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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Are Caenorhabditis elegans receptors useful targets for drug discovery: pharmacological comparison of tyramine receptors with high identity from C. elegans (TYRA-2) and Brugia malayi (Bm4).

The biogenic amine, tyramine (TA), modulates a number of key processes in nematodes and a number of TA-specific receptors have been identified. In the present study, we have identified a putative TA receptor (Bm4) in the recently completed Brugia malayi genome and compared its pharmacology to its putative Caenorhabditis elegans orthologue, TYRA-2, under identical expression and assay conditions. TYRA-2 and Bm4 are the most closely related C. elegans and B. malayi BA receptors and differ by only 14aa in the TM regions directly involved in ligand binding. Membranes from HEK-293 cells stably expressing Bm4 exhibited specific, saturable, high affinity, [(3)H]LSD and [(3)H]TA binding with K(d)s of 18.1+/-0.93 and 15.1+/-0.2 nM, respectively. More importantly, both TYRA-2 and Bm4 TA exhibited similar rank orders of potencies for a number of potential tyraminergic ligands. However, some significant differences were noted. For example, chloropromazine exhibited an order of magnitude higher affinity for Bm4 than TYRA-2 (pK(i)s of 7.6+/-0.2 and 6.49+/-0.1, respectively). In contrast, TYRA-2 had significantly higher affinity for phentolamine than Bm4. These results highlight the utility of the nearly completed B. malayi genome and the importance of using receptors from individual parasitic nematodes for drug discovery.

SER-7, a Caenorhabditis elegans 5-HT7-like receptor, is essential for the 5-HT stimulation of pharyngeal pumping and egg laying.

Serotonin (5-HT) stimulates both pharyngeal pumping and egg laying in Caenorhabditis elegans. Four distinct 5-HT receptors have been partially characterized, but little is known about their function in vivo. SER-7 exhibits most sequence identity to the mammalian 5-HT7 receptors and couples to a stimulation of adenyl cyclase when expressed in COS-7 cells. However, many 5-HT7-specific agonists have low affinity for SER-7. 5-HT fails to stimulate pharyngeal pumping and the firing of the MC motorneurons in animals containing the putative ser-7(tm1325) and ser-7(tm1728) null alleles. In addition, although pumping on bacteria is upregulated in ser-7(tm1325) animals, pumping is more irregular. A similar failure to maintain "fast pumping" on bacteria also was observed in ser-1(ok345) and tph-1(mg280) animals that contain putative null alleles of a 5-HT2-like receptor and tryptophan hydroxylase, respectively, suggesting that serotonergic signaling, although not essential for the upregulation of pumping on bacteria, "fine tunes" the process. 5-HT also fails to stimulate egg laying in ser-7(tm1325), ser-1(ok345), and ser-7(tm1325) ser-1(ok345) animals, but only the ser-7 ser-1 double mutants exhibit an Egl phenotype. All of the SER-7 mutant phenotypes are rescued by the expression of full-length ser-7gfp translational fusions. ser-7gfp is expressed in several pharyngeal neurons, including the MC, M2, M3, M4, and M5, and in vulval muscle. Interestingly, 5-HT inhibits egg laying and pharyngeal pumping in ser-7 null mutants and the 5-HT inhibition of egg laying, but not pumping, is abolished in ser-7(tm1325);ser-4(ok512) double mutants. Taken together, these results suggest that SER-7 is essential for the 5-HT stimulation of both egg laying and pharyngeal pumping, but that other signaling pathways can probably fulfill similar roles in vivo.

Genes encoding putative biogenic amine receptors in the parasitic nematode Brugia malayi.

Filarial nematodes, such as Brugia malayi, cause major health problems worldwide. The lack of a vaccine against B. malayi, combined with ineffective chemotherapy against the adult has prompted the examination of biogenic amine receptors (BARs) as possible targets for drug discovery. We employed bioinformatics to identify genes encoding putative B. malayi BARs. Surprisingly, the B. malayi genome contains half of the genes predicted to encode BARs in the genomes of free-living nematodes such as Caenorhabditis elegans or C. briggsae; however, all of the predicted B. malayi receptors have clear orthologues in C. elegans. The B. malayi genes encode each of the major BAR subclasses, including three serotonin, two dopamine and two tyramine/octopamine receptors and the structure of orthologous BAR genes is conserved. We find that potential G-protein coupling and ligand-specificity of individual BARs may be predicted by phylogenetic comparisons. Our results provide a framework for how G-protein coupled receptors may be targeted for drug development in medically important parasitic nematodes.

Role of dihydrolipoyl dehydrogenase (E3) and a novel E3-binding protein in the NADH sensitivity of the pyruvate dehydrogenase complex from anaerobic mitochondria of the parasitic nematode, Ascaris suum.

The pyruvate dehydrogenase complex (PDC) plays changing roles during the aerobic-anaerobic transition in the life cycle of the parasitic nematode, Ascaris suum. However, the dihydrolipoyl dehydrogenase (E3) subunit appears to be identical in all stages, despite the fact that the PDC is less sensitive to NADH inhibition in anaerobic muscle. Therefore, we have cloned cDNAs encoding E3 and a novel anaerobic-specific E3-binding protein (E3BP) that lacks the terminal lipoyl domain found in E3BPs from yeast and mammals, and functionally expressed E3 and E3 mutants designed to have decreased dimer stability on the assumption that the binding of E3 to an anaerobic-specific E3BP might stabilize the E3 dimer interface and decrease E3 sensitivity to NADH inhibition. As predicted, the mutants exhibited decreased thermal stability, increased sensitivity to NADH and the binding of E3(Y18F) to the E3-depleted core of the pig heart PDC increased E3 activity and decreased E3 sensitivity to NADH inhibition. However, although the free A. suum E3 was less sensitive to NADH inhibition than the pig heart E3, both E3s were significantly more sensitive to NADH inhibition when assayed with dihydrolipoamide than their corresponding PDCs assayed with pyruvate. More importantly, the binding of rE3 to its core complex had little effect on its apparent K(m) for NAD(+), K(i) for NADH inhibition, or the NADH/NAD(+) ratio yielding 50% inhibition. These data suggest that although binding to the core stabilizes the E3 dimer interface, it does not play a significant role in reducing the sensitivity of the A. suum PDC to NADH inhibition during anaerobiosis.

Secretion of a novel developmentally regulated chitinase (family 19 glycosyl hydrolase) into the perivitelline fluid of the parasitic nematode, Ascaris suum.

The early development of the parasitic nematode, Ascaris suum, occurs within a chitinous eggshell and an abundant chitinase (As-p50) has been identified in the perivitelline fluid (PVF) surrounding the infective larva prior to hatching. A cDNA encoding As-p50 was cloned, sequenced and the protein expressed in Escherichia coli. As-p50 is a member of glycosyl hydrolase family 19, previously identified only in plants, making the characterization of As-p50 the first family 19 glycosyl hydrolase from any animal species. As expected, the chitinase activity of recombinant As-p50 or isolated PVF was insensitive to allosamidin. As-p50 expression was developmentally regulated. As-p50 mRNA appeared between days 5 and 8 of development prior to the formation of the first-stage larva (L1). The As-p50 protein and chitinase activity appeared later between days 8 and 15 and remained at constant levels until hatching. GFP-promoter constructs of C08B6.4, the most closely related Caenorhabditis elegans As-p50 homologue, were expressed in hypodermal cells of 3-fold stage larvae and L1s with a timing similar to that of As-p50 and the fusion protein was secreted into the space between the hypodermis and the cuticle. Taken together, these results suggest that As-p50 is involved in the formation of the L1 cuticle and/or the initial molt; however, As-p50 may be multifunctional and also responsible for the digestion of the eggshell during hatching.

Biogenic amine receptors in parasitic nematodes: what can be learned from Caenorhabditis elegans?

The biogenic amines, serotonin, octopamine, tyramine and dopamine regulate many essential processes in parasitic nematodes, such as pharyngeal pumping, muscle contraction, and egg-laying, as well as more complex behaviors, such as mechanosensation and foraging, making biogenic amine receptors excellent targets for drug discovery. This review is designed to summarize our knowledge of nematode biogenic amine signaling and preliminarily identify some of the key receptors involved in the regulation of biogenic amine-dependent behaviors through an analysis of the free-living nematode, Caenorhabditis elegans.