Sensory neurons couple arousal and foraging decisions in C. elegans

Reviewer #1 (Public Review):

Genetic, physiological, and environmental manipulations that increase roaming increase leaving rates. The connection between increased roaming and increased leaving is lost when tax4-expressing sensory neurons are inactivated. This study is conceptually important in its characterization of worm behaviors as time-series of discrete states, a promising framework for understanding behavioral decisions as algorithms that govern state transitions. This framework is well-established in other animals, thanks to Berman and others, but relatively new to worms.

A key discovery is that lawn leaving behavior is probabilistically favored in states of behavioral arousal. I like the use of response-triggered averages (triggered on leaving events) that illustrate a "state-dependent receptive field" of the behavioral response. Response-triggered averages are common in sensory neuroscience, used, for example, to characterize the diverse "stimulus-dependent receptive fields" of different retinal ganglion cell types. It's nice to adapt the idea to illustrate the state-dependence of behavioral state transitions.

The simplest metric of arousal state is crawling speed. When animals crawl faster, they are more likely to leave lawns. A more sophisticated metric of behavioral context is whether the animal is in a "roaming" or "dwelling" state, two-state HMM modeling from previous work (Flavell et al., 2013). Roaming animals are more likely to leave lawns than dwelling animals. Different autoregressive HMM tools can segment worm behavior into 4-states. Also with ARHMMs, the most aroused state is again the state that promotes lawn-leaving.

(With the AR-HMM, I have a small quibble in its characterization as "orthogonal" to the 2-state HMM. Orthogonal has a precise mathematical meaning, but here orthogonal is taken loosely to only mean "very different". I'd prefer the authors just call them "very different" and not use mathematical terms so loosely.)

HMM analysis seems to disentangle effects that were lumped by the simpler metric of overall speed. Crawling speed before lawn leaving events, when analyzed only within roaming periods, is only higher for ¡1 min before the event. I presume that the higher speed that is observed for several minutes before lawn leaving when all states are taken into account (e.g., Fig 1J, Fig 2A, and others) reflects the tendency to be in the faster roaming state than the slower dwelling state for several minutes before lawn leaving? If this is correct, it would be nice for the authors to be explicit about this interpretation, to help the reader understand what is going on.

My principal worry is about the possible artifact if worms are more likely to be at lawn boundaries when moving quickly or in an arousal state (roaming in the 2-state HMM or in state 3 in the AR-HMM)? Lawn-leaving events only occur when the animal is at lawn boundaries. If animals are more likely to be at lawn boundaries when aroused, this should artificially increase the likelihood that these states precede lawn-leaving behaviors for a trivial environment-dependent reason instead of their interesting internal state-dependent reason. The authors might consider trying to disentangle the state-dependent statistics of lawn edge proximity when assessing by how much arousal states precede lawn-leaving events. I realize this is could be a formidable analytical challenge.

One recourse is to align speed, HMM, and AR-HMM states to the other behavioral events that only happen at lawn boundaries. When they do this for head poke-reversals in Figure 2-supplement 3, they also observe an (albeit modest) increase in arousal states before head poke-reversals. It should be easy to also compare what happens with head poke-forward and head poke-pause to better understand potential artifacts in quantifying edge-associated events. In any case, this concern and their strategies to address it should be discussed for clarity and transparency.

The authors use diverse environmental, genetic, and optogenetic perturbations to regulate the roaming state, thereby regulating the statistics of leaving in the expected manner. One surprise is that feeding inhibition evokes roaming and lawn-leaving in both pdfr-1 and tph-1 mutants, even though the tph-1-expressing NSM neurons have been shown to sense bacterial ingestion and food availability. I'm curious, is there anything in these new results that is inconsistent with previous claims by Rhoades et al., 2019, or did Rhoades et al. simply not do these tests?

Another surprise is that evoking roaming does not evoke leaving in tax-4 mutants (which is something of an internal control that argues against the worry that roaming artificially increases the likelihood of leaving, see above). Without sensory neuron activity, worms are only more likely to roam for a minute before leaving rather than roaming for several minutes before leaving like wild-type (Figure 6C). ASJ seems to be the most important sensory neuron in this coupling between roaming and leaving (which is uncoupled when sensory neurons are inactivated).

I'm a little puzzled why the wild-type animals shown in Figure 6C show elevated roaming for several minutes before leaving events, whereas the wild-type animals shown in Figures 4I,J,K show elevated roaming for only about a minute, not much different than tax-4 mutants. Am I missing something? What is different about these different wild-type animals?

Reviewer #2 (Public Review):

In this manuscript, Scheer and Bargmann investigate how behavioral arousal state affects foraging decisions in the nematode C. elegans. Previous work has shown that when placed on a lawn of bacterial food, C. elegans spontaneously switch between two behavioral states, termed roaming and dwelling, during which they exploit or explore the food environment, respectively. It has also been shown that animals spontaneously leave bacterial lawns depending on factors such as food quality or mate availability.

Here, the authors use quantitative behavioral analyses to describe in unprecedented detail the various behavioral choices animals make when encountering the lawn edge. They report that leaving the lawn is a rare outcome compared to other choices such as pausing or reversing back into the lawn. It occurs predominantly out of the roaming state and has a characteristic preceding fast crawling profile. They developed a refined analysis method, the result of which suggests that the arousal state of animals on food can be described by a 4-state behavior (as opposed to the 2-state roaming - dwelling classification); leaving the lawn occurs predominantly from "state 3", which corresponds to the highest level of arousal during roaming. They further show that various manipulations, such as optogenetic inhibition of feeding, stimulation of RIB neurons, or mutations of neuromodulator pathways, all of which have previously been reported to affect crawling speed and/or roaming/dwelling, maintain the coupling between roaming states and leaving, suggesting a dedicated mechanism for coupling leaving to the roaming state. Finally, they use genetics to implicate chemosensory neurons as neuronal circuit elements mediating this coupling.

How arousal states affect decision making is an active area of neuroscience research; therefore, the current manuscript will impact the field beyond the small community of C. elegans researchers. Also, in the past, roaming/dwelling and leaving have been treated as independent behaviors; the current manuscript is very intriguing, demonstrating both the interconnectedness of different behavioral programs and the importance of the animal's behavioral context for specific decisions.

Reviewer #3 (Public Review):

Scheer and Bargmann use a combination of computational and experimental approaches in C. elegans to investigate the neuronal mechanisms underlying the regulation of foraging decisions by the state of arousal. They showed that, in C. elegans, the decision to leave food substrates is linked to a high arousal state, roaming, and that an increase in speed at different timescale preceded the food leaving decisions. They found that mutants that exhibit increased roaming also leave food substrates more frequently and that both behaviors can be triggered if food intake is inhibited. They further identify a set of chemosensory neurons that express the transduction channel tax-4 that couple the roaming state and the food-leaving decisions. The authors postulate that these neurons integrate foraging decisions with behavioral states and internal feeding cues.

The strength of the paper relies on using quantitative and detailed behavioral analysis over multiple time scales in combination with the manipulation of genes and neurons to tackle the state-dependent control of behavioral decisions in C. elegans. The evidence is convincing, the analysis rigorous, and the writing is clear and to the point.