Invertebrate serotonin receptors: a molecular perspective on classification and pharmacology

Serotonin (5-hydroxytryptamine, 5-HT; see Glossary) and its receptors (see Glossary) form one of the oldest and most widely distributed signalling systems found in nature. An extended period of evolution, unfolding over an estimated 750 million years, has yielded an array of 5-HT receptors mediating myriad functions. In vertebrates, 5-HT acts as a central nervous system (CNS) transmitter and modulator, and its role in mood, cognitive processes and numerous behaviours is well documented. 5-HT and its receptors are the focus of much effort to understand psychological disorders in humans and to identify new pharmaceutical treatments (Hoyer et al., 2002; Pithadia and Jain, 2009; Nautiyal and Hen, 2017). In addition to its actions in the CNS, 5-HT is important throughout the body, modulating function in all physiological systems in adulthood and during development. 5-HT is likewise important in invertebrate behaviour and physiology (Gillette, 2006; Blenau and Thamm, 2011; Wu and Cooper, 2012). The study of 5-HT in diverse invertebrates has contributed insight into the evolutionary and molecular underpinnings of this ubiquitous signalling system. Many experiments address serotonergic function (see Glossary) in model organisms which are important for elucidating general principles relevant to human biology and health.

In mammals, seven 5-HT receptor families (5-HT₁ to 5-HT₇) have been identified comprising 14 receptors, 13 of which are G-protein coupled and one of which (5-HT₃) is ionotropic (see Glossary). According to estimates, the major 5-HT G-protein-coupled receptor families differentiated prior to the separation of vertebrates and invertebrates 600 million years ago, and hence the same major receptor families are thought to exist in the two groups (Peroutka and Howell, 1994; Walker et al., 1996). During subsequent vertebrate evolution, further differentiation occurred within some receptor families, yielding five 5-HT₁ (5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, 5-HT_1F), three 5-HT₂ (5-HT_2A, 5-HT_2B, 5-HT_2C) and two 5-HT₅ (5-HT_5A, 5-HT_5B) receptors. The receptor families, which are recognized based on gene organization and sequence homology, can also be grouped by their primary transduction mechanism (Nichols and Nichols, 2008). 5-HT₁ and 5-HT₅ couple preferentially to G_i/o proteins, leading to decreased production of cAMP, whereas 5-HT₄, 5-HT₆ and 5-HT₇ couple preferentially to G_s proteins, leading to increased cAMP production. 5-HT₂ receptors couple to G_q proteins, inducing elevated Ca²⁺ levels (Fig. 1). The mammalian receptors display different responses to agonist and antagonist drugs, and hence they can also be recognized by pharmacological properties (Hoyer et al., 2002; Pytliak et al., 2011).

In invertebrates, early molecular studies confirmed the presence of 5-HT receptor genes orthologous to mammalian 5-HT₁, 5-HT₂ and 5-HT₇, as determined by similarities in sequence and transduction mechanisms (Tierney, 2001). The first invertebrate receptor cloned, 5-HT-dro1 (Witz et al., 1990) later termed 5-HT_7Dro (Colas et al., 1995), was also the first 5-HT₇ receptor to be discovered; the mammalian 5-HT₇ receptor was subsequently cloned and found to be homologous (see Glossary) to 5-HT_7Dro (Shen et al., 1993; Bard et al., 1993). 5-HT₁ receptors were discovered next, first in Drosophila (Saudou et al., 1992) and then in other insects (Von Nickisch-Rosenegk et al., 1996), gastropods (Mollusca, see Glossary) (Sugamori et al., 1993; Angers et al., 1998) and Caenorhabditis elegans (Nematoda, see Glossary) (Olde and McCombie, 1997). Early studies also demonstrated the presence of 5-HT₂ receptors in Drosophila (Colas et al., 1995), molluscs (Gerhardt et al., 1996) and nematodes (Hamdan et al., 1999; Huang et al., 1999). The preferential G-proteins mediating transduction were G_s, G_i and G_q for 5-HT₇, 5-HT₁ and 5-HT₂, respectively, indicating conservation with mammalian receptors in each class.

Since the 1990s, researchers have cloned numerous additional invertebrate 5-HT receptors, providing new data on gene structure, transduction mechanisms and pharmacology. Here, I review this information and, based on the proposed evolutionary relationships, consider the classification of invertebrate receptors. Pharmacological profiles are summarized, with the aim of identifying ligands that can be used to define receptor subtypes or be useful in investigations of receptors in native tissue. However, comparisons among invertebrate receptors are hampered by the lack of a common nomenclature. Names currently assigned to the growing number of invertebrate receptors are highly variable and often do not reflect receptor relationships. Mammalian researchers confronted a similar problem in the 1980s when the historical order of receptor discovery and characterization led to labelling that was irregular and confusing. This problem was addressed by the IUPHAR Receptor Nomenclature Committee, which proposed a standardized way to name receptors and provided updates to accommodate new findings (Humphrey et al., 1993; Hoyer, 2017). Invertebrate 5-HT receptors await a similar collaborative effort. In the meantime, this paper follows the customary nomenclature used for vertebrates that recognizes proposed evolutionary and functional relationships among receptors.

The nomenclature used to identify vertebrate receptors indicates the relevant endogenous ligand first, followed by a numbered subscript to indicate the receptor class. For invertebrate 5-HT receptors (e.g. 5-HT₁, 5-HT₂, 5-HT₇), these subscripts indicate proposed homology with mammalian receptor families. Species identification is important as invertebrate 5-HT receptors can be expected to vary significantly owing to the long periods of independent evolution in different phyla. Where relevant for mammalian receptors, species is denoted by a single letter, e.g. h 5-HT₇ and r 5-HT₇ for human and rat receptors, respectively (Vanhoutte et al., 1996). However, invertebrate receptors have not traditionally been identified in this manner and a single letter for a common name provides insufficient information for invertebrate species identification. Here, species is indicated by the first three letters of the genus in the subscript (e.g. 5-HT_1Apl to denote an Aplysia receptor); if these letters provide insufficient information for species identification, the first two letters of a species name are added (e.g. 5-HT_1Aplca to denote an Aplysia californica receptor).

The expansion of knowledge of invertebrate receptors from diverse phyla presents additional and significant challenges for the development of precise nomenclature. Ideally, receptors should be labelled in a manner that illuminates proposed orthologous relationships among receptors and precisely identifies genes that differ as a result of recent duplications or alternative splicing. In mammalian 5-HT receptor nomenclature, the uppercase letters that follow subscript numbers indicate different genes within a receptor family (e.g. 5-HT_1A, 5-HT_1B, 5-HT_1D) and orthologues (see Glossary) across different species (e.g. the h 5-HT_1B receptor is orthologous to the r 5-HT_1B receptor). However, letters associated with invertebrate 5-HT receptor subscripts do not signify orthology to vertebrate receptor subtypes as the latter emerged after the evolutionary separation of vertebrates and invertebrates (Peroutka and Howell, 1994). Also, additional uppercase, lowercase and Greek letters or numbers have been applied inconsistently and do not unequivocally characterize evolutionary relationships among invertebrate receptor genes or identify isoforms of the same gene. In this review, we follow researchers (Clark et al., 2004; Thamm et al., 2013) who have used Greek letters, α and β, to identify different genes within receptor families, thereby avoiding the implication of orthology with well-established mammalian receptor subtypes. If paralogues (see Glossary), arising owing to recent gene duplication events, exist within a receptor subtype, they are identified by lowercase letters (e.g. 5-HT_1αa and 5-HT_1αb). Multiple genes within the 5-HT₇ receptor family are likewise distinguished by lowercase letters pending additional information on the evolutionary origins of these genes. Different sequences identified as functional splice variants are identified by lowercase letters in parentheses (e.g. 5-HT_2(a) and 5-HT_2(b)). Names used in this paper according to the above criteria are specified in Table 1. Note that this table includes receptors with known functional properties, and is not intended to provide an exhaustive catalogue of all possible or putative 5-HT receptors.

Unlike mammalian receptors, which come from a single class of organisms, invertebrate receptors are from multiple classes and distantly related phyla. Hence, orthologous relationships within receptor families may occur within phyla, but not necessarily across phyla. The classification of 5-HT receptors described below and depicted in Table 1 is provisional and will be subjected to revision as knowledge of invertebrate receptors expands and evolutionary relationships are further elucidated. At the present time, two 5-HT₁ subtypes have been distinguished in species from the phylum Arthropoda (see Glossary): 5-HT_1α and 5-HT_1β. The gene duplication that formed these subtypes is proposed to have taken place prior to the separation of insects and crustaceans, and hence α and β subscripts represent orthologues within this group of animals (Dacks et al., 2006; Watanabe et al., 2011). The prototypical separation of the Drosophila 5-HT₁ receptor gene into two genes labelled A and B (5-HT-dro2A and 5-HT-dro2B later termed 5-HT_1ADro and 5-HT_1BDro, respectively; Saudou et al., 1992) does not reflect the current classification of 5-HT₁ receptor subtypes. Instead, most phylogenetic analyses place both of these genes in the α subgroup (Troppmann et al., 2010; Qi et al., 2017), suggesting a recent duplication event in Drosophila (Watanabe et al., 2011). The two genes are therefore identified as 5-HT_1αaDro and 5-HT_1αbDro. At the present time, most insect receptors fall into the 1α clade, as do 5-HT₁ receptors from crustaceans and the tick Boophilus microplus (Table 1); four 1β receptors have been identified from the moths Bombyx mori (Von Nickisch-Rosenegk et al., 1996) and Manduca sexta (Dacks et al., 2006), the field cricket Gryllus bimaculatus (Watanabe et al., 2011) and the butterfly Pieris rapae (Qi et al., 2017).

In other phyla, fewer 5-HT₁ receptors have been characterized and subtypes have not yet been defined. In molluscs, however, two distinct genes appear to exist. Barbas et al. (2002) described a receptor in A. californica that differed from the 5HT₁ receptor previously described by Angers et al. (1998), and that more closely resembled the sequence from the 5-HT₁ receptor (5-HT_1Lym) from Lymnaea stagnalis (Sugamori et al., 1993). Phylogenetic analyses (Mapara et al., 2008; Panasophonkul et al., 2009; Tanabe et al., 2010) indicate that additional molluscan 5-HT₁ receptors listed in Table 1 are also more closely related to 5-HT_1Lym than to the first receptor cloned from Aplysia (Angers et al., 1998), and the latter may derive from a recent gene duplication (Nagakura et al., 2010). It would be premature to delineate receptor subtypes in molluscs, and hence Greek letters were not applied and the two Aplysia receptors are distinguished by lowercase letters. Two previously described Aplysia receptors are not listed because they are no longer definitively identified as 5-HT receptors (Li et al., 1995, 2003). Thus far, two 5-HT₁ receptors have been characterized in nematodes (Olde and McCombie, 1997; Smith et al., 2003) and are classified by a number subscript only.

5-HT₂ receptors are also divided into two subtypes in arthropods: 5-HT_2α and 5-HT_2β. The first receptor, cloned by Colas et al. (1995) from Drosophila, is currently considered a member of the 5-HT_2α group. Orthologous 2α receptors have been characterized in G. bimaculatus (Watanabe et al., 2011), the blowfly Calliphora vicina (Röser et al., 2012), M. sexta (Dacks et al., 2013) and the honeybee Apis mellifera (Thamm et al., 2013). A second 5-HT₂ sequence was reported in Drosophila (Brody and Cravchik, 2000; Clark et al., 2004) and later cloned and characterized by Gasque and colleagues (2013); it was named 5-HT_2βDro (Clark et al., 2004) or 5-HT2B (Gasque et al., 2013). Orthologous 5-HT_2β receptors have been described in G. bimaculatus (Watanabe and Aonuma, 2012), A. mellifera (Thamm et al., 2013), the insect Rhodnius prolixus (Paluzzi et al., 2015) and the crustaceans Panulirus interruptus (Clark et al., 2004), Procambarus clarkii (Spitzer et al., 2008) and Macrobrachium rosenbergii (Vázquez-Acevedo et al., 2009). The 5-HT₂ receptors characterized in molluscs (Gerhardt et al., 1996; Nagakura et al., 2010) and nematodes (Hamdan et al., 1999; Huang et al., 1999, 2002) are identified by number subscripts only. Receptor subtypes in these groups have not yet been described, although functional splice variants have been shown to occur in both nematode species.

In both mammals and invertebrates, the 5-HT₄, 5-HT₆ and 5-HT₇ receptors each comprise a single subtype (Hoyer et al., 2002; Nichols and Nichols, 2008) and are identified by a subscript number. Few 5-HT₄ and 5-HT₆ receptors have been reported in invertebrates. However, these receptors do occur as a 5-HT₄ and a 5-HT₆ receptor were identified in Aplysia (Nagakura et al., 2010) and C. elegans (Carre-Pierrat et al., 2006; Hapiak et al., 2009), respectively. By contrast, 5-HT₇ receptors occur widely and have been characterized in a number of insects, gastropod molluscs, and C. elegans (Table 1). In addition, 5-HT₇ receptors have been described in flatworms (phylum Platyhelminthes; see Glossary), and this receptor family appears to be the dominant clade in these organisms. Multiple sequences have been found in the planarian Dugesia japonica (Saitoh et al., 1997; Nishimura et al., 2009), the parasitic worm Schistosoma mansoni (Patocka et al., 2014; Chan et al., 2016a) and the cestodes Echinococcus granulosus and Mesocestoides corti (Camicia et al., 2018). Genome searches predict the presence of additional 5-HT receptors in Platyhelminthes (Chan et al., 2015; Patocka et al., 2014), but they have not yet been characterized. Patocka et al. (2014) was unable to find 5-HT₂ receptors in S. mansoni and suggested that they may have been lost in this organism or in the phylum.

An additional receptor, MOD-1, is a serotonin-gated ion channel found in C. elegans with a predicted protein structure similar to that of ionotropic receptors gated by acetylcholine, GABA, glycine and 5-HT (Ranganathan et al., 2000). These ligand-gated ion channels all belong to the Cys-loop receptor superfamily (see Glossary), which includes the mammalian 5-HT₃ receptor. While MOD-1 and 5-HT₃ share a distant evolutionary past and gating by 5-HT, they differ in function in that MOD-1 is a chloride channel whereas 5-HT₃ is a non-selective cation channel (Yaakob et al., 2018). MOD-1 is important in C. elegans, contributing to the control of behaviours such as locomotion, feeding, decision making and aversive learning (Churgin et al., 2017; Iwanir et al., 2016; Zhang et al., 2005). This receptor also occurs in the parasitic nematode Haemonchus contortus and is predicted to be present in several additional nematodes (Komuniecki et al., 2012; Beech et al., 2013), but it has not been reported in other invertebrate clades. In the butterfly P. rapae, a novel receptor has been described by Qi et al. (2014) that is proposed to belong to a new family of receptors designated 5-HT₈. 5-HT activation of this receptor in HEK293 cells induced an increase in Ca²⁺, but the sequence and pharmacology do not resemble 5-HT₂ or other insect 5-HT receptors. Further research is needed to confirm whether 5-HT is the endogenous ligand and to characterize the receptor in additional species.

It has been clear for many years that responses to serotonergic drugs differ between invertebrate and mammalian receptors (Tierney, 2001). Given the substantial increase in characterized invertebrate receptors, it is timely to consider how pharmacology varies within and between invertebrate phyla themselves. The heterologous expression (see Glossary) of cloned receptors has offered much valuable information on the pharmacology of receptors definitively identified at the molecular level. However, interpretation of data and comparisons across receptor subtypes and species are complex for several reasons. First, researchers use a variety of host cells (see Glossary), each of which possesses its own regulatory mechanisms and membrane environment, potentially affecting the methodology required for expression (see Glossary), receptor structure and receptor–ligand interactions (Kenakin, 1997; Thomas and Smart, 2005). Thus, the same receptor might function differently depending on the expression system used or in comparison with the endogenous receptor in native tissue. Second, different assays are used to determine pharmacological properties, including outcomes of transduction pathways (see Glossary), inhibition of radioligand binding and changes in ion currents. Different assays yield disparate information on drug potency and efficacy, limiting direct and quantitative comparisons of drug effects. Third, the battery of drugs tested varies from experiment to experiment, and few drugs have been tested in multiple receptor subtypes. Finally, the heterologous expression of receptors and screening of many drugs is a lengthy process, and detailed data on receptor pharmacology are available for relatively few species. Hence, it is difficult to discern whether differences in receptor responses arise from species differences, structural differences among receptor subtypes or artifacts of methodologies used. Despite these caveats, the information acquired by research to date provides a useful guide for future studies and allows some preliminary conclusions to be drawn.

To characterize receptor subtypes across species, it is essential that the same drugs be tested on multiple organisms. In the arthropod 5-HT₁ receptor, this has been achieved with several agonists, including 5-MeOT, 5-CT, 2-methyl-5-HT, α-methyl-5-HT and 8-OH-DPAT (Table S1; Fig. 2). 5-MeOT, a non-selective vertebrate 5-HT receptor agonist, elicited responses across all receptors tested (5-HT_1αMan, 5-HT_1βMan, 5-HT_1αApi, 5-HT_1αPer, 5-HT_1αPie, 5-HT_1βPie and 5-HT_1αPan). 5-CT is also a non-selective agonist with high affinity for vertebrate 5-HT_1/5/7 receptors. It had variable effects at arthropod 5-HT₁ receptors, displaying activity at some receptors (5-HT_1αApi, 5-HT_1αTri and 5-HT_1αPan) and weak or no activity at others (5-HT_1αMan, 5-HT_1βMan, 5-HT_1αPer, 5-HT_1αPie and 5-HT_1βPie). Likewise, the actions of both 2-methyl-5-HT and α-methyl-5-HT were variable, but one or both displayed relatively high potency at certain receptors (5-HT_1αTri, 5-HT_1αPan and 5-HT_1αPro). 8-OH-DPAT is a well-known agonist at the vertebrate 5-HT_1A receptor, but it had low potency or was completely ineffective in all of the arthropod 5-HT₁ receptors. Interestingly, methysergide is an antagonist at vertebrate and Drosophila 5-HT receptors (Saudou et al., 1992), but displayed agonist activity in several receptors (Table S1). The most commonly tested antagonists were methiothepin (Fig. 2), which is non-selective at vertebrate receptors, and WAY 100635, a vertebrate 5-HT_1A antagonist. Methiothepin was a relatively potent antagonist in most arthropod receptors tested (5-HT_1βMan, 5-HT_1αApi, 5-HT_1αPer, 5-HT_1αTri, 5-HT_1αPie and 5-HT_1βPie). WAY 100635 was an active antagonist in several receptors (5-HT_1αMan, 5-HT_1βMan and 5-HT_1αTri) and acted as an inverse agonist (see Glossary) at 5-HT_1αPer.

Results thus far allow very limited comparisons between 5-HT_1α or 5-HT_1β receptors across species. However, within species, Dacks et al. (2013) reported that methiothepin was an antagonist at 5-HT_1βMan receptors, but had no activity at 5-HT_1αMan receptors, and Qi et al. (2017) found that 8-OH-DPAT at high concentrations had greater efficacy at 5-HT_1αPie than at 5-HT_1βPie. Within-species comparisons also point to possible drug specificity across other receptor families. For example, methysergide acted as an agonist at 5-HT_1αMan (and 5-HT_7Man) receptors, but was inactive at 5-HT_2αMan receptors (Dacks et al., 2013). In crustaceans, mCPP was an agonist of 5-HT_1αPro and 5-HT_1αPan receptors, but inactive at 5-HT_2β receptors in each species. In addition, quipazine was an agonist at 5-HT_1αPro receptors, but inactive at 5-HT_2βPro receptors (Spitzer et al., 2008).

In molluscs, pharmacological profiles are available for 5-HT₁ receptors from Lymnaea (5-HT_1Lym) and Aplysia (5-HT_1aAplca and 5-HT_1bAplca), and they display notable consistency between the two species (Table S1). 5-CT and PAPP were relatively potent agonists at all three receptors, whereas 8-OH-DPAT and NAN-190 were less potent. Methiothepin was the most potent antagonist, and metergoline also displayed high potency in all three receptors. The molluscan profiles resemble those of arthropods in that methiothepin displays high affinity for all three receptors, but the response to agonists is quite different, with 5-CT and 8-OH-DPAT displaying greater potency at molluscan compared with arthropod receptors. Another difference between the two groups lies in the relative potency of 5-HT. In arthropods, 5-HT is typically much more potent and effective than synthetic ligands (Vleugels et al., 2015), but in molluscan 5-HT₁ receptors this is not the case as several drugs act much more potently than does 5-HT itself. Only two nematode 5-HT₁ receptors have been examined pharmacologically: 5-HT_1Cae and 5-HT_1Hae. Ligands shown to be active in both receptors included lisuride, 5-OMeDMT, methiothepin, methysergide and clozapine, but affinity for the chemicals differed considerably. 5-HT_1Cae resembled the molluscan 5-HT₁ receptors in that 5-HT displayed less affinity than many synthetic drugs (Olde and McCombie, 1997). However, affinity for 5-HT was much higher at 5-HT_1Hae­ than at 5-HT_1Cae, and only a single drug (PAPP) displayed slightly higher affinity than 5-HT (Smith et al., 2003).

The pharmacology of arthropod 5-HT₂ receptors has been examined in four 5-HT_2α and five 5-HT_2β receptors. Agonists displaying activity in multiple receptors resembled the previous list for 5-HT₁ receptors: 5-MeOT, 8-OH-DPAT, α-methyl-5-HT, 2-methyl-5-HT and 5-CT (Table S1). The affinity or rank order of potency varied among species, but none acted with sufficient specificity to distinguish 5-HT_2α from 5-HT_2β receptors or to distinguish either from receptors in other families. Among antagonists, methiothepin was active at 5-HT_2αDro, 5-HT_2βDro, 5-HT_2αCal, 5-HT_2αApi, 5HT_2βPro and 5HT_2βPan receptors, confirming its status as a non-selective antagonist. Mianserin and cyproheptadine, vertebrate 5-HT₂ antagonists, were active in all receptors tested (5-HT_2αDro, 5-HT_2αCal, 5-HT_2αApi, 5-HT_2βApi, 5-HT_2βRho and 5-HT_2βDro), and additional antagonists (clozapine, yohimbine, ketanserin, methysergide and spiperone) had variable effects among 5-HT_2α/β receptors. Limited testing suggests that these antagonists were also active at some 5-HT₁ and 5-HT₇ receptors, indicating that they lack specificity across species.

However, within species, some drugs displayed actions that point to a selectivity that would be useful to examine in other species. In A. mellifera, Thamm et al. (2013) reported that SB-200646, methiothepin and methysergide displayed high potency at 5-HT_2αApi receptors, but were inactive at 5-HT_2βApi receptors, and ketanserin was active at 5-HT_2βApi but not 5-HT_2αApi receptors. Although not widely tested, 5-nonyl-5-HT and DOI acted as agonists at 5-HT_2αMan receptors, whereas they displayed no activity at other Manduca 5-HT receptors (Dacks et al., 2013). Röser et al. (2012) found that methiothepin and methysergide were antagonists at 5-HT_2αCal receptors, but at 5-HT_7Cal receptors the former was inactive and the latter acted as an agonist. In P. interruptus, ritanserin, (+)-butaclamol and cinanserin were antagonists at 5-HT_2βPan receptors, but inactive at 5-HT_1αPan receptors; in P. clarkii, methiothepin and cinanserin were antagonists at 5-HT_2βPro receptors, but inactive at 5-HT_1αPro receptors (Spitzer et al., 2008).

In Lymnaea, mCPP and DOB acted as agonists at 5-HT_2Lym receptors, and a number of active antagonists were identified (Gerhardt et al., 1996). As in 5-HT_1Lym, several synthetic ligands were more potent at 5-HT_2Lym receptors than 5-HT itself. Detailed pharmacology is not available for other molluscan 5-HT₂ receptors, but Nagakura et al. (2010) reported that pirenperone acted as an antagonist in A. californica, whereas spiperone was inactive. The nematode receptors 5-HT_2(a)Cae and 5-HT_2Asc displayed considerable overlap in their pharmacological profiles. Both had high affinity for the agonist lisuride and the antagonists (+)-butaclamol, methiothepin, cyproheptadine, clozapine and metergoline, whereas both had relatively low affinity for ketanserin, DOI and 8-OH-DPAT. The receptors differed in their affinity for 5-HT, which was much higher for 5-HT_2Asc relative to 5-HT_2(a)Cae. This difference, also observed in nematode 5-HT₁ receptors, might reflect the difference in size and life history between C. elegans and the parasitic species, H. contortus and Ascaris suum. The parasites are huge compared with C. elegans, yet all of the worms have the same body structure and a surprisingly similar neuronal wiring pattern. Possibly the high receptor affinity for 5-HT facilitates signalling within the much larger worms (Huang et al., 2002; Komuniecki et al., 2012).

The pharmacology of insect 5-HT₇ receptors, currently described in seven species, resembles that of other families in that 5-HT was more potent than other synthetic agonists. Other agonists tested across species included 5-CT, which acted with variable potency at all receptors tested (5-HT_7Aed, 5-HT_7Api, 5-HT_7Cal, 5-HT_7Tri and 5-HT_7Pie), 5-MeOT (active in 5-HT_7Cal, 5-HT_7Tri and 5-HT_7Pie) and 8-OH-DPAT, which was a relatively poor agonist in most receptors (5-HT_7Api, 5-HT_7Cal, 5-HT_7Tri and 5-HT_7Pie). Methiothepin was a potent antagonist or inverse agonist where tested (5-HT_7Api, 5-HT_7Tri, 5-HT_7Pie), whereas other drugs, including the selective mammalian 5-HT₇ antagonist SB-269970, clozapine and ketanserin, had variable effects across species. In C. vicina, results highlight possible drug selectivity: the agonists R(+)-lisuride and AS-19, and the antagonist spiperone were active at 5-HT_7Cal but not 5-HT_2αCal receptors (Röser et al., 2012).

Data on 5-HT₇ pharmacology are limited in molluscs and nematodes. Lee et al. (2009) reported that, in the Aplysia receptor 5-HT_7Aplku, methiothepin was by far the most potent antagonist, followed by clozapine. Other antagonists were much less effective or inactive. 5-HT_7Cae resembled 5-HT₁ and 5-HT₂ receptors from C. elegans in the high potency displayed by methiothepin and clozapine, and the only agonist tested (tryptamine) was less potent than 5-HT (Hobson et al., 2003). As noted above, 5-HT₇ receptors have been characterized in several species from the phylum Platyhelminthes, and pharmacology has been investigated in the human parasite Schistosoma mansoni. In 5-HT_7aSch, the agonist o-methyl-5-HT and 5-HT were similar in potency, and additional agonists (buspirone, tryptamine and 8-OH-DPAT) were much less potent than either o-methyl-5-HT or 5-HT, whereas cyproheptadine, chlorpromazine and mianserin were active antagonists (Patocka et al., 2014). In a further study, Chan et al. (2016c) screened a large battery of compounds with the aim of comparing drug activity at 5-HT_7aSch receptors with that at the human 5-HT₇ receptor. Most drugs displayed higher potency at the human receptor, but some were more active at 5-HT_7aSch (bromocriptine, rotundine, tetrabenazine and tetrandrine), thus identifying these drugs as potential targets for the development of new anthelmintics. Receptors cloned from cestodes were activated by the non-specific agonists egotamine and LSD, though the latter had antagonist rather than agonist activity at 5-HT_7bEch receptors. Two cestode receptors, 5-HT_7bEch and 5-HT_7Mes, responded to 5-HT with EC₅₀ values in the picomolar range, indicating unusually high sensitivity to the transmitter (Camicia et al., 2018).

Across receptors and species, MeOT and methiothepin emerged as the most consistently effective agonist and antagonist, respectively. Hence, should a research question call for engaging multiple 5-HT receptors simultaneously, these two drugs could be useful in many species. Certain well-established vertebrate agonists such as 8-OH-DPAT had low potency at most invertebrate receptors, especially in insects. For some ligands, data are incomplete because they have not yet been tested in more than a single receptor subtype. To determine whether ligands possess selectivity, it would be useful if those shown to be effective in one receptor subtype (e.g. PAPP in molluscan and nematode 5-HT₁ receptors) were systematically tested in other subtypes. In a few cases, ligands tested on multiple receptors within a species show promising selectivity, but thus far these findings do not appear to extend to other species. Instead, ligands that have been relatively widely tested show different effects across receptor subtypes in different species, even within a single phylum. Hence, at the present time, no selective ligands for any invertebrate 5-HT receptor subtypes have been definitively identified. In contrast to the variability of pharmacological profiles, transduction mechanisms associated with 5-HT₁, 5-HT₂ and 5-HT₇ receptors appear to be very well conserved across invertebrate phyla and between invertebrates and mammals.

Expression in heterologous systems can introduce significant changes in receptor function, and hence it is important to confirm that properties displayed in host cells are replicated in native tissue. In model organisms, confirming the function of cloned receptors can be addressed directly by techniques that isolate a receptor of interest in native tissue. For example, following a feeding assay to screen many drugs, Gasque et al. (2013) examined responses to methiothepin in Drosophila 5-HT receptors expressed in HEK293 cells and also used null mutants of each receptor subtype to examine responses to the drug in larvae. These experiments confirmed that all five receptors, whether expressed or endogenous, were blocked by methiothepin and identified 5-HT_2αDro as the only receptor mediating the effect of the drug on larvae feeding. In C. elegans, use of null mutants has provided information on how receptor subtypes contribute to feeding, locomotion and egg-laying behaviours (Hobson et al., 2006; Hapiak et al., 2009). Furthermore, C. elegans itself can be used as a heterologous expression system, allowing receptors from parasitic worms to be pharmacologically screened in nematode tissue, which mimics native tissue more closely than do mammalian cells (Welz et al., 2011; Komuniecki et al., 2012). Law et al. (2015) used this approach to examine individual 5-HT receptor subtypes from H. contortus and other species in C. elegans made null for all five endogenous 5-HT receptors. In the flatworm Dugesia tigrina, Zamanian et al. (2012) developed a loss-of-function technique that could be used to assess the role of specific receptors in drug responses in native tissue. In this approach, responses to drugs were compared in control tissue and following RNAi knockdown (see Glossary) of a 5-HT₇ receptor, allowing identification of drugs dependent on a single receptor (Zamanian et al., 2012). These techniques in model organisms have not yet been used in a comprehensive comparison of heterologously expressed and in vivo receptors, but they offer powerful options for future comparisons of pharmacology in each situation.

Because 5-HT is so widely distributed and important in numerous behavioural and physiological processes, determining the function of 5-HT extends well beyond model organisms and strictly molecular approaches. Many studies have used 5-HT and 5-HT ligands to target receptors in vivo and draw conclusions about their function in physiology (e.g. Smith and Walker, 1974; Tembe et al., 1993; Ali and Orchard, 1994; Zhang and Harris-Warrick, 1994; Leake and Koubanakis, 1995; Dumitriu et al., 2006; Inohara et al., 2015) and behaviour (e.g. Yeh et al., 1997: Tierney et al., 2004; Johnson et al., 2009; Rawls et al., 2010). Receptor subtypes identified in such studies often rely on drug application alone and must be viewed as provisional given the lack of specificity shown by available ligands and uncertainty about the site(s) where drugs acted to produce observable effects. However, these studies are important as they begin to address the question of receptor function in intact tissues and organisms. Also, they provide a foundation for molecular investigations by identifying tissues, receptors or drugs relevant to serotonergic processes. For example, blowfly salivary gland physiology has been investigated for five decades, and the role of 5-HT in mediating increases in both Ca²⁺ and cAMP is well documented (Berridge, 2005). This information pointed to the likely presence of 5-HT₂ and 5-HT₇ receptors, and led to the molecular characterization of 5-HT_2αCal and 5-HT_7Cal and a valuable comparison between the cloned receptors and those in native salivary gland membranes (Röser et al., 2012). Many additional receptors have been cloned following intensive study of 5-HT function in diverse processes, including synaptic plasticity (Barbas et al., 2002; Lee et al., 2009; Nagakura et al., 2010), modulation of motor pattern generation (Clark et al., 2004; Spitzer et al., 2008), reproduction (Ongvarrasopone et al., 2006; Tanabe et al., 2010; Wang and He, 2014), regeneration (Saitoh et al., 1997; Nishimura et al., 2009), movement (Patocka et al., 2014) and other behaviours (Thamm et al., 2010, 2013; Gasque et al., 2013; Hobson et al., 2006).

Data on molecular structure and function should in turn inform current and future research with whole organisms. Indeed, a compelling reason for describing the pharmacology of cloned receptors is to use this information to more accurately manipulate 5-HT receptor subtypes in vivo. For some organisms, within-species data on receptor subtype pharmacology are already available and could shape physiological and behavioural investigations. However, for most invertebrates, the discovery of drugs that act specifically at 5-HT receptor subtypes would be extremely useful. In the meantime, screening large arrays of drugs in intact animals could aid in identifying 5-HT ligands associated with specific phenomena. Some species offer unique whole-animal assays for this purpose. The free-living Platyhelminthe D. japonica displays an extraordinary response to praziquantel in which regenerating worms exposed to the drug invariably develop two heads, a response mediated by 5-HT receptors (Nogi et al., 2009; Chan et al., 2015). The bipolarity response was used to screen serotonergic ligands, yielding ideas about structure–activity relationships at the receptor binding site and contributing to the search for new antihelmintics. More generally, mammalian researchers have long conducted high-throughput screening of drugs using simple, unambiguous behaviours, such as the time rodents struggle to escape during tail suspension or forced swimming (Castagné et al., 2011). Similar approaches are used in invertebrate model species (Nichols et al., 2012; Blazie and Jin, 2018) and could be more widely developed in non-model organisms. Efficient assays may uncover novel invertebrate-specific ligands that can be assessed in detail at the molecular level, and selected for investigations of complex behaviours such as aggression, anxiety and mate choice.

In addition to pharmacology, many molecular investigations yield information about the location of receptor subtypes. The distribution of receptors, mapped using techniques such as immunocytochemistry, in situ hybridization and RT-PCR, can give clues about function that confirm past work and suggest ideas for future focused experiments. 5-HT receptors are often found to be abundant in the nervous system, as expected of a neurotransmitter system important in behaviour. High expression of receptors in particular brain regions or neurons has implicated specific receptor subtypes in processes such as phototaxis (Thamm et al., 2010), modulation of the stomatogastric nervous system (Clark et al., 2004; Troppmann et al., 2010), activity of Aplysia bag cells (Barbas et al., 2002), pharyngeal pumping (Hobson et al., 2003) and olfaction (Dacks et al., 2013). Receptor subtypes have also been localized in ovaries (Tanabe et al., 2010; Wang and He, 2014), Malpighian tubules (Paluzzi et al., 2015) and hindgut (Pietrantonio et al., 2001), consistent with their functional role in these organs.

Much remains to be determined about the role played by individual receptor subtypes in these processes and organs. The 5-HT system, with its ancient origins and evolution over eras of time, is very complex. Despite decades of intensive research, the intricate functioning of mammalian 5-HT receptors is only partly understood, and numerous studies continue to explore the link between 5-HT and human pathologies, especially depression, anxiety and psychosis. The study of 5-HT in invertebrates is no less important to human well-being. Insects, nematodes and flatworms inflict a heavy disease burden on many human populations, and organisms from the same phyla impose economic hardship through their destruction of crops and livestock. Better understanding of 5-HT receptors, especially their pharmacology, is key to the development of more-precise, effective drugs and pesticides (Komuniecki et al., 2012; Chan et al., 2015). A future challenge in this endeavour is to determine how ligand responses are affected by differences in species, receptor subtype and methodology. The study of 5-HT in invertebrates also has much to contribute to our understanding of receptor evolution and how transduction mechanisms and ligand binding sites are conserved or diverge over time. This endeavour would be aided by the development of a common nomenclature, allowing relationships among receptors to be more easily recognized in different species and phyla.

I thank James Wallace and Iona Mackillop for their comments on this review.