The dopamine receptor antagonist haloperidol disrupts behavioral responses of sea urchins and sea stars

The superphylum Deuterostomia includes the phylum Chordata, which includes vertebrates, and the invertebrate phyla Echinodermata and Hemichordata (acorn worms). Despite the common evolutionary ancestry of these animals, the organization of the adult echinoderm nervous system differs dramatically from most of these and other animals because of their lack of a brain and their pentaradial symmetry in the adult forms. Instead of a brain, echinoderms have a circumoral nerve ring from which project five radial nerves, which together have been equated to their central nervous system. The radial nerve cords can be divided into the oral-side ectoneural region, and an aboral side hyponeural region that innervates muscles and tube feet (Cobb, 1985). Regardless of the seemingly gross structural organization of their nervous systems, echinoderms are able to detect and respond to their environment with complex and coordinated behavioral responses. For example, both sea urchins and sea stars will right themselves if inverted, which involves the coordinated directional movement of many tube feet (Parker, 1922; Shah et al., 2018). Other well-characterized behaviors include covering, in which some sea urchin species will cover themselves in nearby oceanic debris (Reese, 1966), phototaxic responses (Yoshida, 1966; Yerramilli and Johnsen, 2010; Shah et al., 2018), and burrowing by some species of sea stars, the heart urchins and sand dollars (Reese, 1966). There is also evidence of learning and memory in echinoderms, particularly in sea stars (Freas and Cheng, 2022).

Any animal behavior requires a motivating drive and the successful coordination of multiple motor neurons. A key neurotransmitter modulating animal behavior is dopamine, which has been shown to modulate motor function in virtually all animals, from arguably the simplest animal nervous systems of cnidarians to the much more complex nervous systems of mammals (Barron et al., 2010). This motor function is a key aspect of behavior and requires not only the coordination of muscles, but also the motivation to perform the intended motor action. The decision, whether conscious or unconscious, to enact a motor response is mediated in part by dopaminergic systems involved in the reward system, with dopaminergic neurons in the ventral striatum that project to the nucleus accumbens in vertebrates being particularly important. In many invertebrates, dopamine has also been shown to have a role in the reward systems, including in mollusks, Drosophila and Caenorhabditis elegans (Barron et al., 2010). Its role in echinoderms is less well studied, although there have been a small number of studies examining the presence and function of dopamine. The genome of the purple sea urchin Strongylocentrotus purpuratus, for example, contains genes encoding tyrosine hydroxylase and monoamine oxidase, which are needed to synthesize and metabolize dopamine, respectively, and seven genes encoding dopamine receptors have been detected (Burke et al., 2006). Dopamine itself has been shown to modulate the stiffness of spine ligaments in the sea urchin Eucidaris tribuloides (Morales et al., 1993), and in sea stars dopamine has been detected in the regenerating arms of Asterina gibbosa (Huet and Franquinet, 1981), and can inhibit the luminescence of photocytes in the brittle star Amphipholis squamata (De Bremaeker et al., 2000). Additionally, it has recently been shown that the dopamine receptor antagonist haloperidol inhibits the righting response in the sea urchin S. purpuratus with an RT50 (drug concentration at which righting time was slowed by 50% of the maximum righting time) of 10.7 µmol l⁻¹ (McDonald et al., 2022). In that study, haloperidol was found to not affect the motility of the tube feet, suggesting a role for dopamine receptors in the neural processing of the behavior rather than actual movement, which is achieved via the water vascular and tube feet systems. The effects of haloperidol contrasted with those of antagonists of glycinergic and adrenergic receptors, which inhibited both the righting response and the motility of the tube feet, suggesting more of a role in the actual mechanics of movement with less relative importance in processing the behavioral response compared with the dopaminergic system. It is not known whether the dopaminergic system is needed to process the behavioral response solely for this unique behavior in this particular species, or whether it is essential for behavioral responses in general in echinoderms. In order to address whether dopamine receptors are needed for different behavioral responses in different echinoderms, we quantified multiple different behaviors in the cold-water purple sea urchin (S. purpuratus), the warm-water green sea urchin (Lytechinus variegatus) and the warm-water gray sea star (Luidia clathrata) and determined whether haloperidol could inhibit these behaviors.

Purple sea urchins, Strongylocentrotus purpuratus (Stimpson 1857), from the Pacific Ocean, approximately 60 mm in test diameter, were obtained from Marinus Scientific (Long Beach, CA, USA) and maintained in circulating aerated tanks at 13°C containing artificial seawater (ASW) (Instant Ocean; That Pet Place, Lancaster, PA, USA) made according to the manufacturer's instructions. All behavioral tests on S. purpuratus were conducted in ASW solutions chilled to 10–15°C.

Green sea urchins, Lytechinus variegatus (Lamarck 1816), approximately 50 mm in test diameter, and gray sea stars, Luidia clathrata (Say 1825), approximately 70 mm diameter, were obtained from the Gulf Coast of Florida via Gulf Specimens (Panacea, FL, USA). Both were maintained in separate circulating aerated tanks containing ASW at room temperature (20°C), and behavioral tests on these species were conducted at 20°C.

All drugs were purchased from Millipore Sigma (St Louis, MO, USA). The following drug stock solutions were made and used immediately or stored at −20°C until required: 100 mmol l⁻¹ hexamethonium in ASW and 100 mmol l⁻¹ haloperidol in methanol. Test solutions were made fresh each day from stocks and diluted in ASW, and in the case of haloperidol methanol to give a final concentration of 0.1% (v/v) methanol. To administer the drugs, animals were immersed in tanks containing ASW, 0.1% methanol or 0.1% methanol plus haloperidol where appropriate, for 1 h and the behavioral tests were conducted in the stated solutions. A 1 h preincubation has previously been found to be sufficient to allow drug absorption and observe behavioral effects of the drug on sea urchins (McDonald et al., 2022).

Righting assays were performed as in McDonald et al. (2022). Briefly, animals were preincubated in test solutions for 1 h, and then were inverted and placed on their aboral side on the base of a tank containing ASW with or without the specified drugs. The time to completely right themselves was recorded, with a cut-off of 15 min applied for the sea urchins and 5 min for the sea stars; for statistical analysis, these cut-off values were used as the time to right. If animals in control conditions failed to right within these time limits, they were excluded from further testing.

A lantern reflex assay was adapted from the method of Brothers and McClintock (2015). An individual urchin was preincubated in test solutions for 1 h, removed from ASW and placed inverted (oral side up) in a plastic tray. A 200 µl plastic pipette tip was used as the probe to elicit the lantern reflex, either as just the tip or coated with an algae paste. To ensure the urchin was aware of the probe's presence, the probe was initially briefly touched on the urchin's mouth at the beginning of the testing. The probe was then repositioned approximately 50 mm away from the urchin's mouth using a flexible clamp. A 3 min video recording (Olympus Tough TG-5 camera, 8.3 MP, 30 frames s⁻¹) was obtained, and the number and duration of each completed lantern reflex (one complete cycle of opening the mouth and then closing the mouth) within the 3 min was determined. The algae paste was made by mixing crushed Tropical Sinking Wafers (Hikari, Hayward, CA, USA) with ASW to a concentration of 0.78 g ml⁻¹. This concentration was found to give a suitable consistency to be able to apply the paste to the probe but also to prevent the paste from dripping onto the urchin.

Animals were preincubated in test solutions for 1 h and then emersed by placing them on a circular glass platform (diameter 70 mm, height 70 mm), with approximately 2 mm of ASW covering the platform to enable continued functioning of their water vascular systems and tube foot movement. Animals were placed oral side down onto the center of the platform, and the time to immerse was recorded as the time when all of the tube feet had detached from the horizontal surface of the platform. If animals in control conditions failed to immerse within 15 min, they were excluded from further testing.

Sea urchins were preincubated in test solutions in individual containers covered with thick black cloth to eliminate all light. After preincubation, 12 olive snail shells (Florida Shells and Gifts, Dunedin, FL, USA) were placed in a circle around each sea urchin (Fig. 4A). The mean size of the shells used was 20.5±0.31 mm (s.d.), N=150, and the containers were left for 1 h in the ambient laboratory light (approximately 300 lux). After this time, each sea urchin was gently lifted and the number of attached shells was counted. The pretest dark acclimatization was found to be necessary to ensure adequate and reproducible covering data.

Multiple comparisons between control and drug conditions were analyzed using two-way ANOVA tests with Bonferroni correction. Where appropriate, Welch's unpaired t-tests were used for comparing two conditions. Error bars indicate s.e.m. Differences were deemed significant at the P<0.05 level.

Previously, we have shown that the dopamine receptor antagonist haloperidol substantially slows the righting response of the cold-water sea urchin S. purpuratus (McDonald et al., 2022). Therefore, we were interested in whether this is a general feature of echinoderm behavior, or whether it is specific to this species and this behavioral response. We chose the sea urchin L. variegatus and the sea star L. clathrata to compare with S. purpuratus. Although both L. variegatus and S. purpuratus are members of the class Echinoidea within the phylum Echinodermata, they are adapted to different ecosystems, with L. variegatus used in these studies from the warm waters of the Gulf of Mexico and the S. purpuratus specimens from the cold waters of the California Pacific Ocean. Under control conditions (ASW or 0.1% methanol vehicle), L. variegatus righted 4 to 5 min after being inverted (Fig. 1A). Application of 100 µmol l⁻¹ haloperidol significantly slowed the righting response approximately 3-fold (two-way ANOVA with Bonferroni correction, P<0.0001). There was no significant effect on righting time of vehicle alone when compared with ASW (P=0.85). To determine whether haloperidol has similar effects on behavior in an echinoderm from a different subphylum as sea urchins (Echinozoa), we utilized the sea star L. clathrata, which, along with all other sea stars and brittle stars, forms the Asterozoa subphylum of Echinodermata. The warm-water sea star L. clathrata righted much faster than the sea urchins, with righting times of approximately 1 min under control (ASW or 0.1% methanol) conditions (Fig. 1B). As with the sea urchins, 100 µmol l⁻¹ haloperidol significantly slowed the righting response (two-way ANOVA with Bonferroni correction, P=0.023), with the vehicle having no significant effect on the righting time (P=0.89). Therefore, animals in two different subphyla of Echinodermata both have their righting responses slowed by haloperidol. These experiments also demonstrated that simple immersion in a drug solution for 1 h is a suitable drug administration protocol for sea stars and is sufficient for the drug to be absorbed, reach its target tissue and elicit its effects.

It may be that haloperidol specifically inhibits just the righting response, but not other behavioral responses of echinoderms. To investigate this in S. purpuratus, we utilized the lantern reflex (Brothers and McClintock, 2015), in which a sea urchin opens and closes its teeth whilst extending them towards a source of offered food (Fig. 2A). We first confirmed that we could quantify the lantern reflex, and that the reflex was occurring in response to food. There was a 9-fold increase in the number of lantern reflexes when a probe coated with algae paste was offered compared with when a probe with no food was offered (Fig. 2B) (Welch's t-test, P<0.0001). We next examined the effect of preincubation in 0.1% methanol vehicle and 100 µmol l⁻¹ haloperidol on the number of lantern reflexes (Fig. 2C). Whereas the inclusion of 0.1% methanol had no effect on the number of lantern reflexes compared with ASW (two-way ANOVA with Bonferroni correction, P=1.0), the inclusion of 100 µmol l⁻¹ haloperidol significantly reduced the mean number of reflexes from 6.3 to 1.8 (P<0.0001). Given that the lantern reflex assay was performed within a set time window (3 min), it may be that if haloperidol was slowing the time it took to complete each lantern reflex, then this could potentially account for the reduced number of reflexes observed in haloperidol. Therefore, we measured the duration of each individual reflex for each individual sea urchin (Fig. 2D), in vehicle and in 100 µmol l⁻¹ haloperidol. Haloperidol led to a slight slowing of the mean duration of each reflex, from 10.6 to 13.3 s (Welch's t-test, P<0.044). However, this 25% increase in lantern reflex duration is not enough to account for the 340% decrease in the number of reflexes seen with 100 µmol l⁻¹ haloperidol. To determine whether the inhibition of the lantern reflex is specific to actions of haloperidol, we also tested the nicotinic acetylcholine receptor antagonist hexamethonium (Fig. 2E,F). At 100 µmol l⁻¹ hexamethonium, there were no significant effects on either the number or the duration of the lantern reflexes (two-way ANOVA with Bonferroni correction, P=0.42 for number and P=0.90 for duration). Repeating the assay using 10 mmol l⁻¹ hexamethonium, a concentration that has previously been shown to dramatically reduce righting times (McDonald et al., 2022), still had no significant effect on either the number (P=0.16) or the duration (P=0.07) of the lantern reflexes.

To determine the effect of haloperidol on a further behavioral response, we developed an immersion assay in which individual animals were preincubated in either ASW, 0.1% methanol vehicle or 100 µmol l⁻¹ haloperidol and then placed on a platform slightly raised above the water level from which they used their tube feet to move back into the water (Fig. 3A). In ASW or vehicle alone, S. purpuratus and L. variegatus both immersed themselves within a few minutes of being emersed, whereas the much more rapidly moving L. clathrata immersed in approximately 20 s in ASW or vehicle (Fig. 3). None of the treatments affected the time to immerse of S. purpuratus (two-way ANOVA with Bonferroni correction, P=1 for ASW versus vehicle, P=0.56 for vehicle versus haloperidol) or L. clathrata (two-way ANOVA with Bonferroni correction, P=1 for ASW versus vehicle, P=0.89 for vehicle versus haloperidol). However, 100 µmol l⁻¹ haloperidol, but not 0.1% methanol only, was found to significantly increase the time needed to immerse of L. variegatus (two-way ANOVA with Bonferroni correction, P=0.25 for ASW versus vehicle, P<0.0001 for vehicle versus haloperidol).

Some sea urchin species are known to utilize a covering behavior in their natural habitat or when suitable objects are provided to them in their aquarium (Millott, 1956; Zhao et al., 2014; Barros et al., 2020). The covering behavior involves the grasping of shells and other debris and holding them close to their test with their tube feet, and this behavior has been documented many times for L. variegatus (Millott, 1956). Therefore, we determined whether this behavior in this species is influenced by haloperidol. We designed a covering assay in which individual sea urchins were provided with sea shells and the number of shells that the urchins grasped following the different treatments was determined. In control and vehicle conditions, L. variegatus typically attached four shells (Fig. 4), and there was no significant difference in the number of shells between these conditions (two-way ANOVA with Bonferroni correction, P=1). However, treatment with 100 µmol l⁻¹ haloperidol significantly decreased the mean number of attached shells (two-way ANOVA with Bonferroni correction, P=0.024). It has not been previously reported as to whether S. purpuratus performs the covering behavior. In control experiments, using just the vehicle alone, 50% of S. purpuratus (N=14) did not use a single shell to cover. Given the low level of covering in this species, we did not pursue this behavior in this species further.

Our results demonstrate that the dopamine receptor antagonist haloperidol can inhibit multiple different behaviors in different echinoderm species, and may indicate a general role for dopamine in modulating motor function, and potentially motivation, in echinoderms. Any echinoderm behavioral response can be broken down into multiple discrete steps. There is the detection of the stimulus, the actual neural processing of the stimulus to come to a ‘decision’ on a course (or not) of action, and the implementation of a motor response coordinated across the nerve ring, all five radial nerves, and subsequent ganglia responsible for tube feet and muscle control. Haloperidol may be affecting one or all of these steps by inhibiting dopamine receptor function. We found that haloperidol inhibited the righting response in the sea star L. clathrata and the sea urchin L. variegatus, and it has previously been shown to inhibit righting in the sea urchin S. purpuratus (McDonald et al., 2022). Given that this behavior is an essential behavioral response in both sea urchins and sea stars in their natural habitat, it may be that common neurobiological pathways are utilized in both of these subphyla, despite the much more rapid righting times of L. clathrata compared with sea urchins. In both cases, the behavior requires the detection of inversion, processing the ‘decision’ to right, coordinating numerous tube feet (and spines in the case of sea urchins) to work in concert to proceed in a direction to right in, and then the actual implementation of motor activity. Previous work has shown that tube foot motility is not affected by haloperidol (McDonald et al., 2022), and thus haloperidol may be interfering with either the detection of inversion, or the processing of the response prior to righting.

Haloperidol was also found to inhibit the lantern reflex of S. purpuratus, with a 3.4-fold decrease in the frequency of the reflex, and a smaller 25% increase in the reflex duration. The lantern reflex involves sensing the nearby food, processing the response, and coordinating the activity of the multiple muscles and ligaments controlling the teeth and movement of the Aristotle's lantern, and the effects of haloperidol suggests that dopamine receptors may be involved in any or all of these steps. Haloperidol was also found to inhibit the covering behavior of L. variegatus. As with the other tested behaviors, covering requires the detection of the nearby stimulus (in this case the presence of the shells), the decision to perform the behavior of holding the shells on the test, the coordinated activity of numerous tube feet to perform the behavior, and the actual motor functions to perform the behavior.

Surprisingly, haloperidol was found to only inhibit immersion in L. variegatus, and not S. purpuratus or L. clathrata. Although S. purpuratus is a cold-water species and the ASW used in these experiments using S. purpuratus was approximately 12°C, temperature is not believed to be the reason for this difference as both L. variegatus and L. clathrata are warm-water species, with the temperature of the ASW used in these experiments at approximately 20°C. The reason for the difference in the effects of haloperidol on the different species is unknown. Testing the effect of haloperidol on immersion on a large number of different species may reveal a systematic pattern of haloperidol effects across different subphyla and classes.

Therefore, in different echinoderm species, haloperidol inhibits behaviors that involve the detection of very different stimuli, namely, inversion, the presence of food, the detection of nearby shells and, in the case of L. variegatus, being emersed from seawater. If, as expected, haloperidol is exerting its effects through inhibition of dopamine receptors, this may mean that dopamine receptors are not involved in all of the varied initial sensory steps of the different behaviors, but instead are required for common steps in these behaviors of neural processing, motivation or coordinating of behavioral responses to a variety of different challenges. We chose to focus on using a dopamine receptor antagonist rather than dopamine itself, as dopamine receptors readily desensitize in the prolonged presence of dopamine. Given this, and the unknown quantitative details regarding the absorption, distribution, metabolism and excretion of dopamine in echinoderms, the ultimate balance of activation compared with desensitization of the receptors in the animals is therefore unclear, and it is feasible that dopamine application may act to either increase or decrease the measured behavioral responses. Genome analysis suggests that in S. purpuratus there are seven D1-like dopamine receptor genes. Although there are limited studies on the localization of dopamine in sea urchins and sea stars, dopamine has been detected in the radial nerves of sea urchins and sea stars (Cottrell, 1967), and dopaminergic neurons are found in the ectoneural but not hyponeural tissue of the sea star Asterias rubens, where it was suggested to function in the control of tube feet (Cottrell and Pentreath, 1970). In non-deuterostome invertebrates, dopamine has been demonstrated to be involved in many different behaviors across different phyla. For example, in C. elegans, it has an inhibitory role in locomotion, feeding, defecation and egg laying, and this inhibition can be removed by haloperidol (Suo et al., 2004; Chase and Koelle, 2007). In Drosophila, dopamine plays a role in fear conditioning and reward (Pribadi and Chalasani, 2022), and in the mollusk Aplysia, dopamine can modulate associative learning behaviors in feeding (Baxter and Byrne, 2006).

Despite the prevalence of dopaminergic signaling in modulating motor activity and motivation in animals across many phyla, it is of particular interest in animals that lack a brain, such as echinoderms. It is uncontroversial to state that the motivation to perform a complex behavior is made within the brain of an organism; however, it is unclear how this applies to an animal lacking a brain, and with a symmetrical, albeit 5-fold symmetrical, nervous system, particularly when the motor output has clear directional components. It would be a of great interest to determine how the neurophysiological steps in the processing of sensory signals in the central nervous system of echinoderms dictates a decisive, coordinated motor response, and this may illuminate neurophysiological facets of decision making in animals that do not have brains.

We thank Madison Reid, Feza Umutoni, Jackson Deneka and Debbie Lim for the care and maintenance of the sea urchins and sea stars during this project.