Topalidou et al., 2017a, 2017b). It appears that recurrent mechanisms are used for slow and rapid rhythmic activities, and perhaps with distinct regulatory programs. For example, during slow oscillatory changes such as sleep and circadian locomotory quiescence, a transcription network including clock genes and the AP2 transcription factor is employed to control the expression of locomotory modulators (e.g., FLP-11 (Turek et al., 2016) and NALCN channel localization factor-1 (Flourakis et al., 2015)). By contrast, for oscillatory behaviors like swimming and crawling, the rapid tuning of rhythmic behaviors is achieved through a connection between fast modulators like dopamine and recurrent quiescence programs used in slow biological rhythms.

Limitations of the study

While this study established a functional link between dopamine and sleep mechanisms, the underlying neural circuits and their regulatory dynamics require further investigation.

Resource availability

Lead contact

Further information and requests for resources and data should be directed to and will be fulfilled by the lead contact, Jihong Bai (jbai@fredhutch.org).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact without restriction.

Data and code availability

Data and code are available from the corresponding author upon request.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.