Receptor-type Guanylyl Cyclases Confer Thermosensory Responses in C. elegans.

Thermosensation is critical for optimal regulation of physiology and behavior. C. elegans acclimates to its cultivation temperature (Tc) and exhibits thermosensitive behaviors at temperatures relative to Tc. These behaviors are mediated primarily by the AFD sensory neurons, which are extraordinarily thermosensitive and respond to thermal fluctuations at temperatures above a Tc-determined threshold. Although cGMP signaling is necessary for thermotransduction, the thermosensors in AFD are unknown. We show that AFD-specific receptor guanylyl cyclases (rGCs) are instructive for thermosensation. In addition to being necessary for thermotransduction, ectopic expression of these rGCs confers highly temperature-dependent responses onto diverse cell types. We find that the temperature response threshold is determined by the rGC and cellular context, and that multiple domains contribute to their thermosensory properties. Identification of thermosensory rGCs in C. elegans provides insight into mechanisms of thermosensation and thermal acclimation and suggests that rGCs may represent a new family of molecular thermosensors.

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.

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.

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.

SER-1, a Caenorhabditis elegans 5-HT2-like receptor, and a multi-PDZ domain containing protein (MPZ-1) interact in vulval muscle to facilitate serotonin-stimulated egg-laying.

Serotonin (5-HT) stimulation of egg-laying in Caenorhabditis elegans is abolished in ser-1 (ok345) animals and is rescued by ser-1 expression in vulval muscle. A PDZ binding motif (ETFL) at the SER-1 C-terminus is not essential for rescue, but facilitates SER-1 signaling. SER-1 binds specifically to PDZ domain 10 of the multi-PDZ domain protein, MPZ-1, based on GST pulldown and co-immunoprecipitation. mpz-1 is expressed in about 60 neurons and body wall and vulval muscles. In neurons, GFP-tagged MPZ-1 is punctate and colocalizes with the synaptic marker, synaptobrevin. The expression patterns of ser-1 and mpz-1 overlap in 3 pairs of neurons and vulval muscle. In addition, MPZ-1 also interacts with other GPCRs with acidic amino acids in the -3 position of their PDZ binding motifs. mpz-1 RNAi reduces 5-HT stimulated egg-laying in wild type animals and in ser-1 mutants rescued by muscle expression of SER-1. In contrast, mpz-1 RNAi has no effect on 5-HT stimulated egg-laying in ser-1 mutants rescued by expression of a truncated SER-1 that lacks the C-terminal PDZ binding motif. The overexpression of MPZ-1 PDZ domain 10 also inhibits 5-HT stimulated egg-laying. These studies suggest that the SER-1/MPZ-1 interaction facilitates SER-1 mediated signaling.

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.

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.