A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior
Summary (4 min read)
Introduction
- Neuromodulators serve critical roles in altering the functions of neurons to elicit alternate behavior.
- To achieve their effects, neuromodulatory systems may act broadly through projections across many brain regions or have circuit-specific actions, based on the GPCRs involved and their cellular expression.
- The experimental tractability of C. elegans, combined with the highly conserved nature of the NLP-12/CCK system, offers a complementary approach for uncovering circuit-level actions underlying neuropeptide modulation, in particular NLP-12/CCK neuropeptide signaling.
- 17–23 ARS responses during food searching in particular are rapid and transient.
- In contrast, NLP-12 signaling through both CKR-1 and CKR-2 GPCRs contribute to NLP-12 regulation of 6 basal locomotion, likely through signaling onto head and body wall motor neurons.
Results
- NLP-12/CCK induced locomotor responses require functional CKR-1 signaling.
- These findings show that nlp-12 and ckr1 act in the same genetic pathway and point to a selective requirement for NLP-12 signaling through CKR-1 in regulating trajectory changes during local searching.
- The authors found that ckr-1 overexpression produced striking increases in turning and large head to tail body bends (Fig. 4A, 6C, Video S4), qualitatively similar to the effects of 11 nlp-12 overexpression (Fig. 1A). ckr-1(OE) animals made steep bends during runs of forward movement, with angles approaching 200°, whereas bending angles in wild type rarely exceeded 75° (Fig. 4B).
- The authors found that ckr-1 is broadly expressed in the nervous system, showing expression in a subset of ventral nerve cord motor neurons, amphid and phasmid sensory neurons, premotor interneurons, and motor neurons in the nerve ring (Fig. 5A-B).
- To investigate how increased SMD activity may impact movement, the authors photostimulated the SMDs in animals expressing Podr-2(16)::Chrimson.44.
Discussion
- Neuropeptidergic systems have crucial roles in modulating neuronal function to shape alternate behavioral responses, but the authors have limited knowledge of the circuit-level mechanisms by which these alternate responses are generated.
- The authors demonstrate that NLP-12 modulation of these circuits occurs through distinct GPCRs, CKR-1 and CKR-2, that primarily act on either head or body wall motor neurons respectively.
- The authors findings may point towards similar utilization of specific CCK-responsive GPCRs to coordinate activity across circuits in mammals.
- SMDs innervate head and neck muscles28,50 and biased activity of dorsal or ventral SMDs is correlated with directional head bending38,39,41,42,51.
- The authors envision that NLP-12 regulation of the SMD neurons acts in parallel with other neural pathways previously shown to promote reversals during local searching.
Strains
- All nematode strains (Table S1) were maintained on OP50 seeded agar nematode growth media (NGM) at room temperature (22–24°C).
- Transgenic animals were generated by microinjection into the germ line and transformation monitored by co-injection markers.
- Multiple independent extrachromosomal lines were obtained for each transgenic strain and data presented from a single representative transgenic line.
- Stably integrated lines were generated by X-ray integration and outcrossed at least four times to wild type.
Molecular Biology
- All plasmids, unless specified, were generated by Gateway cloning (see Supplementary Tables).
- P-ENTR plasmids were generated for all promoters used (Table S5).
- The ckr-1 minigene construct (pRB12/pRB13) was generated by cloning the ckr-1 coding sequence (start to stop), with introns 1, 8 and 9.
- For cell specific overexpression or rescue, the ckr-1 minigene was recombined with entry vectors containing the relevant cell-specific promoters.
Behavioral assays and analyses
- All behavioral assays were carried out using staged 1-day adult animals on Bacto-agar NGM agar plates seeded with a thin lawn of OP50 bacteria (50 µL) unless otherwise noted.
- Body bending angle was measured, at the midbody vertex, as the supplement of the angle between the head, mid-body, and tail vertices (Fig. 1C).
- After one minute, animals were repicked without bacteria and transferred to an unseeded behavior assay plate.
- Digital movies were captured over the first 5 mins (local search) and after 30 mins following removal from food.
- Reorientations were manually scored post hoc from monitoring movement direction, over sequential frames (~200 frames for forward reorientations, ~600 frames for reversal-coupled omega turns) from the start of the reorientation (original trajectory) to when the animal completed the reorientation (new trajectory) (Fig. 3B, S3).
Single worm tracking
- Single worm tracking was carried out using Worm Tracker 2.60 Animals were allowed to acclimate for 30 seconds prior to tracking.
- Movement features were extracted from five minutes of continuous locomotion tracking (Video S7).
- Worm tracker software version 2.0.3.1, created by Eviatar Yemini and Tadas Jucikas (Schafer lab, MRC, Cambridge, UK), was used to analyze 22 movement.
- Absolute midbody bending (Fig. 2A) and head bending (Fig. 2B) angles were quantified.
- Continuous tracking of animals was difficult to achieve using this approach during the numerous steep turns performed during ARS, or with NLP-12 or CKR-1 overexpression.
SMD ablation
- Cell ablation protocol by miniSOG activation was adapted from Xu et. al. 2016.43 MiniSOG activation was achieved by stimulation with repetitive 2 Experiments were performed on unseeded plates using larval stage 4 ckr-1(OE) animals expressing miniSOG and GFP transgenes under the flp-22∆4 promoter.
- Following stimulation, animals were allowed to recover in the dark on NGM OP50 plates for 16 hours prior to behavioral analysis or imaging.
Photostimulation experiments
- Animals were grown on +ATR OP50 plates in dark and L4 animals were transferred to a fresh +ATR plate prior to the day of experiment.
- For ChR2 photostimulation, experiments were conducted using a fluorescent dissecting microscope (Zeiss stereo Discovery.V12) equipped with a GFP filter set.
- Behavior was recorded for a 1- minute period prior to photostimulation and during a subsequent 1-minute period during photostimulation.
- Data are expressed as % change in reorientations across these time intervals.
SMD silencing
- ARS assays were performed on unseeded Histamine (10 mM) and control Bacto-agar NGM plates using staged 1 day adults.
- For SMD silencing, transgenic animals were placed on Histamine plates, seeded with 100 µL OP-50, for 1 hour prior to experiment.
Imaging
- Fluorescent images were acquired using either BX51WI or Yokogawa (Perkin Elmer) spinning disc confocal microscopes.
- Data acquisition was performed using Volocity software.
- Staged one-day adult animals were immobilized using 0.3 M sodium azide on 2% agarose pads.
SMD calcium imaging
- Calcium imaging was performed in behaving transgenic animals, expressing GCaMP6s::SL2::mCherry under flp-22∆4 promoter, on 5% agarose pads on a glass slide.
- Frames where movement artifacts were encountered due to stage movement were not included in analysis.
- The background subtracted calcium signals were plotted as a ratio (GCaMP6s/mCherry).
- After 24 hours, they were shifted to 28°C overnight.
- After loading, the transfected cells were added at a density of 25,000 cells/well, and luminescence was measured for 30 seconds at a wavelength of 469 nm.
Figure S1
- (A) Dendrogram (generated using Phylogeny,fr64) showing the predicted relationship between Drosophila (Dm_CCKLR-1/2), C. elegans (Ce_CKR-1/2), mouse (Mm) and human (Hs) CCK1/2-R GPCRs.
- (B) Boxshade alignment of C. elegans CKR-1 and CKR-2 with Human CCK-1 and CCK-2 receptors.
- Black shading indicates identical amino acids, while grey shading indicates similar amino acids.
- Red bar indicates the amino acids removed by ckr-1(ok2502) deletion.
Figure S2
- NLP-12 peptides activate CKR-1 and CKR-2 in vitro.
- Ratio of total Ca2+ response is calculated as peptide-evoked response normalized to the total Ca2+ response.
Figure S3
- Sequential snapshots of frames from a representative reorientation, for forward reorientations (A) and reversal-coupled omega turn mediated reorientations (B).
- Frame #s and time points are indicated in each panel.
- Frame numbers and time points indicated are relative to first image in each sequence, which represents the start point (frame 0, time 0 s) when the reorientation event began, and the last frame was when the reorientation was completed.
- Black dashed line shows the original trajectory, and white dashed line the new trajectory upon completion of the reorientation.
Figure S4
- (A) Quantification of reorientations during ARS (0-5 minutes following removal from food) compared to animals on food.
- Note the increased number of forward and reversal coupled reorientations.
- Wild type on food: n=9, wild type ARS: n=8 (B) Quantification of reorientations during ARS (0-5 minutes following removal from food) for the genotypes indicated.
Figure S5
- (A) Quantification of reorientations during ARS (0-5 minutes following removal from food) for the genotypes indicated.
- (B) Quantification of reorientations during 0-5 minutes following removal from food for the genotypes indicated.
- Note expression of ckr-1 under the ckr-2 promoter does not rescue reorientations during ARS in ckr-1(ok2502) animals.
Figure S6
- (A) Confocal maximum intensity projections of a segment of the ventral nerve cord of a transgenic animal coexpressing Pckr-1::ckr-1::SL2::GFP and the cholinergic reporter Pacr-2::mCherry.
- White arrowheads denote the SMD cell bodies in all cases.
- (D) Confocal maximum intensity projections of optical sections with SMD fluorescence from the head region of a transgenic animal coexpressing Podr-2(16):: (left panel), and Pckr2::GFP (middle panel).
- Note weak ckr-2 expression in a single SMDD neuron (merge, right panel).
Figure S7
- Confocal maximum intensity projections of transgenic worm expressing Pckr-1::ckr-1::SL2::mCherry and Pckr-2::GFP. (A) ckr-1 and ckr-2 expression in the entire worm.
- Both ckr-1 and ckr-2 are highly expressed in head neurons and ventral nerve cord motor neurons.
Figure S8
- (A) Representative tracks (30 s) for transgenic animals with high levels of cell-specific ckr-1 overexpression (Pflp-22∆4::ckr-1) in wild type (top) or nlp-12 deletion background .
- (D) Single confocal slices of GFP-labeled SMD neurons, following photoactivation compared to control (-photoactivation, left), in transgenic animals without miniSOG expression.
- (E) Photostimulation of SMDs modestly increases body bending amplitude.
Table S2
- Identification (method of ID, marker and strain indicated for each neuron) to determine ckr-1 expressing neurons.
- * Indicated strains were crossed into ufIs141 (Pckr-1::ckr-1::SL2::GFP) to generate strains to determine colocalization.
Table S3
- Promoters used in ckr-1(OE) screen (Fig. 5C) indicating expression pattern.
- **Bold indicates neurons where ckr-1 is expressed.
Table S4
- Plasmid constructs used in cell specific ckr-1(OE) screen or cell-specifc rescue (Fig. 5C, 7A).
- For cell specific overexpression or rescue of ckr-1, ckr-1 minigene was expressed under indicated promoters.
- Entry vectors containing promoters recombined with destination vectors pRB12 or pRB13 for cell-specific overexpression or rescue of ckr-1.
Table S5
- Promoter lengths and primer information for promoters used Pckr-1 3564bp promoter region of ckr-1 amplified from genomic DNA OMF2159 (Forward primer): CTGCAGGATGGAGATTCAATCAGC.
- Podr-2(16) 3.2 kb promoter fragment amplified from genomic DNA OMF1878 (Forward primer): ATGGGAATGGCGGCAAAT OMF1879 (Reverse primer): CGGGCATCCCGACAAACTGT.
- 48 Supplementary Videos Video S1. Representative 20 second video showing locomotion on food of animal overexpressing nlp-12.
- Representative 20 second video showing locomotion of wild type animal during area restricted search (0-5 minutes off food).
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References
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170 citations
"A conserved neuropeptide system lin..." refers background in this paper
...…after food removal, and decrease with prolonged removal from food (>15-20 minutes) as animals transition to global searching (dispersal) (Bhattacharya et al., 2014; Calhoun et al., 2014; Gray et al., 2005; Hills et al., 2004; Hums et al., 2016; Oranth et al., 2018; Wakabayashi et al., 2004)....
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159 citations
"A conserved neuropeptide system lin..." refers background in this paper
...Brain CCK has been increasingly implicated as a key regulator in diverse aspects of behavior, including feeding, satiety, memory, nociception and anxiety (Ballaz, 2017; Chandra and Liddle, 2007; Liddle, 1997; Miyasaka and Funakoshi, 2003; Noble and Roques, 2006; Rehfeld, 2017)....
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154 citations
"A conserved neuropeptide system lin..." refers background in this paper
...Prior studies of worms immobilized using microfluidic chips and freely moving animals noted anti-phasic activity between SMDD and SMDV neurons and opposing head/neck musculature during head bending (or head casting) (Hendricks et al., 2012; Kaplan et al., 2019; Shen et al., 2016; Yeon et al., 2018)....
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...…regulation of SMD activity is complex and involves reciprocal connections with RIA interneurons, reciprocal signaling with RME motor neurons, as well as proprioceptive feedback (Hendricks et al., 2012; Ouellette et al., 2018; Shen et al., 2016; White, 2018; White et al., 1976; Yeon et al., 2018)....
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...They have been previously implicated in directional head bending and steering (Gray et al., 2005; Hendricks et al., 2012; Kaplan et al., 2019; Kocabas et al., 2012; Shen et al., 2016; Yeon et al., 2018)....
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151 citations
"A conserved neuropeptide system lin..." refers background in this paper
...The lgc-55 promoter drives expression in AVB, RMD, SMD and IL1 neurons, as well as neck muscles and a few other head neurons (Pirri et al., 2009), while the odr-2(16) promoter primarily labels the RME and SMD head neurons (Chou et al., 2001) (Supplementary Files 2-3)....
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