A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior
Summary (4 min read)
- 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.
- 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.
- 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.
- 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.
- 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 126.96.36.199, 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.
- 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.
- 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.
- 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.
- 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.
- (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.
- 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.
- 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.
- (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.
- (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.
- (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).
- 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.
- (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.
- 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.
- Promoters used in ckr-1(OE) screen (Fig. 5C) indicating expression pattern.
- **Bold indicates neurons where ckr-1 is expressed.
- 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.
- 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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"A conserved neuropeptide system lin..." refers methods in this paper
...We used the nlp-12 promoter to drive cell-specific expression of Channelrhodopsin-2 (ChR2) (Nagel et al., 2003) in DVA and tracked worm movement during a 1-minute period of blue light (470 nm) photostimulation....
"A conserved neuropeptide system lin..." refers methods in this paper
...…ANOVA with Holms-Sidak post-hoc test. wild type: -Histamine n=8, +Histamine n=7, pSMD::HisCl1::SL2::GFP: -Histamine n=8, +Histamine: n=8 To investigate how increased SMD activity may impact movement, we photostimulated the SMDs in animals expressing Podr-2(16)::Chrimson (Klapoetke et al., 2014)....
"A conserved neuropeptide system lin..." refers background in this paper
...…information about food availability is conveyed by sensory neurons such as AWC and ASK to premotor interneurons (AIA, AIB, AIY) and ultimately transformed into patterns of motor neuron activity that drive reversals (Gray et al., 2005; Hills et al., 2004; Ouellette et al., 2018; Sawin et al., 2000)....
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Q1. What are the contributions mentioned in the paper "A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior" ?
In this paper, the authors explore the GPCR and cellular targets involved in NLP-12 signaling through local and cellular changes.