Pharyngeal Nervous System Research Articles

The enteric nervous system of the C. elegans pharynx is specified by the Sine oculis-like homeobox gene ceh-34.

Overarching themes in the terminal differentiation of the enteric nervous system, an autonomously acting unit of animal nervous systems, have so far eluded discovery. We describe here the overall regulatory logic of enteric nervous system differentiation of the nematode Caenorhabditis elegans that resides within the foregut (pharynx) of the worm. A C. elegans homolog of the Drosophila Sine oculis homeobox gene, ceh-34, is expressed in all 14 classes of interconnected pharyngeal neurons from their birth throughout their life time, but in no other neuron type of the entire animal. Constitutive and temporally controlled ceh-34 removal shows that ceh-34 is required to initiate and maintain the neuron type-specific terminal differentiation program of all pharyngeal neuron classes, including their circuit assembly. Through additional genetic loss of function analysis, we show that within each pharyngeal neuron class, ceh-34 cooperates with different homeodomain transcription factors to individuate distinct pharyngeal neuron classes. Our analysis underscores the critical role of homeobox genes in neuronal identity specification and links them to the control of neuronal circuit assembly of the enteric nervous system. Together with the pharyngeal nervous system simplicity as well as its specification by a Sine oculis homolog, our findings invite speculations about the early evolution of nervous systems.

Structural Analysis of the Caenorhabditis elegans Dauer Larval Anterior Sensilla by Focused Ion Beam-Scanning Electron Microscopy.

At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments.

The connectome of the Caenorhabditis elegans pharynx.

Detailed anatomical maps of individual organs and entire animals have served as invaluable entry points for ensuing dissection of their evolution, development, and function. The pharynx of the nematode Caenorhabditis elegans is a simple neuromuscular organ with a self-contained, autonomously acting nervous system, composed of 20 neurons that fall into 14 anatomically distinct types. Using serial electron micrograph (EM) reconstruction, we re-evaluate here the connectome of the pharyngeal nervous system, providing a novel and more detailed view of its structure and predicted function. Contrasting the previous classification of pharyngeal neurons into distinct inter- and motor neuron classes, we provide evidence that most pharyngeal neurons are also likely sensory neurons and most, if not all, pharyngeal neurons also classify as motor neurons. Together with the extensive cross-connectivity among pharyngeal neurons, which is more widespread than previously realized, the sensory-motor characteristics of most neurons define a shallow network architecture of the pharyngeal connectome. Network analysis reveals that the patterns of neuronal connections are organized into putative computational modules that reflect the known functional domains of the pharynx. Compared with the somatic nervous system, pharyngeal neurons both physically associate with a larger fraction of their neighbors and create synapses with a greater proportion of their neighbors. We speculate that the overall architecture of the pharyngeal nervous system may be reminiscent of the architecture of ancestral, primitive nervous systems.

The pharyngeal nervous system orchestrates feeding behavior in planarians.

Planarians exhibit traits of cephalization but are unique among bilaterians in that they ingest food by means of goal-directed movements of a trunk-positioned pharynx, following protrusion of the pharynx out of the body, raising the question of how planarians control such a complex set of body movements for achieving robust feeding. Here, we use the freshwater planarian Dugesia japonica to show that an isolated pharynx amputated from the planarian body self-directedly executes its entire sequence of feeding functions: food sensing, approach, decisions about ingestion, and intake. Gene-specific silencing experiments by RNA interference demonstrated that the pharyngeal nervous system (PhNS) is required not only for feeding functions of the pharynx itself but also for food-localization movements of individual animals, presumably via communication with the brain. These findings reveal an unexpected central role of the PhNS in the linkage between unique morphological phenotypes and feeding behavior in planarians.

Yeast two-hybrid screening identifies MPZ-1 and PTP-1 as candidate scaffolding proteins of metabotropic glutamate receptors in Caenorhabditis elegans

The metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptor that undergo extensive interactions with scaffolding proteins, and this is intrinsic to their function as an important group of neuromodulators at glutamatergic synapses. The Caenorhabditis elegans nervous system expresses three metabotropic glutamate receptors, MGL-1, MGL-2 and MGL-3. Relatively little is known about how the function and signalling of these receptors is organised in C. elegans. To identify proteins that scaffold the MGL-1 receptor, we have conducted a yeast two-hybrid screen. Three of the interacting proteins, MPZ-1, NRFL-1 and PTP-1, displayed motifs characteristic of mammalian mGluR scaffolding proteins. Using cellular co-expression criterion, we show mpz-1 and ptp-1 exhibited overlapping expression patterns with subsets of mgl-1 neurons. This included neurones in the pharyngeal nervous system that control the feeding organ of the worm. The mGluR agonist L-CCG-I inhibits the activity of this network in wild-type worms, in an MGL-1 and dose-dependent manner. We utilised L-CCG-I to identify if MGL-1 function was disrupted in mutants with deletions in the mpz-1 gene. The mpz-1 mutants displayed a largely wild-type response to L-CCG-I, suggesting MGL-1 signalling is not overtly disrupted consistent with a non-obligatory modulatory function in receptor scaffolding. The selectivity of the protein interactions and overlapping expression identified here warrant further investigation of the functional significance of scaffolding of metabotropic glutamate receptor function.

Pharyngeal pumping in Caenorhabditis elegans depends on tonic and phasic signaling from the nervous system.

Rhythmic movements are ubiquitous in animal locomotion, feeding, and circulatory systems. In some systems, the muscle itself generates rhythmic contractions. In others, rhythms are generated by the nervous system or by interactions between the nervous system and muscles. In the nematode Caenorhabditis elegans, feeding occurs via rhythmic contractions (pumping) of the pharynx, a neuromuscular feeding organ. Here, we use pharmacology, optogenetics, genetics, and electrophysiology to investigate the roles of the nervous system and muscle in generating pharyngeal pumping. Hyperpolarization of the nervous system using a histamine-gated chloride channel abolishes pumping, and optogenetic stimulation of pharyngeal muscle in these animals causes abnormal contractions, demonstrating that normal pumping requires nervous system function. In mutants that pump slowly due to defective nervous system function, tonic muscle stimulation causes rapid pumping, suggesting tonic neurotransmitter release may regulate pumping. However, tonic cholinergic motor neuron stimulation, but not tonic muscle stimulation, triggers pumps that electrophysiologically resemble typical rapid pumps. This suggests that pharyngeal cholinergic motor neurons are normally rhythmically, and not tonically active. These results demonstrate that the pharynx generates a myogenic rhythm in the presence of tonically released acetylcholine, and suggest that the pharyngeal nervous system entrains contraction rate and timing through phasic neurotransmitter release.

Multiple excitatory and inhibitory neural signals converge to fine-tune Caenorhabditis elegans feeding to food availability.

How an animal matches feeding to food availability is a key question for energy homeostasis. We addressed this in the nematode Caenorhabditis elegans, which couples feeding to the presence of its food (bacteria) by regulating pharyngeal activity (pumping). We scored pumping in the presence of food and over an extended time course of food deprivation in wild-type and mutant worms to determine the neural substrates of adaptive behavior. Removal of food initially suppressed pumping but after 2 h this was accompanied by intermittent periods of high activity. We show pumping is fine-tuned by context-specific neural mechanisms and highlight a key role for inhibitory glutamatergic and excitatory cholinergic/peptidergic drives in the absence of food. Additionally, the synaptic protein UNC-31 [calcium-activated protein for secretion (CAPS)] acts through an inhibitory pathway not explained by previously identified contributions of UNC-31/CAPS to neuropeptide or glutamate transmission. Pumping was unaffected by laser ablation of connectivity between the pharyngeal and central nervous system indicating signals are either humoral or intrinsic to the enteric system. This framework in which control is mediated through finely tuned excitatory and inhibitory drives resonates with mammalian hypothalamic control of feeding and suggests that fundamental regulation of this basic animal behavior may be conserved through evolution from nematode to human.

Distinct expression patterns and functions of the pygopus genes in amphioxus and zebrafish early embryogenesis

The Wnt/β-catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machine to β-catenin to regulate the transcription of target genes. Previous studies revealed that the Pygo genes were broadly involved in the embryonic development of Drosophila, Xenopus and mouse, however, their functions in the embryonic development of amphioxus and zebrafish remain to be investigated. Here, we cloned and characterized one Pygo gene from amphioxus Branchiostoma belcheri (BbPygo) and two from zebrafish Danio rerio (DrPygo1 and DrPygo2). The spatial–temporal expression profiles showed that all these genes were maternally expressed. BbPygo expression was distributed ubiquitously in the amphioxus embryos before the gastrula stage and became restricted in the pharyngeal region in late stages. Similar to BbPygo, DrPygo2 was also expressed throughout whole embryos before the early gastrula stage, but limited in the pharyngeal region and central nervous system (CNS) in subsequent developing stages. Further knockdown of the DrPygo2 resulted in developmental defects of the body axis, CNS and cartilage segments in zebrafish. In contrast, the expression signal of the DrPygo1 gene was not detectable using the in situ hybridization method, which is consistent with its extremely low expression level detected by RT-qPCR. The expression patterns and function investigations revealed in this study suggest that the three Pygo genes probably function differentially though all of them show a conserved maternal expression pattern during the early embryonic stages of amphioxus and zebrafish.

It's all in the wiring

To study the relationship between synaptic connectivity and circuit function, the authors compared network connectivity in the pharyngeal nervous system (PNS) of two related nematode species with different feeding behaviours, one microbivore and one predatory. A graph theoretical approach revealed that the two nematodes showed greatly different wiring of neurons that are homologous in terms of cell body position and neurite anatomy. The data suggest that the increased behavioural complexity of the predatory nematode is associated with increased complexity of PNS connectivity.

Selective Toxicity of the Anthelmintic Emodepside Revealed by Heterologous Expression of Human KCNMA1 inCaenorhabditis elegans

Emodepside is a resistance-breaking anthelmintic of a new chemical class, the cyclooctadepsipeptides. A major determinant of its anthelmintic effect is the calcium-activated potassium channel SLO-1. SLO-1 belongs to a family of channels that are highly conserved across the animal phyla and regulate neurosecretion, hormone release, muscle contraction, and neuronal network excitability. To investigate the selective toxicity of emodepside, we performed transgenic experiments in which the nematode SLO-1 channel was swapped for a mammalian ortholog, human KCNMA1. Expression of either the human channel or Caenorhabditis elegans slo-1 from the native slo-1 promoter in a C. elegans slo-1 functional null mutant rescued behavioral deficits that otherwise resulted from loss of slo-1 signaling. However, worms expressing the human channel were 10- to 100-fold less sensitive to emodepside than those expressing the nematode channel. Strains expressing the human KCNMA1 channel were preferentially sensitive to the mammalian channel agonists NS1619 and rottlerin. In the C. elegans pharyngeal nervous system, slo-1 is expressed in neurons, not muscle, and cell-specific rescue experiments have previously shown that emodepside inhibits serotonin-stimulated feeding by interfering with SLO-1 signaling in the nervous system. Here we show that ectopic overexpression of slo-1 in pharyngeal muscle confers sensitivity of the muscle to emodepside, consistent with a direct interaction of emodepside with the channel. Taken together, these data predict an emodepside-selective pharmacophore harbored by SLO-1. This has implications for the development of this drug/target interface for the treatment of helminth infections.

AutoEPG: Software for the Analysis of Electrical Activity in the Microcircuit Underpinning Feeding Behaviour of Caenorhabditis elegans

BackgroundThe pharyngeal microcircuit of the nematode Caenorhabditis elegans serves as a model for analysing neural network activity and is amenable to electrophysiological recording techniques. One such technique is the electropharyngeogram (EPG) which has provided insight into the genetic basis of feeding behaviour, neurotransmission and muscle excitability. However, the detailed manual analysis of the digital recordings necessary to identify subtle differences in activity that reflect modulatory changes within the underlying network is time consuming and low throughput. To address this we have developed an automated system for the high-throughput and discrete analysis of EPG recordings (AutoEPG).Methodology/Principal FindingsAutoEPG employs a tailor made signal processing algorithm that automatically detects different features of the EPG signal including those that report on the relaxation and contraction of the muscle and neuronal activity. Manual verification of the detection algorithm has demonstrated AutoEPG is capable of very high levels of accuracy. We have further validated the software by analysing existing mutant strains with known pharyngeal phenotypes detectable by the EPG. In doing so, we have more precisely defined an evolutionarily conserved role for the calcium-dependent potassium channel, SLO-1, in modulating the rhythmic activity of neural networks.Conclusions/SignificanceAutoEPG enables the consistent analysis of EPG recordings, significantly increases analysis throughput and allows the robust identification of subtle changes in the electrical activity of the pharyngeal nervous system. It is anticipated that AutoEPG will further add to the experimental tractability of the C. elegans pharynx as a model neural circuit.

A comparison of electrically evoked and channel rhodopsin-evoked postsynaptic potentials in the pharyngeal system of Caenorhabditis elegans

Dissecting the function of neural circuits requires the capability to stimulate and record from the component neurones. Optimally, the methods employed should enable precise activation of distinct elements within the circuit and high-fidelity readout of the neuronal response. Here we compare two methods for neural stimulation in the pharyngeal system of Caenorhabditis elegans by evoking postsynaptic potentials (PSPs) either by electrical stimulation or by expression of the channelrhodopsin [ChR2(gf)] in cholinergic neurones of the pharyngeal circuit. Using a dissection that isolates the pharynx and its embedded neural system of 20 neurones permits analysis of the neurotransmitter pathways within this microcircuit. We describe protocols for selective electrically evoked or ChR2-mediated cholinergic synaptic events in this circuit. The latter was achieved by generating strains, punc-17::ChR2(gf);yfp, that express ChR2(gf) in cholinergic neurones. PSPs evoked by both electrical and light stimulation exhibited a rapid time-course and were blocked by cholinergic receptor antagonists and rapidly reversed on cessation of the stimulus. Electrically evoked PSPs were also reduced in a hypomorphic mutant for the synaptic vesicle acetylcholine transporter, unc-17, further indicating they are nicotinic cholinergic PSPs. The pharyngeal nervous system is exquisitely sensitive to both electrical and light activation. For the latter, short light pulses of 200 mus delivered to punc-17::ChR2(gf);yfp are capable of generating full muscle action potentials. We conclude that the application of optogenetic approaches to the C. elegans isolated pharynx preparation opens the way for a precise molecular dissection of synaptic events in the pharyngeal microcircuit by providing a molecular and system level analysis of the synapses that control the feeding behaviour of C. elegans.

Functions of flatworm neuropeptides NPF, GYIRF and FMRF in course of pharyngeal regeneration of anterior body fragments of planarian,Girardia tigrina

The development of the new pharynx in anterior body fragments of G. tigrina was followed for 7 days by immunocytochemical (ICC) study using antiserum to neuropeptide F (NPF) and muscle staining with phalloidin. ICC investigation revealed the presence of NPF in pharyngeal nervous system, peripheral nerve plexuses, in central nervous system of intact planarians. NPF-immunoreactive (IR) nerve fibres were found at the site of regeneration surrounding pharyngeal rudiment. Restoration of the pharynx function during regeneration was analysed by appearance of food response in anterior fragments. Stimulating effects of NPF and FMRF on the pharyngeal regeneration has been observed. The data indicates an important role of neuropeptides in morphogenetic processes.

Behavioral and synaptic defects in C. elegans lacking the NK‐2 homeobox gene ceh‐28

C. elegans pharyngeal behavior consists of two distinct types of muscle contractions, termed pumping and peristalsis. Pumping ingests and concentrates bacteria in the anterior pharyngeal lumen, and it is occasionally followed by a transient peristaltic contraction that carries ingested bacteria through the posterior pharyngeal isthmus. These behaviors are controlled by a small pharyngeal nervous system consisting of 20 neurons that is almost completely independent of the extra-pharyngeal nervous system. The cholinergic motor neuron M4 controls peristalsis via synapses with the posterior isthmus muscles. Here we show that the NK-2 family homeobox gene ceh-28 is expressed in M4, where it regulates synapse assembly and peristalsis. ceh-28 mutants exhibit frequent and prolonged peristalses, and treatment with agonists or antagonists of muscarinic acetylcholine receptors can phenocopy or suppress ceh-28 mutant defects, respectively. Synapses in ceh-28 mutant M4 cells are irregularly spaced and sized, and they are abnormally located along the full length of the isthmus. We suggest that CEH-28 inhibits synaptogenesis, and that ceh-28 mutant behavioral defects result from excessive or ectopic stimulation of muscarinic acetylcholine receptors in the isthmus muscles.

Role of a FMRFamide‐like family of neuropeptides in the pharyngeal nervous system of Caenorhabditis elegans

The nervous system of C. elegans has a remarkable abundance of flp genes encoding FMRFamide-like (FLP) neuropeptides. To provide insight into the physiological relevance of this neuropeptide diversity, we have tested more than 30 FLPs (encoded by 23 flps) for bioactivity on C. elegans pharynx. Eleven flp genes encode peptides that inhibit pharyngeal activity, while eight flp genes encode peptides that are excitatory. Three potent peptides (inhibitory, FLP-13A, APEASPFIRFamide; excitatory, FLP-17A, KSAFVRFamide; excitatory, FLP-17B, KSQYIRFamide) are encoded by flp genes, which, according to reporter gene constructs, are expressed in pharyngeal motoneurons. Thus, they may act through receptors localized on the pharyngeal muscle. The two other potent peptides, FLP-8 (excitatory AF1, KNEFIRFamide,) and FLP-11A (inhibitory, AMRNALVRFamide), appear to be expressed in extrapharyngeal neurons and are therefore likely to act either indirectly or as neurohormones. Intriguingly, a single neuron can express peptides that have potent but opposing biological activity in the pharynx. Only five flp genes encode neuropeptides that have no observable effect on the pharynx, but none of these have shown reporter gene expression in the pharyngeal nervous system. To examine the roles of multiple peptides produced from single precursors, a comparison was made between the bioactivity of different neuropeptides for five flp genes (flp-3, flp-13, flp-14, flp-17, and flp-18). For all but one gene (flp-14), the effects of peptides encoded by the same gene were similar. Overall, this study demonstrates the impressive neurochemical complexity of the simple circuit that regulates feeding in the nematode, C. elegans.

Embryonic development of the nervous system of the temnocephalid flatworm Craspedella pedum.

The nervous system of temnocephalid flatworms consists of the brain and three pairs of longitudinal connectives extending into the trunk and tail. The connectives are crosslinked by an invariant number of regularly spaced commissures. Branches of the connectives innervate the tentacles of the head and the sucker organ in the tail. A set of nerve rings encircling the pharynx and connected to the brain and connectives constitute the pharyngeal nervous system. The nervous system is formed during early embryogenesis when the embryo represents a multilayered mesenchymal mass of cells. Gastrulation and the formation of separate epithelial germ layers that characterize most other animal groups are absent. The brain arises as a bilaterally symmetric condensation of postmitotic cells in the deep layers of the anterior region of the embryonic mesenchyme. The pattern of axon outgrowth, visualized by labeling with anti-acetylated tubulin (acTub) antibody, shows marked differences from the pattern observed in other flatworm taxa in regard to the number of neurons that express the acTub epitope. Acetylated tubulin is only expressed in neurons that form long axon tracts. In other flatworm species, such as the typhloplanoid Mesostoma and the polyclad Imogine, which were investigated by us with the acTub antibody (Hartenstein and Ehlers [2000] Dev. Genes Evol. 210:399-415; Younossi-Hartenstein and Hartenstein [2000] Dev. Genes Evol. 210:383-398), only a small number of "pioneer neurons" become acTub positive during the embryonic period. By contrast, in temnocephalids, most, if not all, neurons express acTub and form long, large-diameter axons. Initially, the brain commissure, pharyngeal nerve ring, and the connectives are laid down. Commissural tracts and tentacle nerves branching off the connectives appear later. We speculate that the precocious differentiation of the nervous system may be related to the fact that temnocephalids move by muscle action, and possess a massive and complex muscular system when they hatch. In addition, they have muscular specializations such as the anterior tentacles and the posterior sucker that are used as soon as they hatch. By contrast, juveniles of Mesostoma and larvae of polyclads move predominantly by ciliary action that may not require a complex neural circuitry for coordination.

The Caenorhabditis elegans peb-1 Gene Encodes a Novel DNA-Binding Protein Involved in Morphogenesis of the Pharynx, Vulva, and Hindgut

Gene expression in the Caenorhabditis elegans pharynx is regulated in part by organ-specific signals, which in the myo-2 gene target a regulatory sequence called the C subelement. C subelement activity requires the organ specification factor PHA-4, a winged-helix transcription factor expressed in all pharyngeal cells. To identify additional factors involved in pharyngeal organogenesis, we performed a yeast one-hybrid screen for C subelement binding proteins. Here we describe the novel factor PEB-1, which is coexpressed with PHA-4 in many pharyngeal cell types, including muscles, epithelial cells, marginal cells, and glands, but is undetectable in the pharyngeal nervous system. PEB-1 is also detected outside the pharynx in cells surrounding the rectum and vulva, as well as in the germ line. Reduction of peb-1 function using RNAi results in morphological defects in the somatic tissues in which peb-1 is expressed. We have mapped the PEB-1 DNA-binding domain to a 158-residue region, which is unrelated to known DNA-binding proteins but shares some sequence similarity to the Drosophila Mod(mdg4) proteins. PEB-1 specifically recognizes a site in the C subelement that partially overlaps the PHA-4 binding site. Both the PEB-1 and the PHA-4 binding sites are necessary for strong C subelement enhancer activity in some cells in which these factors are coexpressed. In contrast the PEB-1 site is dispensable for C subelement activity in pharyngeal neurons. We propose that PEB-1 functions with PHA-4 to activate target gene expression in cells in which they are coexpressed.

Caenorhabditis elegans rab-3Mutant Synapses Exhibit Impaired Function and Are Partially Depleted of Vesicles

Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterized Caenorhabditis elegans rab-3 mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore, rab-3 animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed in rab-3 mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.

Serotonergic Nervous System of Two Macrostomid Species: Recent or Ancient Divergence?

Serotonergic Nervous System of Two Macrostomid Species: Recent or Ancient Divergence?

Interacting genes required for pharyngeal excitation by motor neuron MC in Caenorhabditis elegans.

We studied the control of pharyngeal excitation in Caenorhabditis elegans. By laser ablating subsets of the pharyngeal nervous system, we found that the MC neuron type is necessary and probably sufficient for rapid pharyngeal pumping. Electropharyngeograms showed that MC transmits excitatory postsynaptic potentials, suggesting that MC acts as a neurogenic pacemaker for pharyngeal pumping. Mutations in genes required for acetylcholine (ACh) release and an antagonist of the nicotinic ACh receptor (nAChR) reduced pumping rates, suggesting that a nAChR is required for MC transmission. To identify genes required for MC neurotransmission, we screened for mutations that cause slow pumping but no other defects. Mutations in two genes, eat-2 and eat-18, eliminated MC neurotransmission. A gain-of-function eat-18 mutation, ad820sd, and a putative loss-of-function eat-18 mutation, ad1110, both reduced the excitation of pharyngeal muscle in response to the nAChR agonists nicotine and carbachol, suggesting that eat-18 is required for the function of a pharyngeal nAChR. Fourteen recessive mutations in eat-2 fell into five complementation classes. We found allele-specific genetic interactions between eat-2 and eat-18 that correlated with complementation classes of eat-2. We propose that eat-18 and eat-2 function in a multisubunit protein complex involved in the function of a pharyngeal nAChR.