A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior

  1. Shankar Ramachandran
  2. Navonil Banerjee
  3. Raja Bhattacharya
  4. Michele L Lemons
  5. Jeremy Florman
  6. Christopher M Lambert
  7. Denis Touroutine
  8. Kellianne Alexander
  9. Liliane Schoofs
  10. Mark J Alkema
  11. Isabel Beets
  12. Michael M Francis

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Abstract

Neuromodulators 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 Caenorhabditis 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.

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  1. Version published to 10.7554/elife.71747 on eLife
  2. eLife

    ###Reviewer #3:

    In this manuscript, Ramachandran and colleagues describe how cholecystokinin-related NLP-12 neuropeptide signalling in C. elegans can regulate two different behavioural programmes, area-restricted search (ARS) and basal locomotion, by conditionally engaging different specific receptors that are expressed in different neuronal targets. They thoroughly characterise the CKR-1 receptor which had not been described previously, and place its function in context with that of the previously known NLP-12 receptor CKR-2. The manuscript gives new insight into an interesting and likely conserved mechanisms of how neuromodulatory systems enable adaptive behaviour by coordinating the action of neural circuits even when they are not directly connected. The conclusions drawn appear solid and are justified by the data presented, and the experimental approaches and results are well documented.

    The main problem with the work is a certain lack of clarity regarding the separation of the roles of the CKR-1 and CKR-2 receptors on basal locomotion/body bending and head bending/reorientations. Overexpression of NLP-12 places animals in a chronic ARS state, as described in a previous publication. Is the NLP-12 overexpression model representative of the increased reorientation in area restricted search, or of control of undulations in basal locomotion, or both? If it is primarily representative of area restricted search, this would mean that CKR-2, similarly to CKR-1, mediates the chronic ARS state induced by NLP-12 overexpression, because in fig. 1B and C its mutation causes a reduction in the phenotype, and deletion of both ckr-1 and ckr-2 causes a stronger reduction.

    Also, it is unconvincing that SMD neurons do not express ckr-2 (see S3D); no comparison of ckr-1 and ckr-2 expression levels in SMD is provided and in fact the CeNGEN data of single cell RNAseq of C. elegans neurons shows similar expression of both receptors in SMDD (accessible at cengen.shinyapps.io/SCeNGEA). On the other hand, loss of ckr-2 on its own does not cause a significant reduction in ARS (fig 3A). To clarify this, the authors could measure the reorientation rate in the nlp-12OE ckr-2 mutant strain.

    Given that ckr-1 overexpression as shown in figs 4-6 increases both body bending amplitude (and ARS-like high reorientation rate, the authors offer the interesting possibility that SMD may also affect basal locomotion. I would suggest an experiment that clarifies whether SMD also controls body bending in basal locomotion using the single-worm tracking assay shown in fig 2A with the SMD-specific ckr-1 rescue strains in a ckr-1 mutant background (as used in figure 7). Also they could measure body bending in the existing data on the SMD::Chrimson optogenetics.

  3. eLife

    ###Reviewer #2:

    Ramachandran et al. report the discovery of a C. elegans GPCR - CKR-1 - that mediates some of the effects of the cholecystokinin-like neuropeptide NLP-12 on posture and foraging behavior. The discovery of this receptor permits further study of this neuropeptide signaling system, which is conserved from worms to vertebrates. Although CKR-1 is expressed in many neurons, the authors show that its function in SMD head-motorneurons is especially important for control of posture and foraging. The manuscript's strengths include: (1) rigorous characterization of receptor-ligand interactions in vitro, using a cell-based assay for GPCR activation, and in vivo, using genetic analysis, (2) compelling data in support of a model in which NLP-12 regulates SMD neurons to control foraging, (3) high-resolution analysis of C. elegans posture during foraging, which illustrates the complexity and richness of this behavior, and (4) the circuit model, i.e. a role for SMDs, is tested using a number of independent methods and clearly indicated.

    The manuscript does have some weaknesses. In addition to specific technical points listed below, the manuscript discussed neuropeptides derived from a single source, the DVA pre-motor neuron, acting on distinct targets via distinct receptors in a conditional manner. This interesting model is suggested by the title and the abstract and comes up plainly in the introduction and discussion. However, the model is not clearly supported by the data, which primarily focus on the characterization of CKR-1 as a relevant receptor for NLP-12 peptides. Another weakness in the manuscript arises from the authors' switching between various assays for posture during locomotion, which makes it difficult for the reader to compare data between figures. Rich kymography data are relegated to supplementary figures, and data from only a subset of relevant genotypes are shown as kymographs. The manuscript would be strengthened by more uniform analysis of posture and foraging. Finally, while the data clearly show that effects of NLP-12 on posture and foraging require SMD neurons, the manuscript does not investigate how NLP-12 affects SMD activity. The manuscript would be strengthened by experiments showing a functional connection between DVA and SMD neurons, e.g. functional imaging of SMDs during optogenetic manipulation of DVAs.

    Specific comments:

    1. One premise of the work is that DVA neurons are the sole source in vivo of NLP-12 peptides. A recent study (Tao et al. 2019, Dev. Cell) shows that there is an alternate source of NLP-12, the PVD nociceptors. The authors should address the possibility that their assays also detect a contribution of PVD neurons to posture/foraging.

    2. The text associated with Figure 1B-C is tentative with respect to assigning redundant functions to CKR-1 and CKR-2. Why? The data are clear; these receptors function redundantly.

    3. The very nice in vitro analysis of NLP-12 receptors should include negative controls. Ideally, the authors would use a scrambled neuropeptide or a related neuropeptide to demonstrate specificity of the interactions between NLP-12 and CKR-1/2.

    4. The different 'bending angles' used in Figures 1 and 2 make it difficult to compare data between figures. Also, the schematics used to explain the bending angles have small fonts and are hard to read.

    5. Figure 3E shows the results of a nice experiment in which optogenetic activation of NLP-12-expressing cells - presumably DVA - causes reorientations. The authors assert that this effect requires CKR-1 but not CKR-2. The data, however, suggest that CKR-2 might have an effect. The variance of the data does not allow the authors to reject a null hypothesis, but they err in then assuming that this means that CKR-2 plays no role in the phenomenon. This experiment should be repeated to determine whether there is indeed a specific or privileged role for CKR-1 in mediating NLP-12-dependent reorientations.

    6. Also, Figure 3E should show raw data - don't show proportional changes - and all Figure 3 should be scatter plots allowing the reader to assess the variance of the data.

    7. The authors show that effects of receptor overexpression are suppressed by loss of NLP-12 peptides. Is there precedent for this kind of genetic interaction in the literature?

    8. Also, the authors assert that suppression of effects of CKR-1 overexpression by loss of NLP-12 shows that NLP-12 peptides are the sole ligands for this receptor (page 9, line 17). It is not clear why the authors reach this conclusion.

    9. There are some very nice data that are assigned to supplementary figures but might be better placed in main figures. Fig. S3A-B shows data that are integral to the authors' model and could be presented in a main figure. Also, the localization of NLP-12::Venus in DVA axons near SMD processes would be appropriate to show in a main figure. It would be ideal to mark SMDs with a red fluor so that NLP-12::Venus colocalization with SMD processes could be assessed.

    10. The kymography data are nice but incomplete. The authors should show kymographs from strains of all relevant genotypes. This would include: (1) ckr-1(oe); nlp-12, (2) nlp-12, ckr-1, and ckr-2 single mutants, and (3) ckr-1; ckr-2 double mutants.

    11. Page 12, last paragraph indicates that 'low levels' of expression rescue ckr-1 phenotype - how has the expression level been determined? I guess that the authors refer to the amount of DNA used for transgenesis, not a direct measure of transgene expression - this should be reworded.

    12. The manuscript would be strengthened by experiments that measured the effect of DVA activation on SMD physiology and what contribution NLP-12 signaling makes to any functional connection between these neurons. One potential impact of this work is that it establishes a nice paradigm for new molecular genetic analyses of neuropeptide signaling. Direct observation of the effects of NLP-12 peptides on SMD neuron physiology would further strengthen the authors' conclusions and suggest mechanisms by which CKR-1 regulates cell physiology.

    13. Minor comment: Fig S1C is a little confusing w/ respect to how the ligand is indicated - it implies that there exists a ligand-binding site at the amino terminus of the receptors.

  4. eLife

    ###Reviewer #1:

    In this manuscript Ramachandran et al. provide a C. elegans behavioral genetics study focused on the worm cholecystokinin-like neuropeptide-receptor system. They show that nlp-12 neuropeptides released from the DVA neuron fulfill a dual role in controlling body posture as well as head-bending mediated area restricted search (ARS). Previous work showed that DVA controls body posture via nlp-12 signaling to ckr-2 receptor in ventral cord motor neurons. Moreover, nlp-12 signaling was implicated in ARS; but the exact circuit mechanisms and targets of nlp-12 remained elusive. The present work shows in a pretty straight forward way that ckr-1 in SMD head motor neurons is the missing link. In worms, ARS is composed of quiet complex body movements including high angle turns during the worm's forward crawling state. Nlp-12 and ckr-1 mutants show reduced head bending during ARS, while overexpression leads to a stark ectopic ARS like behavior. The authors convincingly show that SMDs are the site of action for ckr-1 and implicated in ARS. They show both requirement and sufficiency of SMDs for ARS like behaviors. The regulation of ARS vs. dispersive behaviors has been extensively studied at the levels of sensory and interneurons in the worm, but how the switch is implemented at motor circuits was largely unknown. Conceptually, this is one of only a few studies investigating the selective control of head versus body movements and provides some interesting insights into the underlying mechanisms; therefore, the study is definitely important and timely. But, it is unclear still how upper sensory circuits transmit the switch between ARS and dispersal to the DVA-SMD circuit. Moreover, the present study does not investigate the signaling pathway of ckr-1 in SMDs and its role in controlling neuronal activity, e.g. via Ca++ imaging. As a sole behavioral genetics study, however, I find the manuscript quite complete. The experiments logically build upon each other and the paper is well written. My only major critique is that parts of the behavioral analyses are described with insufficient detail so that it is unclear to the expert how and what exact movements were quantified. This should be addressed by providing more detailed figure captions, methods sections, more supplemental figures and movies.

    1. The authors should exclude (or separate) reversal states and post-reversal turns in their analyses when measuring head bending, body bending and turn events, but it is unclear if they did so.

    2. Fig 1C and methods: it is unclear what defines a singular bending event as marked on the y-axis. Did the authors measure the maximum angle during each half-oscillation? If yes, this should be explained and how maxima were calculated etc. Or do the histograms represent all values from all recording frames. In the latter case, the y-axis labelling is misleading, and I suggest use "fraction of frames".

    3. Fig 1C: these are averaged histograms of n=10-12 worms, but what is the average number of events per worm and in total?

    4. Fig 1B-C, 2A etc.: to perform the measurements as depicted in upper panels is not really trivial, and I have the impression that the authors used their software packages in a black-box manner. What are the exact image processing steps to implement these measurements, i.e. how was vertex and sides of the angles exactly positioned? The authors should provide a time-series of individual examples alongside with movies demonstrating how accurately the pipeline performs during complex ARS postures.

    5. Fig 2B: the angles and body segments describing the head and head-bending angels should be unambiguously defined. The cartoon in 2B looks like they just measured nose movements.

    6. Fig 3B: reorientation events are not sufficiently defined here. During ARS, worms frequently switch between forward-backward movement, perform post-reversal turns and in a continuous manner exhibit curved trajectories. From a trajectory like the red one in 3A, it is again not trivial to identify and discretize individual turning events with a start and an end and distinguish them from reversals and post reversal turns.

    -- The procedure needs to be explained in greater detail with justification of parameter choice.

    -- How did the authors validate that the procedure performed well, especially during the complex ARS behaviors?

    -- Again, example trajectories and movies should be shown.

    1. All histogram panels lack statistics, e.g. KS test or appropriate alternatives.
  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    The reviewers find your work very interesting and acknowledge its importance in understanding the role of cholecystokinin signaling in differentially controlling aspects of locomotion behavior in C. elegans. In its current form, it represents a near complete and well done behavioral genetics study that could improve further with addressing some of the comments below and also harmonizing the behavior metrics that were used for quantifications. The work could be brought to another level though if the authors performed new lines of experiments that give further mechanistic insights, e.g. via physiological methods, into how ckr-1 signaling controls SMD activity.

  6. Version published to 10.1101/2020.04.27.064550 on bioRxiv