RESEARCH ARTICLE

Neuroligin tuning of pharyngeal pumping reveals extrapharyngealmodulation of feeding in Caenorhabditis elegansFernando Calahorro*, Francesca Keefe, James Dillon, Lindy Holden-Dye and Vincent O’Connor

ABSTRACTThe integration of distinct sensory modalities is essential forbehavioural decision making. In Caenorhabditis elegans, thisprocess is coordinated by neural circuits that integrate sensorycues from the environment to generate an appropriate behaviour atthe appropriate output muscles. Food is a multimodal cue thatimpacts the microcircuits to modulate feeding and foraging drivers atthe level of the pharyngeal and body wall muscle, respectively. Whenfood triggers an upregulation in pharyngeal pumping, it allows theeffective ingestion of food. Here, we show that a C. elegansmutant inthe single gene orthologous to human neuroligins, nlg-1, is defectivein food-induced pumping. This was not due to an inability to sensefood, as nlg-1 mutants were not defective in chemotaxis towardsbacteria. In addition, we found that neuroligin is widely expressed inthe nervous system, including AIY, ADE, ALA, URX and HSNneurons. Interestingly, despite the deficit in pharyngeal pumping,neuroligin was not expressed within the pharyngeal neuromuscularnetwork, which suggests an extrapharyngeal regulation of this circuit.We resolved electrophysiologically the neuroligin contribution tothe pharyngeal circuit by mimicking food-dependent pumping andfound that the nlg-1 phenotype is similar to mutants impaired inGABAergic and/or glutamatergic signalling. We suggest thatneuroligin organizes extrapharyngeal circuits that regulate thepharynx. These observations based on the molecular and cellulardeterminants of feeding are consistent with the emerging role ofneuroligin in discretely impacting functional circuits underpinningcomplex behaviours.

KEY WORDS: nlg-1, Sensory cues, EPG, Electropharyngeogram,Sensory modulation

INTRODUCTIONComplex cognitive function is underpinned by discretemicrocircuits that are integrated to control behaviour in a mannerappropriate for the environmental context (LeDoux, 2000; Barkuset al., 2010; O’Connor et al., 2010). Arguably one of the mostimportant environmental stimuli for an animal is its food. Thisrelationship has been subjected to extensive investigation for themodel genetic animal Caenorhabditis elegans with a view toproviding insight into the fundamental behavioural relationship ofan organism with its food source (Shtonda and Avery, 2006; Greeneet al., 2016; Thutupalli et al., 2017). For C. elegans, this food isbacteria, which it ingests by increasing its rate of pharyngeal

pumping in a process controlled by pharyngeal motor neurons thatdirect coordinated cycles of pharyngeal radial muscle contractionand relaxation (Franks et al., 2006). Intrinsic control of the pharynxmodulated byMC andM3 neurons is required for an efficient intakeof bacteria (Avery, 1993a; Avery, 1993b). In addition, there is anextrinsic control pivoting on the single RIP neuron that connects thepharyngeal circuit to the central nervous system. Worms in whichRIP has been ablated pump normally on food. This suggests thatneuroanatomical connectivity is not essential for the regulation offeeding and that neurohormonal signalling is sufficient to sustainpharyngeal pumping in response to food (Albertson and Thomson,1976; Dalliere et al., 2016). These mechanisms provide a meanswhereby the rate of pharyngeal pumping is influenced by thepresence of food (Horvitz et al., 1982; Rogers et al., 2001).

MC neurons act as pacemakers to control the rapid activation ofthe pumping rate in C. elegans (Avery and Horvitz, 1989; Raizenand Avery, 1994). This is facilitated by the motorneuron M3releasing glutamate onto the muscle to facilitate its relaxation. Thisrelaxation of the metacorpus and isthmus is critical in trappingthe bacteria in the corpus (Albertson and Thomson, 1976).Electrophysiological recording of the pharyngeal neural networkvia electropharyngeogram (EPG), provides a measurement of finefeatures that underpin pumping (Avery et al., 1995). This allowsrecording of the electrophysiological signature reflecting theactivity of both M3 and MC neurons and has identified neuronalsignalling and neuromodulatory components of the pharyngealmicrocircuit (Raizen and Avery, 1994; Dillon et al., 2009). Twoserotonergic neurons play a key role in mediating the pharyngealresponse to the availability of food: the pharyngeal secretory neuronNSM and the chemosensory neuron ASD (Li et al., 2012). Theneuron NSM is embedded within the pharyngeal circuit andimplicated in the acute response to newly encountered food (Iwaniret al., 2016). Although functional NSM neurons are required forbursts of fast pumping in the presence of food, serotonin productionin HSN neurons is sufficient to trigger food-dependent pumpingdynamics when serotonin production in NSM is perturbed. Thissuggests that the HSN neurons, located in the middle of the wormbody and sending no processes into the pharynx, are involved in theregulation of feeding (Lee et al., 2017). Finally, the role played byADF in feeding regulation is related to sensing fluctuations in theenvironment (Lee et al., 2017).

Food availability also modifies theworm’s locomotory behaviourand induces high probability of dwelling. This behaviour promotesthe worm’s residence on a patch of food. This dwelling behaviour isinterspersed with roaming, a behaviour that allows the worms toexplore their environment (Flavell et al., 2013). Thus, C. elegansexhibits a repertoire of behavioural responses to food that requirescoordination of precisely organised neural circuits. How theupstream sensory inputs are integrated and modulated to bringabout this coordinated behavioural response is important inunderstanding the systems-level control of foraging and feeding.Received 26 July 2018; Accepted 7 December 2018

Biological Sciences, Building 85, University of Southampton,Southampton SO17 1BJ, UK.

*Author for correspondence (F.Calahorro@soton.ac.uk)

F.C., 0000-0003-0659-7728

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In this study, we have focused on the role of neuroligin in theregulation of this food-dependent behaviour. Neuroligin is asynaptic protein intimately involved in the molecular organizationof discrete circuits (Calahorro, 2014; Bemben et al., 2015). Inparticular, in its absence, there is an impairment in the organizationof the excitatory–inhibitory balance in neuronal circuits(Varoqueaux et al., 2006). nlg-1 is the C. elegans orthologue ofhuman NLGN1; the neuroligin-1 protein is present at most synapsesand has a conserved organization in its functional domains(Calahorro, 2014). C. elegans nlg-1 mutants show a range ofsensory deficits (Calahorro et al., 2009; Hunter et al., 2010;Calahorro and Ruiz-Rubio, 2012). These investigations highlightthe evolutionarily conserved role of neuroligin in the organisation ofhigher-level function, which underpins the association of neuroliginmutations with autism spectrum disorder in humans. Recent studieshighlight the significance of neuroligin in the molecularorganization of synapses; mutations in neuroligin impart synapticdysfunction that, in turn, has consequences for discrete microcircuitfunction (Zhang et al., 2018; Polepali et al., 2017). This isparticularly pertinent in autism spectrum disorders where thebehavioural complexity underlying such disorders is related to‘fractionable’ circuits (Williams and Bowler, 2014). Discretedisruption of individual circuits could be used to modify thefunction in downstream circuits not reliant on neuroligin, leading toan altered synaptic function and consequently inducing neuro-atypical behaviours (Rothwell et al., 2014; Bey et al., 2018). Thisprinciple is reinforced in animal models in which neuroliginmutation may selectively impact disease-related behaviour.Indeed, C. elegans provide an excellent opportunity to delineate

the role of neuroligin in the context of discrete functionalmicrocircuit organization of sensory-driven behaviour. This isbecause C. elegans express one orthologue of neuroligin and can beinvestigated using behavioural outputs with well-establishedmicrocircuit dependence. In C. elegans, nlg-1 is selectivelyexpressed in a subset of neurons. Here, we provide evidence thatneuroligin functions in an extrapharyngeal circuit that coordinates afeeding response by modulating the cue promoting the pharyngealfunction. This further supports its fundamental role in theintegration of sensory information and co-ordination of activityvia discrete microcircuits.

MATERIALS AND METHODSC. elegans culture and strainsC. elegans strains were maintained under standard conditions(Brenner, 1974; Molin et al., 1999). Worms were synchronized bypicking L4 larval stage to new plates 18 h prior to performingbehavioural experiments. The strains used were: nlg-1 (ok259) X(6× outcrossed); nlg-1 (ok259) X, Ex [pPD95.77 (Pnlg-1::nlg-1Δ#14); Pmyo-3::gfp]; nlg-1 (ok259) X, Ex [pPD95.77; Pmyo-3::gfp]; RM371, pha-1(e2123), sIs [Pnlg-1::yfp+pCI( pha-1)];MT6308 eat-4 (ky5) III (2× outcrossed); CB156, unc-25 (e156)III (1× outcrossed); CB382, unc-49 (e382) III (2× outcrossed).We identified neurons that express nlg-1 reporter constructs based

on their cell position and co-labelling with mCherry, red fluorescentprotein (RFP) or DsRed-based reporters. The co-labelled reportertransgenes were: (1) oyls 51 (also known as Psrh-142::rfp). Aspecific marker for ADF chemosensory neuron (Xu et al., 2015);(2) oyIs44, oyIs44 [Podr-1::rfp+lin-15(+)]. A specific marker forAWB, AWB odor sensory neurons (Sarafi-Reinach et al., 2001);(3) ofEx205 [pBHL98 (lin-15ab+); Ptrx-1::DsRed]. A specificmarker for ASJ sensory neuron (Miranda-Vizuete et al., 2006);(4) dbEx719 [Pnpr-5::mCherry+Punc-122::gfp]. A specific marker

for sensory neurons ADF, ASE, ASG, ASI, ASJ, ASK, AWA,AWB, IL2; interneurons AIA, AUA; phasmids sensory neuronsPHA, PHB (Cohen et al., 2009); (5) sIs [Peat-4::mrfp]. A specificmarker for neurons expressing the vesicular glutamate transportereat-4 (Serrano-Saiz et al., 2013); (6) otIs151 [Pceh-36::rfp+rol-6(su1006)]. A specific marker for chemosensory neurons AWC andASE (Chang et al., 2003); (7) otIs181 [Pdat-1::mCherry+Pttx-3::mCherry]. Specific markers for both dopaminergic neurons (Pdat-1),ADE, PDE, CEP; and for the sensory interneuron AIY (Pttx-3)(Flames and Hobert, 2009); (8) vsIs108 [Pthp-1::rfp+Punc-13::gfp+lin-15(+)]. A specific marker for HSM andNSM serotonergic neurons(Tanis et al., 2008).

Cloning and transgenic methodscDNA cloning of the C. elegans nlg-1 transcriptThe genomic sequence of nlg-1 was used to design the followingspecific primers: nlg-1 forward, 5′-GGCATggatccCATTTATC-TTCTTCTCC-3′ and nlg-1 reverse, 5′-GTAgaattcGTTAGACCT-GTATCTCTTCC-3′. These were used to PCR amplify the codingregion corresponding to the Δ#14 transcript, the dominantneuroligin isoform in the adult stage (Calahorro et al., 2015),from the cDNA clone yk1657a10 (from Yuji Kohara, NationalInstitute of Genetics, Mishima, Japan).

Rescue constructs and transgenic methods.The 2.5 kb promoter region of nlg-1 was amplified using the Pnlg-1forward 5′-TtctagaCATATTTTTGGGGAGGCTTTC-3′ and Pnlg-1reverse 5′-GAAGGAGAAGAAGATAAATGggatccATGC-3′primers from the C40C9 cosmid clone (from Sanger Institute,UK). The promoter sequence was cloned using the XbaI/BamHIsite in a pDD95.77 vector where the GFP was removed. Finally, thenlg-1 Δ#14 sequence was fused to the nlg-1 promoter using theBamHI/EcoRI sites. The promoter was authenticated by sequencingusing the following primers: 5′-AAGCTTGCATGCCTGCAG-GTCGAC-3′, 5′-AAGCTTGCATGCCTGCAGGTCGAC-3′; andthe following primers for nlg-1: 5′-AATGCAGACTGGAGAAA-CTTTG-3′, 5′-CTATTACCAGAGCAAGACGATG-3′, 5′-GCTT-CTCTGGTTTCTCTTCTTATG-3′, 5′-CTGTTTCCTTTCCATTC-TTGTGC-3′, 5′-AGAATGGAAAGGAAACAGAGCC-3′, 5′-GT-GCGATGCGGATAGTAAGGG-3′.

Transgenic methodsnlg-1 (ok259) X 1-day-old adults were microinjected with nlg-1plasmid (prepared as described above; 50 ng/μl) together with the‘marker’ plasmid Pmyo-3::gfp (30 ng/μl), as previously described.For controls, nlg-1 (ok259) X animals were microinjected with‘empty rescue’ plasmid and the marker plasmid Pmyo-3::gfp at thesame concentration used for generating the rescue transgenic lines.Microinjection mixes were prepared using double-distilled water.

Behavioural assaysFeeding behaviourFeeding behaviour was visually scored by counting the number ofpharyngeal pumps for 1 min using a binocular dissectingmicroscope (Nikon SMZ800; ×63). A single pharyngeal pumpwas defined as one contraction–relaxation cycle of the terminalbulb of the pharyngeal muscle. To count pharyngeal pumps onfood, 1-day-old adult worms (L4+1) grown at 20°C, in standardNGM E. coli OP50 plates, were gently picked onto the middle ofthe bacterial lawn (OD600 of 0.8 AU seeded the day before). After10 min, the pharyngeal pumping was recorded using a handcounter. Five consecutive measurements (1 min each) were made

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and the mean of pharyngeal pumping rate for this time period wasthen calculated.To count pharyngeal pumps off food, 1-day-old adult worms

grown at 20°C, in standard NGM E. coli OP50 plates, were gentlypicked onto the middle of a 9-cm-diameter non-food plate and leftfor 5 min to clean themselves of attached bacteria. Animals werefinally transferred onto a second clean non-food plate, wherepumping rate was scored after 10 min. Five consecutivemeasurements (1 min each) were made and the mean ofpharyngeal pumping rate for this time period was then calculated.During this experiment, worms dried out after migration off theedge of the agar plate were not counted.

Chemotaxis: food race assaysNGM plates (9 cm) were poured the day before the assay andkept overnight at room temperature. These plates were seeded witha spot of 50 µl E. coli OP50 at an optical density of 0.8 AU(OD600nm) displaced 2 cm from the edge of the plate and incubatedovernight. The day before the assay 50–100 L4 animals were pickedonto NGM E. coli OP50 plates and kept at 20°C for 18 h. These L4+1 worms were washed twice in M9 buffer to remove residualbacteria adhered to the body. The washed worms were addedin a minimal volume 2 cm from the edge opposite the food patch.The number of animals reaching food was recorded every 10 minfor 2 h. The cumulative number that reached the food spot wascalculated.

Electropharyngeogram (EPG) recordingsThe extracellular electrophysiological activity in the pharyngealnetwork was measured using the microfluidic device Neurochip aspreviously described (Hu et al., 2013). L4+1 worms were loadedinto the NeuroChip in Dent’s saline (in mmol l−1: 10 D-glucose, 140NaCl, 1 MgCl2, 3 CaCl2, 6 KCl, and 10 HEPES; pH 7.4)

supplemented with 0.01% BSA (w/v). Extracellular voltagerecordings were made in ‘bridge’ mode, and the extracellularpotential was set to 0 mV using the voltage offset immediately priorto recording. Data were acquired using Axoscope (AxonInstruments) and stored for subsequent offline analysis. EPGtraces were annotated with AutoEPG software (Dillon et al., 2009).Statistical analysis was performed using one-way ANOVA withBonferroni multiple comparisons post-test. Recordings were madein both Dent’s saline alone or when loaded into the device in Dent’ssaline and 5 mmol l−1 5-hydroxytryptamine (5HT; serotonincreatinine sulfate monohydrate used for electrophysiologyexperiments was obtained from Sigma-Aldrich). EPGs wererecorded for 3–5 min from when the animal was trapped in therecording microchannel, conditions under which the 5 mmol l−1

serotonin induced pumping (Hu et al., 2013). A single pump EPGrecording corresponds to the coordinated contraction and relaxationof the pharynx. Recordings were analysed offline using a signalpeak detection and measurements made to define: frequency;average duration of single EPGs; number of P waves per EPG,regulated by activity of the inhibitory motorneuronM3; shape of theEPG using the R to E ratio, closely associated with muscledepolarization (contraction) at the beginning of a pump andrepolarization (relaxation) at the end of a pump; pattern ofactivity, under neural control.

ImagingImaging wormsA Nikon Eclipse (E800) microscope was used to image fluorescencefrom cells expressing GFP or RFP proteins under cell-specificpromoters (see above). Worms were placed in 0.5 μl M9 buffercontaining 10 mmol l−1 levamisole on a thin 2% agarose pad.Immobilized worms were covered with a 24×24 mm cover slip, andviewed with 40× or 63× objective magnification for no more than

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Fig. 1. A. Neuroligin-deficient mutants have lower pharyngeal pumping rates on food. (A) Bars represent the mean pharyngeal pumping rate ±s.e.m.The means were calculated from data collected from repeats of the same experiment conducted on different days, giving the specified ‘n’ number, but ineach case the experiment was paired with both wild-type N2 and nlg-1mutant. Rescue plasmid Pnlg-1::nlg-1 Δ#14 (nlg-1 rescue) was co-injected with themarkerplasmid Pmyo-3::gfp. To generate control lines, the marker plasmid Pmyo-3::gfp was co-injected together with the empty plasmid used to build the rescueplasmids. Data were subjected to one-way ANOVA followed by a Bonferroni’s post hoc test. There was a significant difference between nlg-1 mutant and thecontrol line means compared with the human nlg-1 rescue line (***P≤0.001). No significant difference was found between the means of nlg-1 mutant and thecontrol line (ns, P>0.05). (B) nlg-1 mutants are not chemotaxis-deficient for food. Data are expressed as the means±s.e.m. of ‘n’ experiments where eachexperiment is a single food race. Statistical analysis was performed using Student’s unpaired t-test. There were no significant differences between N2 and nlg-1mutants at any of the individual time points (n=3).

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Fig. 2. NLG-1 modulates the contraction–relaxation cycle of the pharyngeal neuromuscular system that underpins feeding. (A) (i) Representativeunfiltered electropharyngeogram (EPG) traces recorded using the NeuroChip with whole C. elegans, comparing the EPG waveform from both the wild type andnlg-1 (ok259) in the presence or absence of 5-HT. Excitation (E), relaxation (R) andP spikes are annotated in green and the pump duration of an EPG is indicated.(ii) Representative unfiltered EPG signatures from N2, nlg-1 mutant, control empty transgene and nlg-1 rescue. The asterisks mark the trace to denote thepresence of P spikes. In the nlg-1 and control transgenic strains, respectively, # indicates where P spikes should arise but are absent. (B) (i) Neuroligin mutantshave a longer pump duration (top) and a subsequent decreased pump rate (bottom). In the presence of 5 mmol l−1 5-HT despite a comparable increase inpump rate, the pump duration of the nlg-1 mutant is longer. (ii) Quantification of pump duration in N2 and ngl-1 mutant, comparing to control line (carrying theempty transgene) and nlg-1 rescue line. Pump duration is reduced relative to control by introducing the nlg-1 rescue transgene (***P≤0.001). (C) nlg-1 mutantsshowa significant reduction in the number ofP spikes per EPG (***P≤0.001) and it is partially restored in the presence of the nlg-1 rescue transgene (***P≤0.001).ns, not significant.

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10 min after addition of levamisole. At least 10 independent wormsper strain were analysed. The position of cell bodies relative toneuronal ganglia, as well as the dominant visible neuronal processwere imaged by both DIC optical and epifluorescence microscopy.Images were acquired through a Hamamatsu Photonics camerasoftware, and were cropped to size, assembled, and processed usingAbode PhotoShop® (Adobe Systems) and ImageJ (NIH) software.

DiI stainingA stock solution of 2 mg ml−1 DiI in dimethyl formamide wasprepared, and then a 1:200 dilution in M9 was used for staining.Animals were transferred into a microtitre well containing 150 µlstain and incubated for 2–3 h in the dark at room temperature. Usinga pipette, the animals were transferred to a fresh plate and left tocrawl on the bacterial lawn for about 1 h to destain. Finally, animalswere immobilized on an agar pad with sodium azide and visualizedby fluorescence microscopy.

Pharynx dissectionAround 10 L4+1 animals were transferred into a 3 cm Petri dishcontaining 2 ml M9 supplemented with BSA and incubated for 1 hat 4°C to immobilize the animals. Then, animals were observedunder a normal dissection microscope (40× magnification).Dissection was performed as previously described (Franks et al.,2009). The isolated pharynxes were pipetted onto a 2% agarose padand covered with a cover slip prior to experimental observation.A total of five isolated pharynxes were checked.

Statistical analysisData are expressed as means±s.e.m. and were analysed usingGraphPad Prism version 7.00 (GraphPad Software Inc.) using anunpaired Student’s t-test, one-way ANOVA or two-wayANOVA, asindicated in figure legends. For ANOVA, the data were subjected topost hoc analysis using the method indicated in the figure legends.Significance level was set at P<0.05.

RESULTSNeuroligin is required to upregulate feeding in the presenceof bacteriaIn a comparison of pharyngeal pumping with the N2 wild type(244±7 pumps min−1), the nlg-1 mutant worms showed a reductionof ≈26% (180±12 pumps min−1) (Fig. 1A). This was selective tothe on-food context, as the pharyngeal pumping rate of nlg-1mutants and wild-type animals was not significantly different15 min after transferring to a non-food plate (39±4 min−1 for wildtype and 38±4 min−1 for nlg-1; n=18 animals for each strain;P>0.05; data not shown). This food context-dependent reduction inpharyngeal pumping in nlg-1 was rescued by expressing a cDNAencoding the nlg-1 Δ#14 isoform from the nlg-1 promoter. Thetranscript Δ#14 is the dominant isoform in the adult stage (Calahorroet al., 2015). This fully rescued the pharyngeal deficit in nlg-1mutants (Fig. 1A). These results indicate a selective role for NLG-1-dependent signalling in maintaining a high rate of pharyngealpumping in the presence of food. It is unlikely that this deficit can beexplained by an inability of nlg-1 worms to sense food as they show

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Fig. 3. Glutamate and GABA signalling mutants exhibit a similar pharyngeal phenotype to nlg-1. (A) Representative unfiltered EPG traces recordedusing the NeuroChip comparing N2 signature with nlg-1, eat-4, unc-25 and unc-49 mutants. EPG parameters comparison between neuroligin and GABA/glutamate mutants: eat-4 (encodes a vesicular glutamate transporter), unc-25 (encodes a GABA neurotransmitter biosynthetic enzyme), unc-49 (encodes asubunit of a heteromeric GABA receptor). (B) Relative to N2, nlg-1 (*P≤0.05), eat-4 (*P≤0.05), unc-25 (***P≤0.001) and unc-49 (***P≤0.001) mutants showlonger pump duration. In addition nlg-1 (***P≤0.001), eat-4 (***P≤0.001), unc-25 (***P≤0.001) and unc-49 (***P≤0.001) have fewerP spikes. Finally, both unc-25and unc-49mutants show a reduced pump rate relative to N2, nlg-1 and eat-4 subsequent with the increase in pump duration. In basal conditions, no EPGs weredetected during 30 min recordings for unc-25 and/or unc-49 mutants. These parameters were extracted from 2 min recording in presence of 5-HT (5 mmol l−1).Statistical analysis was performed using one-way ANOVA with Bonferroni multiple comparisons post-test. ns, not significant.

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chemotaxis towards a point source of bacteria at the same rate aswild-type worms do (Fig. 1B).

NLG-1, GABA and glutamate signalling act as positivemodulators of feedingExperiments in which the RIP neuron is ablated indicate there maybe important extrapharyngeal determinants that drive thepharyngeal microcircuit via volume transmission. However, theseRIP ablations may also highlight a clear potential for intrinsicsignalling pathways embedded within the pharyngeal nervoussystem to act as regulators of the food-induced pumping (Dalliere

et al., 2016). Insight into the contribution of distinct signallingpathways to pharyngeal regulation can be obtained from analysis ofan electrophysiological recording called the electropharyngeogramor EPG (Avery et al., 1995; Cook et al., 2006; Franks et al., 2006),and by comparing the EPG waveform between wild-type andmutants that are defective in the signalling pathways of interest.Therefore, we made EPG recordings to provide insight into theneural mechanisms underpinning the nlg-1-dependent pharyngealbehaviour. Pump rate and pump duration are interlinked in that thepump rate cannot be higher than allowed by the pump duration i.e.another contraction–relaxation cycle cannot begin until the pharynxhas fully relaxed. Nonetheless, within these constraints, it ispossible to have distinct changes in the pattern of activity. Each EPGwaveform represents the activity of the pharyngeal neuromuscularcircuit during a single contraction–relaxation cycle of thepharyngeal muscle that underlies a pharyngeal pump and carriesinformation about the activity of specific neurons that regulate pumpfrequency, pump duration and the discrete functional organisationof each pharyngeal pump (Franks et al., 2006). The typical EPGharbours potentials that relate to the MC synaptic activity (‘e’), thesynchronous contraction of the muscle (‘E’), the inhibitory signalsfrom M3 (‘P’), and the rapid relaxation of the muscle (‘R’) and therepolarization of the terminal bulb (‘r’) (Fig. 2Ai).

In the absence of 5-hydroxytryptamine (5-HT or serotonin),the frequency of EPGs is low for both N2 wild type and nlg-1 (N2,6.19±1.65 pumps min−1; nlg-1, 4.18±1.32 pumps min−1; P>0.05),reflecting the fact that this is an ‘off-food’ pumping rate andtherefore feeding rate is down-regulated (Dalliere et al., 2016). Thisis consistent with the low pump rate seen in animals off food.However, despite the similarity in EPG frequency between N2 andnlg-1, the EPG analysis revealed discrete changes in the EPGwaveform when N2 and nlg-1 mutants were compared: the nlg-1mutant exhibited a marked increase in pump duration (Fig. 2A,B)and an absence of transient potentials called P waves that aredependent on the activity of the inhibitory glutamatergic neuron M3(Avery, 1993b) (Fig. 2C). We next analysed the EPG waveform inthe presence of 5-HT. This is a well-characterized positive regulatorof pharyngeal pumping which is engaged in the presence of food(Niacaris and Avery, 2003). In the presence of 5-HT, EPG rate waselevated in both N2 and nlg-1 (Fig. 2B). Despite this 5-HT-mediated increase in pumping, the nlg-1 EPG waveform showed asignificant reduction in the number of P waves, as well as anincreased pump duration relative to recordings made from 5-HT-

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Fig. 4. Neuroligin is expressed in a subset of extrapharyngeal neuronsin the head ganglia. (A) Neuroligin is expressed in a subset of neuronsaround the pharynx. An isolated pharynx from a transcriptional reporter strainshows no expression of neuroligin in neurons of the pharynx. Two differenttranscriptional reporter strains were used to compare neuroligin expressionlevel in pharyngeal neurons: RM371 where the expression is driven by anintegrated array carrying ∼3.5 kb of nlg-1 upstream sequence as well asregulatory elements present in the first exons of the neuroligin gene fusedwith YFP (top panel) and strain BC13535 where the expression is driven byan integrated array carrying ∼2.9 kb upstream to the ATG fused with GFP(bottom panel). The cartoon above the isolated pharynx images showsdetail of the transcriptional reporters for each strain. (B) Neuroligin is notexpressed in ADFor NSMneurons, the twomajor pharynx neuronsmodulatingthe feeding efficacy extrinsic and intrinsically, respectively. However,neuroligin is expressed in HSN neuron, which triggers food-dependentpumping thus regulating the feeding extrapharyngeally. Colocalization withPthp-1::rfp shows HSN expression around the vulva. A characteristicprocess to identify the NSM neuron is detailed in the box inset on the image.Scale bars: 50 μm.

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treated N2 pharynxes (Fig. 2A). The loss of these key signatureswas rescued in the strain expressing the nlg-1 rescue transgene fromthe nlg-1 promoter (Fig. 2B,C).To gain further insight into the neural basis of the effect of nlg-1

on the EPG waveform, we made a systematic comparison of EPGsin nlg-1 compared with mutants of known regulators of EPGs. It iswell established that the absence of P waves provides a readout ofactivity of the inhibitory glutamatergic motorneuron M3 (Avery,1993b), which regulates the duration of the pharyngeal contraction–relaxation cycle by accelerating muscle repolarisation. The loss ofglutamate release from M3, as observed in the mutant eat-4 (Raizenand Avery, 1994), results in fewer P waves and increased pumpduration (Raizen andAvery, 1994). nlg-1mutants showa similar EPGsignature to eat-4 mutants (Fig. 3). This is consistent withan involvement of glutamate signalling that is intrinsic to thepharyngeal circuit, as the increased pump duration and loss of Pwaves is characteristic of a role for the glutamatergic pharyngealneuron M3.Although GABA is not known to be a neurotransmitter within

the pharyngeal microcircuit (Franks et al., 2006), GABAergicsignalling has been reported to modulate pumping (Dalliere et al.,2016). Recent observations show a nlg-1 dependence ofGABAergic signalling in both C. elegans (Maro et al., 2015; Tuet al., 2015) and mammals (Budreck and Scheiffele, 2007; Fu andVicini, 2009). Motivated by these findings, we next compared theEPG waveform for the GABAergic signalling mutants unc-25(e156) and unc-49 (e382) with nlg-1. Surprisingly, given thatneither GABA nor GABA receptors are present in the pharyngealneurons, the EPG phenotype is even more marked in unc-25 andunc-49 (Fig. 3A,B) than in eat-4, with a very extended pump

duration and absence of P spikes. This therefore identifies apreviously unreported GABAergic extrapharyngeal regulation ofpharyngeal physiology. Moreover, the similarity in the EPGphenotype between the GABAergic mutants and nlg-1 suggestsneuroligin signalling exerts its organization of pharyngeal functionat an extrapharygneal locus rather than at a site intrinsic to thepharyngeal circuitry. Therefore, we next investigated the expressionpattern of nlg-1 in the pharyngeal and extrapharyngeal circuits.

nlg-1 is expressed in a subset of sensory neurons andinterneuronsnlg-1 is widely expressed in the nervous system of C. elegans,including the head region (Hunter et al., 2010). Although detailedexpression has been ascribed to AIY, DAs, VAs, PVD, URA and anundefined subset of cholinergic and GABAeregic neurons (Hunteret al., 2010; Hu et al., 2012; Maro et al., 2015; Tu et al., 2015), thereis no definitive information on expression in the pharyngealmicrocircuit. Therefore, an important first step was to resolve nlg-1expression in the pharyngeal nervous system. This consists of 20neurons that are embedded within the pharyngeal muscle andseparated from the central nervous system of the worm by a basallamina (Albertson and Thomson, 1976). We dissected and isolatedpharynxes from two nlg-1 transcriptional reporter strains andestablished that there is no nlg-1 expression in the pharynx or in itsassociated microcircuit (Fig. 4A). Therefore, neuroligin exerts itsregulation of pharyngeal behaviour through extrapharyngealcircuits.

To place nlg-1 in the context of neural circuits that link food-related sensory inputs to pharyngeal behaviour (Fig. 1A), weconducted detailed mapping of its expression pattern.

Table 1. Results of colocalization experiments showing the strains used in this study, as well as the rationale to use specific markers labellingspecific sensory and interneurons*

Colocalization strain Rationalenlg-1 Co-expression Pre- and postsynaptic targets

srh-142::RFP; nlg-1::GFP Upon food cues, 5-HT is released from ADF,stimulating pharyngeal pumping

No ADF: 1 presynaptic synapse to URX, 1presynaptic synapse to AIY, 1 postsynapticsynapse to AIY

tph-1::RFP; nlg-1::GFP 5-HT released from the pharyngeal neuronNSM, whichacts in a neurohumoral manner in food-relatedlocomotion behaviours. HSN triggers food-dependent pumping in an extrapharyngeal manner

Yes Non-direct synapses to nlg-1-dependenttargets

odr-1::RFP; nlg-1::GFP Local area search behaviour is triggered by olfactoryAWC/AWB neurons

No AWC: 23 presynaptic synapses to AIY, 2postsynaptic synapses to AIY

trx-1::RFP; nlg-1::GFP ASJ sensory neuron is involved in chemosensation anddietary restriction

No Non-direct synapses to nlg-1-dependenttargets

ceh-36::RFP; nlg-1::GFP Olfactory AWC and gustatory ASE neurons directlyinvolved in food-dependent behaviours

No ASE: 37 presynaptic synapses to AIY

npr-5::RFP; nlg-1::GFP Gustatory ASK neuron is involved in local area searchbehaviour; inner labial sensory neuron IL2 is involvedin food detection; AIA interneuron and gustatory ASEare involved in circuits associated with feeding; AUAinterneuron involved in dopamine and food-relatedbehaviours

Yes AUA: 1 presynaptic synapse to URX, 1presynaptic synapse to AIY, 9 presynapticsynapses to ADF, 9 presynaptic synapse toURX

dat-1::RFP; ttx-3::RFP; nlg-1::GFP Role of dopaminergic sensory neurons (ADE, PDE,CEP) and AIY interneuron controlling pharyngealpumping

Yes AIY: 1 pre- and postsynaptic synapse to ADF,37 postsynaptic synapses to ASE, 23postsynaptic synapses to AWC

eat-4::RFP; nlg-1::GFP Role of glutamate signalling in regulating feeding iscontext dependent

Yes AUA: 1 presynaptic synapse to URX, 1presynaptic synapse to URX, 9postsynaptic synapses to ADF, 9postsynaptic synapses to URX

*Pre- and postsynaptic targets for each specific neurons analysed in this study based on the relevance to the nlg-1-dependent interactors identified in this study,related to food-dependent cues sensory processing.

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Approximately 20 neurons express neuroligin in the anteriorganglia. We focused our attention on extrapharyngeal neurons thatare known to regulate food-related behaviours (Table 1). To

facilitate this, we carried out colocalization experiments using arraysexpressing RFP in specific sensory neurons (Table 1). ADF is asensory neuron that provides a serotonergic drive to increase

A B

C D

Fig. 5. Neuroligin is expressed in a subset of sensory neurons and interneurons. (A) A representative image from DiI staining of the strain carrying thePnlg-1::yfp array. The green signal corresponds to cells expressing YFP under the nlg-1 promoter, and the red signal corresponds to DiI staining in amphidsensory neurons. There is no neuroligin expression in the DiI stained ADL, ASH, ASI, ASJ, ASK, AWB amphid neurons. However, there is expression ofYFP in ALA, URX and AVJ neurons. The box inset shows a detail of YFP and DiI staining in a subset of neurons. The neurons indicated with arrows and greenlabels correspond to positive neuroligin expression. The neurons indicated with arrowheads and white labels correspond to amphid neurons that are DiI stained.(B) Overlapping specific expression of neuroligin in a subset of dopaminergic ADE neurons, as well as the AIY interneuron. Representative images from thestrain carrying the double promoter Pttx-3::mCherry/Pdat-1::mCherry and Pnlg-1::yfp arrays are shown. The top image corresponds to a merged compositionbetween both green and red channels. Bottom panel shows detail of neurons where neuroligin is expressed in both ADE and AIY neurons (white arrows).(C) Overlapping expression of neuroligin in a subset of glutamatergic neurons expressing eat-4. Neuroligin is expressed in an unidentified subset of eat-4 neuronsin the head ganglia, as well as in the PHC phasmid neuron in the tail ganglia. The location of these ganglia is highlighted in the cartoon on the top of the panel.Representative images from the strain carrying both arrays Peat-4::mCherry; Pnlg-1::yfp in two different orientated planes are shown. The bottom panelscorrespond to detail view of eat-4/nlg-1 matched expression. The white arrows indicate colocalized cell bodies. The dotted circles show colocalized cell bodies.(D) The expression of neuroligin is matched to mCherry expression driven by the npr-5 promoter. The npr-5 promoter drives expression in a subset of sensoryneurons (ADF, ASE, ASG, ASI, ASJ, ASK, AWA, AWB, IL2) and interneurons (AIA, AUA). Neuroligin is expressed in the AUA interneuron as well as in otherunidentified subset of sensory neurons. A representative image from the strain carrying both arrays [Pnpr-5::mCherry]; Pnlg-1::YFP is shown. The bottompanel shows detail of a subset of neurons where neuroligin is expressed. Thewhite arrows indicate colocalized cell bodies. The dotted circles indicate colocalizedcell bodies. Scale bars: 35 μm (A), 50 μm (B,D), 40 μm (C).

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pumping rate on food (Song et al., 2013). We found noco-expression of neuroligin in ADF (Fig. 4B) and no expressionin major sensory neuron classes, using specific markers for thefollowing neurons: ASJ, AWB, AWC and ASE neurons (Fig. 4,Fig. S1). However, although neuroligin is not expressed in NSM, wefound expression in HSN neurons in the vulva area (Fig. 4B).Finally, to further investigate expression in a subset of sensoryneurons we tried to identify neuroligin expression in the DiI-labelled amphid neurons: ADL, ASH, ASI, ASJ, ASK, AWB.Again, we found no neuroligin expression (Fig. 5A).

However, nlg-1 is expressed in a subset of eat-4 glutamatergicneuron classes (Fig. 5, Fig. S1). In this context, it is noteworthy thatglutamatergic signals are required for both foraging and feedingbehaviours (Hills et al., 2004; Dalliere et al., 2016). In addition,neuroligin expression was localized specifically in a subset ofdopaminergic sensory neurons, ADEs (Fig. 5B), which also areinvolved in food-dependent behaviours (Hills et al., 2004), as wellas in the bilateral interneurons AIY (Fig. 5B), that function to extendfood-seeking periods (Shtonda and Avery, 2006). Finally, bypositional identification of cell bodies, we found expression of nlg-1in the sensory neurons ALA and URX, as well as the interneuronAVJ (Fig. 5A).

DISCUSSIONThe C. elegans neuroligin loss-of-function mutant is unable tomaintain the same high rate of pharyngeal pumping in the presenceof food compared with thewild type. This suggests that neuroligin isrequired in the circuitry that regulates feeding behaviour in responseto sensory cues arising from bacteria. The activity of the pharynx isregulated by local and hormonal excitatory and inhibitory systemsthat converge to fine-tune food intake in a context-dependentmanner (Dillon et al., 2015, 2016; Dalliere et al., 2016). Insight intopharyngeal regulation came from a study in which it was shown thatlaser ablation of all pharyngeal neurons does not completely abolishpharyngeal pumping (Avery and Horvitz, 1989). It has beenproposed that there is an intrinsic myogenic rhythm that ismodulated by the pharyngeal nervous system. This systemis embedded underneath the pharyngeal basal membrane and isconnected to the extrapharyngeal network via the RIP neurons(Bhatla et al., 2015). This provides a neural pathway for regulationof feeding behaviour by the extrapharyngeal nervous system(Trojanowski et al., 2016) in response to sensory cues arisingfrom food (Bhatla et al., 2015). However, ablation of RIP does notremove all food-dependent pharyngeal behaviours, suggesting thatother pathways, either intrinsic to the pharynx (e.g. sensing ofbacteria in the pharyngeal lumen) or arising throughextrapharyngeal neurohormonal signals, are important (Dalliereet al., 2016). The involvement of neuroligin in the first mechanismis unlikely as there is no evidence for nlg-1 expression in thepharynx. This suggests neuroligin is required in an extrapharyngealcircuit for robust up-regulation of pharyngeal pumping in thepresence of food.

Our data suggest that neuroligin-dependent extrapharyngealprocessing of the food cue modifies the manner in which thepharynx responds to the presence of food (Fig. 6) and that it isrequired to decrease the duration of a pharyngeal pump, most likelyby increasing activity of the glutamatergic neuron M3. An inabilityto execute this control in the presence of food is consistent with theobservation that nlg-1 is unable to sustain a high level of pumping inthe presence of food as a long pump duration is incompatible withfast pumping. We considered where in the extrapharyngeal circuitryneuroligin may mediate this effect by mapping the expression

NLG-1

GABA

GABAGLU

DAACh

ACh

Feeding behaviour

URXAD3

ALA

Sensory

Integration

AVJAUA

AIY

Foodcues

Fig. 6. Hypothetical model where neuroligin-expressing cells act to senseand integrate food cues to up-regulate feeding. The proposed modelrecognizes a modulatory role of neuroligin in organizing the synapses thatsupport pharynx function. The primary observation is that neuroligin has aconsequence on pharyngeal pumping and in the way it responds to bacteria.However, as neuroligin is only expressed in the extrapharyngeal nervoussystem, this molecular determinant of synaptic function must act in upstreamcircuits that are remote from the pharyngeal nervous system. As depicted,neuroligin is located in distinct subset of indicated identified sensory neurons(orange circles) and interneurons (green circles). These neurons belong todistinct neurotransmitter classes (orange, blue, red and violet) implicated in thedetection of food related cues, and/or known to underpin integration of theinputs that mediate food-dependent behaviours beyond pharyngeal pumping.The absence of neuroligin likely shifts the balance of inter-neuronal signallingwithin these food-modulated circuits supported by neuroligin-expressing cells.In vivo, the nlg-1 mutant is unlikely to completely remove transmission butrather modifies the weight of transmission mediated by the nlg-1-expressingsynapses. Based on current knowledge, this would be distributed across thecircuit rather than confined to any one of the indicated neuroligin-dependentsynapses. As these food-dependent circuits lie upstream of pharyngealpumping, they modify its function and this is expressed as a blunted feedingresponse in the presence of food. ACh, acetylcholine; DA, dopamine; GABA,gamma-aminobutyric acid; Glu, glutamine.

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pattern of nlg-1. It may function in circuits detecting food-relatedstimuli from the environment through sensory neurons such us ALAand URX and those processing information through interneuronssuch us AIY and AUA (Coates and de Bono, 2002; Chalasani et al.,2007). In addition, our data show a co-expression of neuroligin inextrapharyngeal glutamatergic neurons and this mirrors neuroligincontrol of glutamate transmission in mammals (Graf et al., 2004;Budreck et al., 2013). Neuroligin is also expressed in the ADEdopaminergic neurons and this could indicate a deficit in thedopaminergic contribution to food-related behaviours in nlg-1(Hills et al., 2004). In this context, it is interesting to note that inmice neuroligin-3 modulates inhibition onto dopaminergic neurons(Rothwell et al., 2014).We observed nlg-1 expression in ALA and AVJ which are

GABA-positive cells (Gendrel et al., 2016). This goes hand in handwith the electrophysiological data showing that GABA signalling-deficient mutants phenocopy the neuroligin pharyngeal EPGphenotype. This indirectly suggests a pivotal role for regulation ofGABAergic signalling by neuroligin, which is required to sustain ahigh level of feeding in the presence of food. A role for neuroligin incontrolling GABAergic signalling in the context of a food-drivenbehaviour has parallels with recent evidence showing neuroliginorganizes C. elegans GABAergic postsynapses (Maro et al., 2015;Tong et al., 2015; Tu et al., 2015) and the neuroligin dependence ofGABA cellular signalling at the body wall neuromuscular junction(Tu et al., 2015).In conclusion, we provide evidence for the altered processing of

food cues in a neuroligin-deficient mutant, nlg-1, which results in aninability to maintain a sustained level of feeding in the presence ofan abundant food source. This altered processing of sensory cuesimparted by neuroligin deficiency is interesting in the broadercontext of the role of neuroligin in human autism spectrum disorder,which features dysfunctional sensory processing (Beker et al.,2018). In addition, the data highlight how molecular determinantswhen restricted to remote circuits can impact distal circuits that donot express the key molecular determinant but have significantconsequence for the prime output of that circuit. This distribution ofcause and effect will be an important consideration wheninvestigating the behavioural consequence of genetic determinantsin the context of interacting microcircuits.Our study delivers new insight into the way in which neuroligin is

required for context-dependent behavioural responses (Calahorroet al., 2009; Hunter et al., 2010; Calahorro andRuiz-Rubio, 2012).Wesuggest a model in which neuroligin functions in the extrapharyngealnervous system and links sensory detection of food to feedingbehaviour through communication between sensory neurons andinterneurons impacting in downstream pharyngeal circuits. Thus,neuroligin is required to organize the circuit(s) that integrates foodsensory cues to generate an appropriate behavioural response.

AcknowledgementsWe thank James Rand for kindly sharing strains and unpublished data; AntonioMiranda-Vizuete for sharing the trx-1::DsRed strain. Additional strains were providedby the CGC, which is funded by NIH Office of Research Infrastructure Programs(P40 OD010440).

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: F.C., L.H., V.O.; Methodology: F.C., J.D., L.H., V.O.; Formalanalysis: F.C., F.K.; Investigation: F.C., F.K.; Resources: L.H., V.O.; Writing - originaldraft: F.C., L.H., V.O.; Supervision: L.H., V.O.; Project administration: L.H., V.O.;Funding acquisition: F.C., L.H., V.O.

FundingThis study was supported by a grant from Wessex Medical Trust, UK (V05) to F.C.

Supplementary informationSupplementary information available online athttp://jeb.biologists.org/lookup/doi/10.1242/jeb.189423.supplemental

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RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb189423. doi:10.1242/jeb.189423

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