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Many chemosensory cues evoke responses of the same valence under widely varying physiological conditions. It remains unclear whether similar or distinct neural mechanisms are involved in the detection and processing of such chemosensory cues across contexts. We show that in Caenorhabditis elegans , a chemosensory cue is processed by distinct neural mechanisms at two different life stages that share the same valence state. Both starved adults and dauer larvae are attracted to carbon dioxide (CO 2 ), but CO 2 evokes different patterns of neural activity and different motor outputs at the two life stages. Moreover, the same interneuron within the CO 2 microcircuit plays a different role in driving CO 2 -evoked motor output at the two life stages. The dauer-specific patterns of CO 2 -evoked activity in this interneuron require a dauer-specific gap junction complex and insulin signaling. Our results demonstrate that functionally distinct microcircuits are engaged in response to a chemosensory cue that triggers the same valence state at different life stages, revealing an unexpected complexity to chemosensory processing.

Animals frequently exhibit the same behavior under different environmental or physiological conditions. To what extent these behaviors are generated by similar vs. distinct mechanisms is unclear. Moreover, the circumstances under which divergent neural mechanisms establish the same behavior, and the molecular signals that regulate the same behavior across conditions, are poorly understood. We show that in C. elegans, distinct neural mechanisms mediate the same chemosensory behavior at two different life stages. Both dauer larvae and starved adults are attracted to carbon dioxide (CO2), but CO2 attraction is mediated by distinct sets of interneurons at the two life stages. Some interneurons mediate CO2 response only in dauers, some show CO2-evoked activity in adults and dauers but contribute to CO2 response only in adults, and some show CO2-evoked activity that opposes CO2 attraction in adults but promotes CO2 attraction in dauers. We also identify a novel role for insulin signaling in establishing life-stage-specific CO2 responses by modulating interneuron activity. Further, we show that a combinatorial code of both shared and life-stage-specific molecular signals regulate CO2 attraction. Our results identify a mechanism by which the same chemosensory behavior can be generated by distinct neural circuits, revealing an unexpected complexity to chemosensory processing.

Summary The extent to which behavior is shaped by experience varies between individuals. Genetic differences contribute to this variation, but the neural mechanisms are not understood. Here, we dissect natural variation in the behavioral flexibility of two Caenorhabditis elegans wild strains. In one strain, a memory of exposure to 21% O2 suppresses CO2-evoked locomotory arousal; in the other, CO2 evokes arousal regardless of previous O2 experience. We map that variation to a polymorphic dendritic scaffold protein, ARCP-1, expressed in sensory neurons. ARCP-1 binds the Ca2+-dependent phosphodiesterase PDE-1 and co-localizes PDE-1 with molecular sensors for CO2 at dendritic ends. Reducing ARCP-1 or PDE-1 activity promotes CO2 escape by altering neuropeptide expression in the BAG CO2 sensors. Variation in ARCP-1 alters behavioral plasticity in multiple paradigms. Our findings are reminiscent of genetic accommodation, an evolutionary process by which phenotypic flexibility in response to environmental variation is reset by genetic change., Highlights • Behavioral flexibility varies across Caenorhabditis and C. elegans wild isolates • A natural polymorphism in ARCP-1 underpins inter-individual variation in plasticity • ARCP-1 is a dendritic scaffold protein localizing cGMP signaling machinery to cilia • Disrupting ARCP-1 alters behavioral plasticity by changing neuropeptide expression, Individuals can vary in their capacity to adapt their behavior to changes in the environment. Beets et al. identify a natural genetic polymorphism that modifies behavioral flexibility by altering the neuromodulatory output of primary sensory neurons.

SUMMARYNeuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here we show that the C. elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits.Impact statementInvestigation of neuromodulatory control of ethologically conserved area-restricted food search behavior shows that NLP-12 stimulation of the head motor circuit promotes food searching through the previously uncharacterized CKR-1 GPCR.

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate the development and host-seeking behavior, we used the entomophilic nematode Pristionchus pacificus to characterize dauer-constitutive mutants (Daf-c) that inappropriately enter developmental diapause to become dauer larvae. We found two Daf-c loci with dauer-constitutive and cuticle exsheathment phenotypes that can be rescued by the feeding of Δ7-dafachronic acid, and that are dependent on the conserved canonical steroid hormone receptor Ppa-DAF-12. Specifically at one locus, deletions in the sole hydroxysteroid dehydrogenase (HSD) in P. pacificus resulted in Daf-c phenotypes. Ppa-hsd-2 is expressed in the canal-associated neurons (CANs) and excretory cells whose homologous cells in Caenorhabditis elegans are not known to be involved in the dauer decision. While in wildtype only dauer larvae are attracted to host odors, hsd-2 mutant adults show enhanced attraction to the host beetle pheromone, along with ectopic activation of a marker for putative olfactory neurons, Ppa-odr-3. Surprisingly, this enhanced odor attraction acts independently of the Δ7-DA/DAF-12 module, suggesting that Ppa-HSD-2 may be responsible for several steroid hormone products involved in coordinating the dauer decision and host-seeking behavior in P. pacificus.

SM Figure 1. Ppa-hsd-2 transcript is absent in csu88. (A) RT-PCR using primers targeting Ppa-hsd-2transcript did not amplify from Ppa-hsd-2(csu88) mixed-stage cDNA, but did amplify from PS312 cDNA. The expected band size was 832 bp using RHL927 and RHL930. For no RT controls, RNA was processed without reverse transcriptase. (B) The Ppa-odr-3 reaction was used as a positive control for the same csu88 cDNA sample. The expected band size was 303 bp using RHL1097 and RHL1100. SM Figure 2. Olfactory responses of Daf-c dauer larvae. (A) Dauer larvae of Daf-c mutants do not respond to insect pheromones known to be attractive to adults (1% E-11-tetradecenyl acetate (E-TDA), 10% hexadecenal, 10% 2-(E)-nonenol) and the plant defense volatile 1% ß-caryophyllene. Sample sizes: dfc-1ETDA = 523, csu60 ETDA = 193, dfc-1 hexadecenal = 175, csu60 hexadecenal = 147, dfc-1 2-(E)-nonenol = 204, csu60 2-(E)-nonenol = 992, dfc-1 ß-caryophyllene = 656, csu60 ß-caryophyllene = 278. For each category, 11-24 assays were performed over at least 2 trials. (B) Dauer larvae of Daf-c mutants also do not respond to small molecular weight compounds that are strong attractants for C. elegans: 1% 2,3-butanedione (diacetyl), 1% 2,3-pentanedione, and 1% isoamyl alcohol. *Ptu391 and csu60. Sample sizes: dfc-1 butanedione = 313, csu60 butanedione = 1658, dfc-1 pentanedione = 261, csu60 pentanedione = 176, dfc-1 isoamyl alcohol = 243, csu60 isoamyl alcohol = 252. For each category, 11-16 assays were performed over at least two trials. Violin plots show medians with quartiles. SM Figure 3. Amphid neuron marker expressions for two reporters are wildtype in Ppa-hsd-2(csu60). Representative overlay images of adult hermaphrodites expressing the Ppa-odr-7p::rfp reporter in AM9(ADF) and AM7(AWC) neurons of (A) wild-type PS312 and (B) hsd-2(csu60) mutant. Overlay images of adult hermaphrodites expressing the Ppa-che-1p::rfp reporter in AM5(ASE) and AM6(ASG) neurons of (C) wild-type PS312 and (D) hsd-2(csu60) mutant. Anterior is left and dorsal is up. Scale bars represent each reporter strain: 20 µm. SM Figure 4. RT-qPCR results of Ppa-odr-3 expression. Over-expression of Ppa-odr-3 in hsd-2(csu60) is ~10-fold more abundant than wild-type PS312 (arbitrarily set as one). Data represents 12 technical replicates from two biological RNA extraction replicates. Error bars denote s.e.m. SM Figure 5. The effect of temperature on dauer formation in Ppa-hsd-2(csu60) and Ppa-hsd-2(csu88).Synchronized eggs from 20°C were transferred to the designated temperatures and scored for pre-dauer J2d, dauer, or J3 larvae. Census was taken on both day 6 and day 11 at 15°C due to the slow development. Wild-type PS312 do not form any dauer larvae under any of these temperatures in the presence of food. Error bars represent s.e.m. See SI Table 1 for details. SM Figure 6. J2d are committed to the dauer larvae fate. Ppa-hsd-2(csu60)(N = 6, n = 109) and Ppa-hsd-2(csu88) (N = 5, n = 127) J2d larvae with dark intestine lacking radial constriction and buccal cap were picked onto new plates with food and scored 24 and 48 hours later. Error bars represent s.e.m.

Carbon dioxide (CO2) is an important sensory cue for many animals, including both parasitic and free-living nematodes. Many nematodes show context-dependent, experience-dependent and/or life-stage-dependent behavioural responses to CO2, suggesting that CO2 plays crucial roles throughout the nematode life cycle in multiple ethological contexts. Nematodes also show a wide range of physiological responses to CO2. Here, we review the diverse responses of parasitic and free-living nematodes to CO2. We also discuss the molecular, cellular and neural circuit mechanisms that mediate CO2 detection in nematodes, and that drive context-dependent and experience-dependent responses of nematodes to CO2.

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate development and behavior, we used the nematode modelPristionchus pacificusto characterize mutants that inappropriately enter developmental diapause to become dauer larvae (Daf-c). We found several key olfactory differences betweenP. pacificusandC. elegansDaf-c dauers. In addition, the twoP. pacificusDaf-c alleles disrupt steroid synthesis required for proper regulation of the conserved canonical steroid hormone receptor DAF-12, whose dauer-constitutive and cuticle exsheathment phenotypes can be rescued by the feeding of Δ7-dafachronic acid. One allele,csu60, has a deletion in the sole HydroxySteroid Dehydrogenase (HSD) inP. pacificus.Bothhsd-2(csu60)adults and dauers show enhanced attraction to a beetle pheromone, possibly due to the heterochronic activation of dauer-specific neuronal development in the adults. Surprisingly, this enhanced odor attraction acts independently ofdaf-12, revealing unexpected targets of steroid hormones regulating ecdysis and olfaction inP. pacificus.Author SummaryThe remarkable evolutionary success of nematodes can be attributed to their dispersal ability as stress-resistant dauer larvae and the equivalent parasitic infective larvae. The decision to enter dauer development is regulated by a conserved steroid hormone receptor that receives multiple external and internal cues, though the extent in which these cues also coordinate other physiological and behavioral processes is not well understood in divergent species. We used the insect-associated nematodePristionchus pacificusto genetically dissect two mutants that form dauers inappropriately, and found that one mutation in a steroidogenic enzyme,Ppa-hsd-2, is predicted to abolish the biosynthesis of the hormones needed to negatively regulate dauer entry when food is available. Unexpectedly,Ppa-HSD-2 is also required to properly complete the dauer larval molt, known as exsheathment, as well as to confer differences in adult versus dauer larvae chemotaxis behavior towards a host pheromone. Given that dauers are the host-seeking stage of parasitic nematodes, hormonal disruption represents a tenable target for biological control.

Summary How sexually dimorphic behaviors are represented in the brain is a long-standing question. Two new studies in C. elegans uncover novel molecular mechanisms that allow neurons shared by opposite sexes to generate distinct sex-specific behaviors.

Animal behaviors are often composed of distinct alternating behavioral states. Neuromodulatory signals are thought to be critical for establishing stable behavioral states and for orchestrating transitions between them. However, we have only a limited understanding of how neuromodulatory systems act in vivo to alter circuit performance and shape behavior. To address these questions, we have investigated neuromodulatory signaling in the context of Caenorhabditis elegans egg-laying. Egg-laying activity cycles between discrete states–short bursts of egg deposition (active phases) that alternate with prolonged quiescent periods (inactive phases). Here using genetic, pharmacological and optogenetic approaches for cell-specific activation and inhibition, we show that a group of neurosecretory cells (uv1) located in close spatial proximity to the egg-laying neuromusculature direct the temporal organization of egg-laying by prolonging the duration of inactive phases. We demonstrate that the modulatory effects of the uv1 cells are mediated by peptides encoded by the nlp-7 and flp-11 genes that act locally to inhibit circuit activity, primarily by inhibiting vesicular release of serotonin from HSN motor neurons. This peptidergic inhibition is achieved, at least in part, by reducing synaptic vesicle abundance in the HSN motor neurons. By linking the in vivo actions of specific neuropeptide signaling systems with the generation of stable behavioral outcomes, our study reveals how cycles of neuromodulation emanating from non-neuronal cells can fundamentally shape the organization of a behavioral program., Author summary Animals have robust mechanisms in place to shape their behavior in a manner that is beneficial both for their survival and for the survival of their progeny. A class of signaling molecules known as neuropeptides have been implicated in driving transitions between behavioral states but we have only a limited understanding of how neuropeptide signaling modulates neural circuit activity in vivo to elicit alternate behavioral outcomes. Egg-laying behavior in the model system C. elegans cycles between clusters of egg-laying and prolonged inactive periods. This temporal organization provides for spatial dispersal of eggs, presumably benefiting progeny by limiting local overcrowding and competition for food. Here we uncover a novel neuromodulatory mechanism that shapes the timing of egg-laying behavior. Specifically, we find that neuromodulatory signaling from a group of non-neuronal cells specifies transitions between active and inactive phases of egg-laying by regulating neurotransmitter release from the motor output neurons of the egg-laying circuit. Advancing our knowledge of how neuropeptides and other modulators act in the context of the circuits in which they are endogenously released will be critical in ongoing efforts to understand how alternate behavioral states, for example those underlying mood or arousal, are encoded.

Establishing and maintaining the appropriate number of GABA synapses is key for balancing excitation and inhibition in the nervous system, though we have only a limited understanding of the mechanisms controlling GABA circuit connectivity. Here, we show that disrupting cholinergic innervation of GABAergic neurons in the C. elegans motor circuit alters GABAergic neuron synaptic connectivity. These changes are accompanied by a reduced frequency and increased amplitude of GABAergic synaptic events. Acute genetic disruption in early development–during the integration of post-embryonic born GABAergic neurons into the circuit–produces irreversible effects on GABAergic synaptic connectivity that mimic those produced by chronic manipulations. In contrast, acute genetic disruption of cholinergic signaling in the adult circuit does not reproduce these effects. Our findings reveal that GABAergic signaling is regulated by cholinergic neuronal activity, likely through distinct mechanisms in the developing and mature nervous system.