Cannabinoids Stimulate the TRP Channel-Dependent Release of Both Serotonin and Dopamine to Modulate Behavior in C. elegans.

Cannabis sativa alters sensory perception and exhibits potential medicinal benefits. In mammals, cannabinoids activate two canonical receptors, CB1/CB2, as well additional receptors/ion channels whose overall contributions to cannabinoid signaling have yet to be fully assessed. In Caenorhabditis elegans, the endogenous cannabinoid receptor agonist, 2-arachidonoylglycerol (2-AG) activates a CB1 ortholog, NPR-19, to modulate behavior (Oakes et al., 2017). In addition, 2-AG stimulates the NPR-19 independent release of both serotonin (5-HT) and dopamine (DA) from subsets of monoaminergic neurons to modulate locomotory behaviors through a complex monoaminergic signaling pathway involving multiple serotonin and dopamine receptors. 2-AG also inhibits locomotion in remodeled monoamine receptor mutant animals designed to measure the acute release of either 5-HT or DA, confirming the direct effects of 2-AG on monoamine release. 2-AG-dependent locomotory inhibition requires the expression of transient receptor potential vanilloid 1 (TRPV1) and TRPN-like channels in the serotonergic or dopaminergic neurons, respectively, and the acute pharmacological inhibition of the TRPV1-like channel abolishes both 2-AG-dependent 5-HT release and locomotory inhibition, suggesting the 2-AG may activate the channel directly. This study highlights the advantages of identifying and assessing both CB1/CB2-dependent and independent cannabinoid signaling pathways in a genetically tractable, mammalian predictive model, where cannabinoid signaling at the molecular/neuronal levels can be correlated directly with changes in behavior.SIGNIFICANCE STATEMENT This study is focused on assessing CB1/CB2-independent cannabinoid signaling in a genetically tractable, whole-animal model where cannabinoid signaling at the molecular/neuronal levels can be correlated with behavioral change. Caenorhabditis elegans contains a cannabinoid signaling system mediated by a canonical cannabinoid receptor, NPR-19, with orthology to human CB1/CB2 (Oakes et al., 2017). The present study has characterized an NPR-19-independent signaling pathway that involves the cannabinoid-dependent release of both serotonin and dopamine and the expression of distinct TRP-like channels on the monoaminergic neurons. Our work should be of interest to those studying the complexities of CB1/CB2-independent cannabinoid signaling, the role of TRP channels in the modulation of monoaminergic signaling, and the cannabinoid-dependent modulation of behavior.

Investigating the function and possible biological role of an acetylcholine-gated chloride channel subunit (ACC-1) from the parasitic nematode Haemonchus contortus.

The cys-loop superfamily of ligand-gated ion channels are well recognized as important drug targets for many invertebrate specific compounds. With the rise in resistance seen worldwide to existing anthelmintics, novel drug targets must be identified so new treatments can be developed. The acetylcholine-gated chloride channel (ACC) family is a unique family of cholinergic receptors that have been shown, using Caenorhabditis elegans as a model, to have potential as anti-parasitic drug targets. However, there is little known about the function of these receptors in parasitic nematodes. Here, we have identified an acc gene (hco-acc-1) from the sheep parasitic nematode Haemonchus contortus. While similar in sequence to the previously characterized C. elegans ACC-1 receptor, Hco-ACC-1 does not form a functional homomeric channel in Xenopus oocytes. Instead, co-expression of Hco-ACC-1 with a previously characterized subunit Hco-ACC-2 produced a functional heteromeric channel which was 3x more sensitive to acetylcholine compared to the Hco-ACC-2 homomeric channel. We have also found that Hco-ACC-1 can be functionally expressed in C. elegans. Overexpression of both cel-acc-1 and hco-acc-1 in both C. elegans N2 and acc-1 null mutants decreased the time for worms to initiate reversal avoidance to octanol. Moreover, antibodies were generated against the Hco-ACC-1 protein for use in immunolocalization studies. Hco-ACC-1 consistently localized to the anterior half of the pharynx, specifically in pharyngeal muscle tissue in H. contortus. On the other hand, expression of Hco-ACC-1 in C. elegans was restricted to neuronal tissue. Overall, this research has provided new insight into the potential role of ACC receptors in parasitic nematodes.

Monoamines differentially modulate neuropeptide release from distinct sites within a single neuron pair.

Monoamines and neuropeptides often modulate the same behavior, but monoaminergic-peptidergic crosstalk remains poorly understood. In Caenorhabditis elegans, the adrenergic-like ligands, tyramine (TA) and octopamine (OA) require distinct subsets of neuropeptides in the two ASI sensory neurons to inhibit nociception. TA selectively increases the release of ASI neuropeptides encoded by nlp-14 or nlp-18 from either synaptic/perisynaptic regions of ASI axons or the ASI soma, respectively, and OA selectively increases the release of ASI neuropeptides encoded by nlp-9 asymmetrically, from only the synaptic/perisynaptic region of the right ASI axon. The predicted amino acid preprosequences of genes encoding either TA- or OA-dependent neuropeptides differed markedly. However, these distinct preprosequences were not sufficient to confer monoamine-specificity and additional N-terminal peptide-encoding sequence was required. Collectively, our results demonstrate that TA and OA specifically and differentially modulate the release of distinct subsets of neuropeptides from different subcellular sites within the ASIs, highlighting the complexity of monoaminergic/peptidergic modulation, even in animals with a relatively simple nervous system.

Cannabinoids Activate Monoaminergic Signaling to Modulate Key C. elegans Behaviors.

Cannabis sativa, or marijuana, a popular recreational drug, alters sensory perception and exerts a range of potential medicinal benefits. The present study demonstrates that the endogenous cannabinoid receptor agonists 2-arachidonoylglycerol (2-AG) and anandamide (AEA) activate a canonical cannabinoid receptor in Caenorhabditis elegans and also modulate monoaminergic signaling at multiple levels. 2-AG or AEA inhibit nociception and feeding through a pathway requiring the cannabinoid-like receptor NPR-19. 2-AG or AEA activate NPR-19 directly and cannabinoid-dependent inhibition can be rescued in npr-19-null animals by the expression of a human cannabinoid receptor, CB1, highlighting the orthology of the receptors. Cannabinoids also modulate nociception and locomotion through an NPR-19-independent pathway requiring an α2A-adrenergic-like octopamine (OA) receptor, OCTR-1, and a 5-HT1A-like serotonin (5-HT) receptor, SER-4, that involves a complex interaction among cannabinoid, octopaminergic, and serotonergic signaling. 2-AG activates OCTR-1 directly. In contrast, 2-AG does not activate SER-4 directly, but appears to enhance SER-4-dependent serotonergic signaling by increasing endogenous 5-HT. This study defines a conserved cannabinoid signaling system in C. elegans, demonstrates the cannabinoid-dependent activation of monoaminergic signaling, and highlights the advantages of studying cannabinoid signaling in a genetically tractable whole-animal model.SIGNIFICANCE STATEMENTCannabis sativa, or marijuana, causes euphoria and exerts a wide range of medicinal benefits. For years, cannabinoids have been studied at the cellular level using tissue explants with conflicting results. To better understand cannabinoid signaling, we have used the Caenorhabditis elegans model to examine the effects of cannabinoids on behavior. The present study demonstrates that mammalian cannabinoid receptor ligands activate a conserved cannabinoid signaling system in C. elegans and also modulate monoaminergic signaling, potentially affecting an array of disorders, including anxiety and depression. This study highlights the potential role of cannabinoids in modulating monoaminergic signaling and the advantages of studying cannabinoid signaling in a genetically tractable, whole-animal model.

Opiates Modulate Noxious Chemical Nociception through a Complex Monoaminergic/Peptidergic Cascade.

UNLABELLED: The ability to detect noxious stimuli, process the nociceptive signal, and elicit an appropriate behavioral response is essential for survival. In Caenorhabditis elegans, opioid receptor agonists, such as morphine, mimic serotonin, and suppress the overall withdrawal from noxious stimuli through a pathway requiring the opioid-like receptor, NPR-17. This serotonin- or morphine-dependent modulation can be rescued in npr-17-null animals by the expression of npr-17 or a human κ opioid receptor in the two ASI sensory neurons, with ASI opioid signaling selectively inhibiting ASI neuropeptide release. Serotonergic modulation requires peptides encoded by both nlp-3 and nlp-24, and either nlp-3 or nlp-24 overexpression mimics morphine and suppresses withdrawal. Peptides encoded by nlp-3 act differentially, with only NLP-3.3 mimicking morphine, whereas other nlp-3 peptides antagonize NLP-3.3 modulation. Together, these results demonstrate that opiates modulate nociception in Caenorhabditis elegans through a complex monoaminergic/peptidergic cascade, and suggest that this model may be useful for dissecting opiate signaling in mammals. SIGNIFICANCE STATEMENT: Opiates are used extensively to treat chronic pain. In Caenorhabditis elegans, opioid receptor agonists suppress the overall withdrawal from noxious chemical stimuli through a pathway requiring an opioid-like receptor and two distinct neuropeptide-encoding genes, with individual peptides from the same gene functioning antagonistically to modulate nociception. Endogenous opioid signaling functions as part of a complex, monoaminergic/peptidergic signaling cascade and appears to selectively inhibit neuropeptide release, mediated by a α-adrenergic-like receptor, from two sensory neurons. Importantly, receptor null animals can be rescued by the expression of the human κ opioid receptor, and injection of human opioid receptor ligands mimics exogenous opiates, highlighting the utility of this model for dissecting opiate signaling in mammals.

Multiple Sensory Inputs Are Extensively Integrated to Modulate Nociception in C. elegans.

Sensory inputs are integrated extensively before decision making, with altered multisensory integration being associated with disorders such as autism. We demonstrate that the two C. elegans AIB interneurons function as a biphasic switch, integrating antagonistic, tonic, and acute inputs from three distinct pairs of sensory neurons to modulate nociception. Off food, animals reverse away from a noxious stimulus. In contrast, on food or serotonin, AIB signaling is inhibited and, although animals initiate an aversive response more rapidly, they continue forward after the initial backward locomotion is complete. That is, animals continue to move forward and feed even when presented with a noxious repellant, with AIB inhibition decreasing the repellant concentration evoking a maximal response. These studies demonstrate that the AIBs serve as an integrating hub, receiving inputs from different sensory neurons to modulate locomotory decision making differentially, and highlight the utility of this model to analyze the complexities of multisensory integration. SIGNIFICANCE STATEMENT: Dysfunctional sensory signaling and perception are associated with a number of disease states, including autism spectrum disorders, schizophrenia, and anxiety. We have used the C. elegans model to examine multisensory integration at the interneuron level to better understand the modulation of this complex, multicomponent process. C. elegans responds to a repulsive odorant by first backing up and then either continuing forward or turning and moving away from the odorant. This decision-making process is modulated extensively by the activity state of the two AIB interneurons, with the AIBs integrating an array of synergistic and antagonistic glutamatergic inputs, from sensory neurons responding directly to the odorant to others responding to a host of additional environmental variables to ultimately fine tune aversive behaviors.

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets.

Serotonin differentially modulates Ca2+ transients and depolarization in a C. elegans nociceptor.

Monoamines and neuropeptides modulate neuronal excitability and synaptic strengths, shaping circuit activity to optimize behavioral output. In C. elegans, a pair of bipolar polymodal nociceptors, the ASHs, sense 1-octanol to initiate escape responses. In the present study, 1-octanol stimulated large increases in ASH Ca(2+), mediated by L-type voltage-gated Ca(2+) channels (VGCCs) in the cell soma and L-plus P/Q-type VGCCs in the axon, which were further amplified by Ca(2+) released from intracellular stores. Importantly, 1-octanol-dependent aversive responses were not inhibited by reducing ASH L-VGCC activity genetically or pharmacologically. Serotonin, an enhancer of 1-octanol avoidance, potentiated 1-octanol-dependent ASH depolarization measured electrophysiologically, but surprisingly, decreased the ASH somal Ca(2+) transients. These results suggest that ASH somal Ca(2+) transient amplitudes may not always be predictive of neuronal depolarization and synaptic output. Therefore, although increases in steady-state Ca(2+) can reliably indicate when neurons become active, quantitative relationships between Ca(2+) transient amplitudes and neuronal activity may not be as straightforward as previously anticipated.

Context-dependent modulation reconfigures interactive sensory-mediated microcircuits in Caenorhabditis elegans.

Caenorhabditis elegans navigates sensory landscapes by integrating inputs from 14 pairs of polymodal sensory neurons. Sensory neurons interact synaptically and through gap junction networks and are modulated by complex local/humoral, nutritionally dependent, monoaminergic and peptidergic signaling cascades that dynamically reconfigure individual sensory-mediated locomotory circuits. Monoaminergic/peptidergic signaling modifies the sensory signal by providing, first, feedback loops between sensory neurons and postsynaptic partners to fine tune inputs, second, crosstalk between sensory neurons to integrate responses and third, local/humoral extrasynaptic signals to facilitate broader, long term system-wide modulation. Overall, these observations highlight the differences between an anatomical wiring diagram and 'functional connectomes' that are essential to generate the alternative circuit configurations required to choose different behavioral outcomes in the face of changing environmental inputs.

Neuropeptides amplify and focus the monoaminergic inhibition of nociception in Caenorhabditis elegans.

Monoamines and neuropeptides interact to modulate most behaviors. To better understand these interactions, we have defined the roles of tyramine (TA), octopamine, and neuropeptides in the inhibition of aversive behavior in Caenorhabditis elegans. TA abolishes the serotonergic sensitization of aversive behavior mediated by the two nociceptive ASH sensory neurons and requires the expression of the adrenergic-like, Gαq-coupled, TA receptor TYRA-3 on inhibitory monoaminergic and peptidergic neurons. For example, TA inhibition requires Gαq and Gαs signaling in the peptidergic ASI sensory neurons, with an array of ASI neuropeptides activating neuropeptide receptors on additional neurons involved in locomotory decision-making. The ASI neuropeptides required for tyraminergic inhibition are distinct from those required for octopaminergic inhibition, suggesting that individual monoamines stimulate the release of different subsets of ASI neuropeptides. Together, these results demonstrate that a complex humoral mix of monoamines is focused by more local, synaptic, neuropeptide release to modulate nociception and highlight the similarities between the tyraminergic/octopaminergic inhibition of nociception in C. elegans and the noradrenergic inhibition of nociception in mammals that also involves inhibitory peptidergic signaling.

Monoaminergic signaling as a target for anthelmintic drug discovery: receptor conservation among the free-living and parasitic nematodes.

This review is designed to summarize the information on monoamine-dependent paralysis as a target for anthelmintic development, examine the conservation of monoamine receptors in the genomes of both free-living and parasitic nematodes, and highlight the utility of the Caenorhabditis elegans model system for dissecting the monoaminergic modulation of locomotory decision-making.

RHGF-2 is an essential Rho-1 specific RhoGEF that binds to the multi-PDZ domain scaffold protein MPZ-1 in Caenorhabditis elegans.

RhoGEF proteins activate the Rho family of small GTPases and thus play a key role in regulating fundamental cellular processes such as cell morphology and polarity, cell cycle progression and gene transcription. We identified a Caenorhabditis elegans RhoGEF protein, RHGF-2, as a binding partner of the C. elegans multi-PDZ domain scaffold protein MPZ-1 (MUPP1 in mammals). RHGF-2 exhibits significant identity to the mammalian RhoGEFs PLEKHG5/Tech/Syx and contains a class I C-terminal PDZ binding motif (SDV) that interacts most strongly to MPZ-1 PDZ domain eight. RHGF-2 RhoGEF activity is specific to the C. elegans RhoA homolog RHO-1 as determined by direct binding, GDP/GTP exchange and serum response element-driven reporter activity. rhgf-2 is an essential gene since rhgf-2 deletion mutants do not elongate during embryogenesis and hatch as short immobile animals that arrest development. Interestingly, the expression of a functional rhgf-2::gfp transgene appears to be exclusively neuronal and rhgf-2 overexpression results in loopy movement with exaggerated body bends. Transient expression of RHGF-2 in N1E-115 neuroblastoma cells prevents neurite outgrowth similar to constitutive RhoA activation in these cells. Together, these observations indicate neuronally expressed RHGF-2 is an essential RHO-1 specific RhoGEF that binds most strongly to MPZ-1 PDZ domain eight and is required for wild-type C. elegans morphology and growth.

Monoamines and neuropeptides interact to inhibit aversive behaviour in Caenorhabditis elegans.

Pain modulation is complex, but noradrenergic signalling promotes anti-nociception, with α(2)-adrenergic agonists used clinically. To better understand the noradrenergic/peptidergic modulation of nociception, we examined the octopaminergic inhibition of aversive behaviour initiated by the Caenorhabditis elegans nociceptive ASH sensory neurons. Octopamine (OA), the invertebrate counterpart of norepinephrine, modulates sensory-mediated reversal through three α-adrenergic-like OA receptors. OCTR-1 and SER-3 antagonistically modulate ASH signalling directly, with OCTR-1 signalling mediated by Gα(o). In contrast, SER-6 inhibits aversive responses by stimulating the release of an array of 'inhibitory' neuropeptides that activate receptors on sensory neurons mediating attraction or repulsion, suggesting that peptidergic signalling may integrate multiple sensory inputs to modulate locomotory transitions. These studies highlight the complexity of octopaminergic/peptidergic interactions, the role of OA in activating global peptidergic signalling cascades and the similarities of this modulatory network to the noradrenergic inhibition of nociception in mammals, where norepinephrine suppresses chronic pain through inhibitory α(2)-adrenoreceptors on afferent nociceptors and stimulatory α(1)-receptors on inhibitory peptidergic interneurons.

The interaction of octopamine and neuropeptides to slow aversive responses in C. elegans mimics the modulation of chronic pain in mammals.

Octopamine (OA) appears to function as the invertebrate counterpart of norepinephrine (NE) in the modulation of a number of key behaviors. In C. elegans, OA signaling is complex, mediated by at least three distinct α-adrenergic-like receptors and appears to activate more global peptidergic signaling cascades that have the potential to dramatically amplify the octopaminergic signal. These OA-dependent peptidergic signaling cascades involve an array of neuropeptides that activate receptors throughout the nervous system and have the potential to both directly and indirectly modulate locomotory decision-making. In this commentary we highlight the use of C. elegans as a model to expand our understanding of noradrenergic signaling in mammals, specifically as it relates to the role of NE in anti-nociception.

Dissecting the serotonergic food signal stimulating sensory-mediated aversive behavior in C. elegans.

Nutritional state often modulates olfaction and in Caenorhabditis elegans food stimulates aversive responses mediated by the nociceptive ASH sensory neurons. In the present study, we have characterized the role of key serotonergic neurons that differentially modulate aversive behavior in response to changing nutritional status. The serotonergic NSM and ADF neurons play antagonistic roles in food stimulation. NSM 5-HT activates SER-5 on the ASHs and SER-1 on the RIA interneurons and stimulates aversive responses, suggesting that food-dependent serotonergic stimulation involves local changes in 5-HT levels mediated by extrasynaptic 5-HT receptors. In contrast, ADF 5-HT activates SER-1 on the octopaminergic RIC interneurons to inhibit food-stimulation, suggesting neuron-specific stimulatory and inhibitory roles for SER-1 signaling. Both the NSMs and ADFs express INS-1, an insulin-like peptide, that appears to cell autonomously inhibit serotonergic signaling. Food also modulates directional decisions after reversal is complete, through the same serotonergic neurons and receptors involved in the initiation of reversal, and the decision to continue forward or change direction after reversal is dictated entirely by nutritional state. These results highlight the complexity of the "food signal" and serotonergic signaling in the modulation of sensory-mediated aversive behaviors.

A genetic survey of fluoxetine action on synaptic transmission in Caenorhabditis elegans.

Fluoxetine is one of the most commonly prescribed medications for many behavioral and neurological disorders. Fluoxetine acts primarily as an inhibitor of the serotonin reuptake transporter (SERT) to block the removal of serotonin from the synaptic cleft, thereby enhancing serotonin signals. While the effects of fluoxetine on behavior are firmly established, debate is ongoing whether inhibition of serotonin reuptake is a sufficient explanation for its therapeutic action. Here, we provide evidence of two additional aspects of fluoxetine action through genetic analyses in Caenorhabditis elegans. We show that fluoxetine treatment and null mutation in the sole SERT gene mod-5 eliminate serotonin in specific neurons. These neurons do not synthesize serotonin but import extracellular serotonin via MOD-5/SERT. Furthermore, we show that fluoxetine acts independently of MOD-5/SERT to regulate discrete properties of acetylcholine (Ach), gamma-aminobutyric acid (GABA), and glutamate neurotransmission in the locomotory circuit. We identified that two G-protein-coupled 5-HT receptors, SER-7 and SER-5, antagonistically regulate the effects of fluoxetine and that fluoxetine binds to SER-7. Epistatic analyses suggest that SER-7 and SER-5 act upstream of AMPA receptor GLR-1 signaling. Our work provides genetic evidence that fluoxetine may influence neuronal functions and behavior by directly targeting serotonin receptors.

The monoaminergic modulation of sensory-mediated aversive responses in Caenorhabditis elegans requires glutamatergic/peptidergic cotransmission.

Monoamines and neuropeptides interact to modulate behavioral plasticity in both vertebrates and invertebrates. In Caenorhabditis elegans behavioral state or "mood" is dependent on food availability and is translated by both monoaminergic and peptidergic signaling in the fine-tuning of most behaviors. In the present study, we have examined the interaction of monoamines and peptides on C. elegans aversive behavior mediated by a pair of polymodal, nociceptive, ASH sensory neurons. Food or serotonin sensitize the ASHs and stimulate aversive responses through a pathway requiring the release of nlp-3-encoded neuropeptides from the ASHs. Peptides encoded by nlp-3 appear to stimulate ASH-mediated aversive behavior through the neuropeptide receptor-17 (NPR-17) receptor. nlp-3- and npr-17-null animals exhibit identical phenotypes and animals overexpressing either nlp-3 or npr-17 exhibit elevated aversive responses off food that are absent when nlp-3 or npr-17 are overexpressed in npr-17- or nlp-3-null animals, respectively. ASH-mediated aversive responses are increased by activating either Galpha(q) or Galpha(s) in the ASHs, with Galpha(s) signaling specifically stimulating the release of nlp-3-encoded peptides. In contrast, octopamine appears to inhibit 5-HT stimulation by activating Galpha(o) signaling in the ASHs that, in turn, inhibits both Galpha(s) and Galpha(q) signaling and the release of nlp-3-encoded peptides. These results demonstrate that serotonin and octopamine reversibly modulate the activity of the ASHs, and highlight the utility of the C. elegans model for defining interactions between monoamines and peptides in individual neurons of complex sensory-mediated circuits.

Three distinct amine receptors operating at different levels within the locomotory circuit are each essential for the serotonergic modulation of chemosensation in Caenorhabditis elegans.

Serotonin modulates behavioral plasticity in both vertebrates and invertebrates and in Caenorhabditis elegans regulates key behaviors, including locomotion, aversive learning and olfaction through at least four different 5-HT receptors. In the present study, we examined the serotonergic stimulation of aversive responses to dilute octanol in animals containing null alleles of these 5-HT receptors. Both ser-1 and mod-1 null animals failed to increase sensitivity to dilute octanol on food/5-HT, in contrast to wild-type, ser-4 or ser-7 null animals. 5-HT sensitivity was restored by the expression of MOD-1 and SER-1 in the AIB or potentially the AIY, and RIA interneurons of mod-1 and ser-1 null animals, respectively. Because none of these 5-HT receptors appear to be expressed in the ASH sensory neurons mediating octanol sensitivity, we identified a 5-HT(6)-like receptor, F16D3.7(SER-5), that was required for food/5-HT-dependent increases in octanol sensitivity. ser-5 null animals failed to increase octanol sensitivity in the presence of food/5-HT and sensitivity could be restored by expression of SER-5 in the ASHs. Similarly, the RNAi knockdown of ser-5 expression in the ASHs of wild-type animals also abolished 5-HT-dependent increases in octanol sensitivity, suggesting that SER-5 modulates the octanol responsiveness of the ASHs directly. Together, these results suggest that multiple amine receptors, functioning at different levels within the locomotory circuit, are each essential for the serotonergic modulation of ASH-mediated aversive responses.

Dual excitatory and inhibitory serotonergic inputs modulate egg laying in Caenorhabditis elegans.

Serotonin (5-HT) regulates key processes in both vertebrates and invertebrates. Previously, four 5-HT receptors that contributed to the 5-HT modulation of egg laying were identified in Caenorhabditis elegans. Therefore, to assess potential receptor interactions, we generated animals containing combinations of null alleles for each receptor, especially animals expressing only individual 5-HT receptors. 5-HT-stimulated egg laying and egg retention correlated well with different combinations of predicted excitatory and inhibitory serotonergic inputs. For example, 5-HT did not stimulate egg laying in ser-1, ser-7, or ser-7 ser-1 null animals, and ser-7 ser-1 animals retained more eggs than wild-type animals. In contrast, 5-HT-stimulated egg laying in ser-4;mod-1 animals was greater than in wild-type animals, and ser-4;mod-1 animals retained fewer eggs than wild-type animals. Surprisingly, ser-4;mod-1;ser-7 ser-1 animals retained the same number of eggs as wild-type animals and exhibited significant 5-HT-stimulated egg laying that was dependent on a previously uncharacterized receptor, SER-5. 5-HT-stimulated egg laying was absent in ser-5;ser-4;mod-1;ser-7 ser-1 animals, and these animals retained more eggs than either wild-type or ser-4;mod-1;ser-7 ser-1 animals. The 5-HT sensitivity of egg laying could be restored by ser-5 muscle expression. Together, these results highlight the dual excitatory/inhibitory serotonergic inputs that combine to modulate egg laying.

Tyramine and octopamine independently inhibit serotonin-stimulated aversive behaviors in Caenorhabditis elegans through two novel amine receptors.

Biogenic amines modulate key behaviors in both vertebrates and invertebrates. In Caenorhabditis elegans, tyramine (TA) and octopamine (OA) inhibit aversive responses to 100%, but not dilute (30%) octanol. TA and OA also abolish food- and serotonin-dependent increases in responses to dilute octanol in wild-type but not tyra-3(ok325) and f14d12.6(ok371) null animals, respectively, suggesting that TA and OA modulated responses to dilute octanol are mediated by separate, previously uncharacterized, G-protein-coupled receptors. TA and OA are high-affinity ligands for TYRA-3 and F14D12.6, respectively, based on their pharmacological characterization after heterologous expression. f14d12.6::gfp is expressed in the ASHs, the neurons responsible for sensitivity to dilute octanol, and the sra-6-dependent expression of F14D12.6 in the ASHs is sufficient to rescue OA sensitivity in f14d12.6(ok371) null animals. In contrast, tyra-3::gfp appears not to be expressed in the ASHs, but instead in other neurons, including the dopaminergic CEP/ADEs. However, although dopamine (DA) also inhibits 5-HT-dependent responses to dilute octanol, TA still inhibits in dop-2; dop-1; dop-3 animals that do not respond to DA and cat-2(tm346) and Pdat-1::ICE animals that lack significant dopaminergic signaling, suggesting that DA is not an intermediate in TA inhibition. Finally, responses to TA and OA selectively desensitize after preexposure to the amines. Our data suggest that although tyraminergic and octopaminergic signaling yield identical phenotypes in these olfactory assays, they act independently through distinct receptors to modulate the ASH-mediated locomotory circuit and that C. elegans is a useful model to study the aminergic modulation of sensory-mediated locomotory behaviors.