A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans

Innate social behaviours emerge from neuronal circuits that interpret sensory information on the basis of an individual’s own genotype, sex and experience. The regulated aggregation behaviour of the nematode Caenorhabditis elegans, a simple animal with only 302 neurons, is an attractive system to analyse these circuits. Wild social strains of C. elegans aggregate in the presence of specific sensory cues, but solitary strains do not. Here we identify the RMG inter/motor neuron as the hub of a regulated circuit that controls aggregation and related behaviours. RMG is the central site of action of the neuropeptide receptor gene npr-1, which distinguishes solitary strains (high npr-1 activity) from wild social strains (low npr-1 activity); high RMG activity is essential for all aspects of social behaviour. Anatomical gap junctions connect RMG to several classes of sensory neurons known to promote aggregation, and to ASK sensory neurons, which are implicated in male attraction to hermaphrodite pheromones. We find that ASK neurons respond directly to pheromones, and that high RMG activity enhances ASK responses in social strains, causing hermaphrodite attraction to pheromones at concentrations that repel solitary hermaphrodites. The coordination of social behaviours by RMG suggests an anatomical hub-and-spoke model for sensory integration in aggregation, and points to functions for related circuit motifs in the C. elegans wiring diagram. Many naturally isolated social strains of C. elegans aggregate into feeding groups with dozens of animals, although other strains, including the laboratory strain N2, are solitary. Aggregating strains show several behavioural changes compared to solitary feeders: they accumulate on the border of a lawn of bacterial food (bordering) and move rapidly on food. Aggregation, bordering and rapid movement are coordinately controlled by the neuropeptide Y receptor homologue NPR-1 (ref. 2). Solitary strains have a high-activity form of NPR-1 (215-valine), whereas aggregating strains have a low-activity form of NPR-1 (215-phenylalanine); npr-1 null mutants also aggregate. Neuropeptide control of aggregation provides an analogy with mammalian social behaviour, which is regulated by the neuropeptides oxytocin and vasopressin. In addition to genetic regulation by npr1, aggregation is sensitive to environmental signals. It is stimulated by URX sensory neurons that detect environmental oxygen, and ASH and ADL sensory neurons that sense noxious stimuli. Attraction to low-oxygen environments promotes accumulation at the lawn border and feeding in groups, which have low oxygen levels compared to the open lawn. Population density, food availability and environmental stressors also modulate aggregation. The site of integration of these diverse cues is unknown. How NPR-1 acts to regulate behaviour is not well understood. A previous report using a genomic npr-1 fragment identified the oxygensensing URX neuron as a site of npr-1 action, but behavioural rescue was incomplete, with rescue of aggregation, partial rescue of bordering, and no rescue of rapid movement. To identify other neurons in which NPR-1 promotes solitary behaviour, we first established that a fulllength npr-1 complementary DNA expressed from the endogenous npr-1 promoter rescued solitary behaviour in the strong loss-offunction mutant npr-1(ad609lf), then refined the essential site of expression using other characterized promoters (Fig. 1a, b). Because promoter expression patterns in C. elegans can vary between transgenes, we used a bicistronic messenger RNA to express both npr-1 and green fluorescent protein (GFP), and identified GFP-positive neurons in each rescued line with solitary behaviour (Supplementary Table 1 and Methods). Only promoters driving expression in the inter/motor neuron RMG showed robust rescue of aggregation, bordering, and locomotion speed (Fig. 1b and Supplementary Fig. 1). We next addressed whether RMG expression of npr-1 is sufficient to suppress aggregation. No RMG-specific promoter is known, so an intersectional strategy was developed to drive npr-1 expression only in cells that express both flp-21 and ncs-1, using Cre-mediated recombination between loxP sites that flanked transcriptional stop sequences. When ncs-1::nCre and flp-21::LoxStopLox::GFP strains were crossed together, the intersection between ncs-1 and flp-21 allowed strong and consistent GFP expression only in RMG and M2 pharyngeal neurons (Fig. 2a). We next inserted the npr-1 cDNA into the flp21::LoxStopLox plasmid (Fig. 2a); in npr-1(lf ) animals expressing both flp-21::LoxStopLox::npr-1 and ncs-1::nCre, aggregation, bordering and high speed on food were strongly suppressed (Fig. 2b). M2 is synaptically isolated from neurons implicated in these behaviours, so we conclude that RMG expression of npr-1 can block aggregation and related behaviours. Mammalian neuropeptide Y receptors generally inhibit neurotransmitter release. To determine whether NPR-1 suppresses aggregation by inhibiting or by activating RMG, we killed RMG in wild-type and npr-1(lf ) animals using a laser microbeam, anticipating an effect on the genotype(s) in which RMG is normally active. Killing RMG in npr-1(lf ) eliminated aggregation, bordering and rapid movement (Fig. 2c, d), whereas killing RMG in solitary wild-type animals had no effect (Fig. 2d). These results show that RMG neurons stimulate aggregation-related behaviours in npr-1 mutants, and indicate that NPR-1 inhibits RMG activity in solitary strains. Inspection of the C. elegans wiring diagram revealed that RMG is the hub of a gap-junction network connecting seven classes of neurons, including the oxygen-sensitive URX neurons and the nociceptive ASH and ADL neurons previously implicated in aggregation behaviour (Fig. 3a). RMG-ablated npr-1 animals were normal in their avoidance of high osmolarity, a behaviour mediated by ASH (Supplementary Fig. 2). Therefore RMG is not essential for all functions of associated sensory neurons, but selectively required for aggregation and related behaviours.