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

Shankar Ramachandran1, Navonil Banerjee1, Raja Bhattacharya1, Denis Touroutine1, Christopher M. Lambert1, Lilianne Schoofs2, Isabel Beets2, Michael M. Francis1 •
University of Massachusetts Medical School1, Katholieke Universiteit Leuven2
28 Apr 2020-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: Investigation of neuromodulatory control of ethologically conserved area-restricted food search behavior shows that NLP-12 stimulation of the head motor circuit promotes food searching through the previously uncharacterized CKR-1 GPCR.
Abstract: Neuromodulators promote adaptive behaviors in response to either environmental or internal physiological changes. These responses are often complex and may involve concerted activity changes across circuits that are not physically connected. It is not well understood how neuromodulatory systems act across circuits to elicit complex behavioral responses. Here we show that the C. elegans NLP-12 neuropeptide system, related to the mammalian cholecystokinin system, shapes responses to food availability by selectively modulating the activity of head and body wall motor neurons. NLP-12 modulation of the head and body wall motor circuits is generated through conditional involvement of alternate GPCR targets. The CKR-1 GPCR is highly expressed in the head motor circuit, and functions to enhance head bending and increase trajectory reorientations during local food searching, primarily through stimulatory actions on SMD head motor neurons. In contrast, NLP-12 activation of CKR-1 and CKR-2 GPCRs regulates body bending under basal conditions, primarily through actions on body wall motor neurons. Thus, locomotor responses to changing environmental conditions emerge from conditional NLP-12 stimulation of head or body wall motor neuron targets.

Summary (4 min read)

Jump to: [Introduction] – [Results] – [Discussion] – [Strains] – [Molecular Biology] – [Behavioral assays and analyses] – [Single worm tracking] – [SMD ablation] – [Photostimulation experiments] – [SMD silencing] – [Imaging] – [SMD calcium imaging] – [Figure S1] – [Figure S2] – [Figure S3] – [Figure S4] – [Figure S5] – [Figure S6] – [Figure S7] – [Figure S8] – [Table S2] – [Table S3] – [Table S4] and [Table S5]

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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Figures (2)

Content maybe subject to copyright    Report

A conserved neuropeptide system links head and body motor circuits
to enable adaptive behavior
Shankar Ramachandran
1,#
, Navonil Banerjee
1,#,a
, Raja Bhattacharya
1,#,b
, Michele L Lemons
2
, Jeremy
Florman
1
, Christopher M. Lambert
1
, Denis Touroutine
1
, Kellianne Alexander
1
, Liliane Schoofs
3
, Mark J
Alkema
1
, Isabel Beets
3
and Michael M. Francis
1
*
1
Department of Neurobiology
715 Lazare Research Building
University of Massachusetts Medical School
364 Plantation St.
Worcester, MA 01605
2
Department of Biological and Physical Sciences
Assumption University
Worcester, MA 01609
3
Department of Biology
University of Leuven (KU Leuven)
Leuven, Belgium
*corresponding author (michael.francis@umassmed.edu)
#
equal contributions
Current address:
a
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los
Angeles, Los Angeles, CA
b
Amity Institute of Biotechnology, Amity University Kolkata, West Bengal, India
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 28, 2021. ; https://doi.org/10.1101/2020.04.27.064550doi: bioRxiv preprint
!
2
SUMMARY
Neuromodulators promote adaptive behaviors that are often complex and involve concerted
activity changes across circuits that are not physically connected. It is not well understood how
neuromodulatory systems act across circuits to elicit complex behavioral responses. Here we
show that the C. elegans NLP-12 neuropeptide system, related to the mammalian
cholecystokinin system, shapes responses to food availability by modulating the activity of head
and body wall motor neurons. NLP-12 modulation is achieved through conditional involvement
of alternate GPCR targets. The CKR-1 GPCR is highly expressed in the head motor circuit, and
enhances head bending and trajectory changes during local food searching, primarily through
stimulatory actions on SMD head motor neurons. Under basal conditions, NLP-12 signaling
regulates body bending, primarily through the CKR-2 GPCR located on body wall motor
neurons. Thus, locomotor responses to changing environmental conditions emerge from
conditional NLP-12 stimulation of head or body wall motor neurons.
Impact statement: Investigation of neuromodulatory control of ethologically conserved area-
restricted food search behavior shows that NLP-12 stimulation of the head motor circuit through
the previously uncharacterized CKR-1 GPCR promotes food searching.
Key words:
Neuropeptide, neuromodulation, neural circuits, adaptive behavior, area-restricted food search,
C. elegans, G protein-coupled receptor
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 28, 2021. ; https://doi.org/10.1101/2020.04.27.064550doi: bioRxiv preprint
!
3
Author contributions
RB and MMF were responsible for original conceptualization and design of the project, NB and
SR further developed the project. SR, NB and RB carried out plasmid and strain building,
experimentation and analysis. DT built plasmids and strains. CML generated molecular biology
constructs. IB and LS performed in vitro experiments and analysis. ML, KA, JF and MJA
assisted with behavior and calcium imaging experiments. MMF was responsible for data
interpretation and writing the manuscript with SR.
Acknowledgements
We thank the Caenorhabditis Genetics Center, which is funded by the National Institutes of
Health National Center for Research Resources, and the Mitani laboratory (National
Bioresource Project) for providing Caenorhabditis elegans strains. We thank Mei Zhen lab for
Matlab script for calcium imaging analysis, Claire Bénard for strains, Michael Gorczyca and
William Joyce for technical support. We thank Francis lab members for helpful comments on the
manuscript.
Funding
This work was supported by NIH R21NS093492 (MMF), European Research Council 340318
and Research Foundation Flanders grant G0C0618N (IB).
Disclosure Statement: The authors have nothing to declare.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 28, 2021. ; https://doi.org/10.1101/2020.04.27.064550doi: bioRxiv preprint
!
4
Introduction
Neuromodulators serve critical roles in altering the functions of neurons to elicit alternate behavior.
Disruptions in neuromodulatory transmitter systems are associated with a variety of behavioral and
neuropsychiatric conditions, including eating disorders, anxiety, stress and mood disorders,
depression, and schizophrenia.
1–3
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. A single neuromodulator may therefore perform vastly different
signaling functions across the circuits where it is released. For example, Neuropeptide Y (NPY)
coordinates a variety of energy and feeding-related behaviors in mammals through circuit-specific
mechanisms. NPY signaling may increase or decrease food intake depending upon the circuit and
GPCR targets involved.
4,5
Due to the varied actions of neuromodulators across cell types and neural
circuits, it has remained challenging to define how specific neuromodulatory systems act in vivo to
elicit alternate behaviors. Addressing this question in the mammalian brain is further complicated by
the often widespread and complex projection patterns of neuromodulatory transmitter systems, and
our still growing knowledge of brain connectivity.
The compact neural organization and robust genetics of invertebrate systems such as Caenorhabditis
elegans are attractive features for studies of neuromodulatory function. Prior work has shown that C.
elegans NLP-12 neuropeptides are key modulatory signals in the control of behavioral adaptations to
changing environmental conditions, such as food availability or oxygen abundance.
6–8
The NLP-12
system is the closest relative of the mammalian Cholecystokinin (CCK) neuropeptide system and is
highly conserved across flies, worms and mammals.
9–11
CCK is abundantly expressed in the
mammalian brain, however a clear understanding of the regulatory actions of CCK on the circuits
where it is expressed is only now beginning to emerge.
12–15
Like mammals, the C. elegans genome
encodes two putative CCK-responsive G protein-coupled receptors (GPCRs) (CKR-1 and CKR-2),
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 28, 2021. ; https://doi.org/10.1101/2020.04.27.064550doi: bioRxiv preprint
!
5
though, to date, direct activation by NLP-12 peptides has only been demonstrated for the CKR-2
GPCR.
9–11,16
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.
Sudden decreases in food availability or environmental oxygen levels each evoke a characteristic
behavioral response in C. elegans where animals limit their movement to a restricted area by
increasing the frequency of trajectory changes (reorientations), a behavior known as local or area-
restricted searching (ARS). ARS is a highly conserved adaptive behavior and is evident across diverse
animal species.
17–23
ARS responses during food searching in particular are rapid and transient.
Trajectory changes increase within a few minutes after food removal, and decrease with prolonged
removal from food (>15-20 minutes) as animals transition to global searching (dispersal).
6–8,24–27
The
clearly discernible behavioral states during food searching present a highly tractable model for
understanding contributions of specific neuromodulatory systems. NLP-12 neuropeptide signaling
promotes increases in body bending amplitude and turning during movement,
6,7
motor adaptations
that are particularly relevant for ARS. Notably, nlp-12 is strongly expressed in only a single neuron, the
interneuron DVA that has synaptic targets in the motor circuit and elsewhere.
6,28
Despite the restricted
expression of nlp-12, there remains considerable uncertainty about the cellular targets of NLP-12
peptides and the circuit-level mechanisms by which NLP-12 modulation promotes its behavioral
effects.
Here we explore the GPCR and cellular targets involved in NLP-12 neuromodulation of local food
searching. Our findings reveal a primary requirement for NLP-12 signaling onto SMD head motor
neurons, mediated through the CKR-1 GPCR, for trajectory changes during local searching. In
contrast, NLP-12 signaling through both CKR-1 and CKR-2 GPCRs contribute to NLP-12 regulation of
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 28, 2021. ; https://doi.org/10.1101/2020.04.27.064550doi: bioRxiv preprint
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01 Jul 2008-Nucleic Acids Research
TL;DR: The Phylogeny.fr platform transparently chains programs to automatically perform phylogenetic analyses and can also meet the needs of specialists; the first ones will find up-to-date tools chained in a phylogeny pipeline to analyze their data in a simple and robust way, while the specialists will be able to easily build and run sophisticated analyses.
Abstract: Phylogenetic analyses are central to many research areas in biology and typically involve the identification of homologous sequences, their multiple alignment, the phylogenetic reconstruction and the graphical representation of the inferred tree. The Phylogeny.fr platform transparently chains programs to automatically perform these tasks. It is primarily designed for biologists with no experience in phylogeny, but can also meet the needs of specialists; the first ones will find up-to-date tools chained in a phylogeny pipeline to analyze their data in a simple and robust way, while the specialists will be able to easily build and run sophisticated analyses. Phylogeny.fr offers three main modes. The ‘One Click’ mode targets non-specialists and provides a ready-to-use pipeline chaining programs with recognized accuracy and speed: MUSCLE for multiple alignment, PhyML for tree building, and TreeDyn for tree rendering. All parameters are set up to suit most studies, and users only have to provide their input sequences to obtain a ready-to-print tree. The ‘Advanced’ mode uses the same pipeline but allows the parameters of each program to be customized by users. The ‘A la Carte’ mode offers more flexibility and sophistication, as users can build their own pipeline by selecting and setting up the required steps from a large choice of tools to suit their specific needs. Prior to phylogenetic analysis, users can also collect neighbors of a query sequence by running BLAST on general or specialized databases. A guide tree then helps to select neighbor sequences to be used as input for the phylogeny pipeline. Phylogeny.fr is available at: http://www.phylogeny.fr/

4,364 citations

Journal Article•DOI•
Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.

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Georg Nagel1, Tanjef Szellas, Wolfram Huhn, Suneel Kateriya, Nona Adeishvili, Peter Berthold, Doris Ollig1, Peter Hegemann, Ernst Bamberg1 •
Max Planck Society1
25 Nov 2003-Proceedings of the National Academy of Sciences of the United States of America
TL;DR: It is demonstrated by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel, and may be used to depolarize small or large cells, simply by illumination.
Abstract: Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane α helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.

2,519 citations

"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....

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Journal Article•DOI•
Independent optical excitation of distinct neural populations

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Nathan C. Klapoetke1, Yasunobu Murata2, Sung Soo Kim3, Stefan R. Pulver3, Amanda Birdsey-Benson2, Yong Ku Cho1, Tania K. Morimoto1, Amy S. Chuong1, Eric J. Carpenter4, Zhijian Tian5, Jun Wang5, Yinlong Xie5, Zhixiang Yan5, Yong Zhang5, Brian Y. Chow6, Barbara Surek7, Michael Melkonian7, Vivek Jayaraman3, Martha Constantine-Paton2, Gane Ka-Shu Wong4, Edward S. Boyden1 •
Massachusetts Institute of Technology1, McGovern Institute for Brain Research2, Howard Hughes Medical Institute3, University of Alberta4, Beijing Genomics Institute5, University of Pennsylvania6, University of Cologne7
01 Mar 2014-Nature Methods
TL;DR: Two channelrhodopsins, Chronos and Chrimson, are described, discovered through sequencing and physiological characterization of opsins from over 100 species of alga, that enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.
Abstract: Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

1,701 citations

"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)....

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Independent optical excitation of distinct neural populations

[...]

Nathan C. Klapoetke1, Yasunobu Murata2, Sung Soo Kim3, Stefan R. Pulver3, Amanda Birdsey-Benson2, Yong Ku Cho1, Tania K. Morimoto1, Amy S. Chuong1, Eric J. Carpenter4, Zhijian Tian5, Jun Wang5, Yinlong Xie5, Zhixiang Yan5, Yong Zhang5, Brian Y. Chow6, Barbara Surek7, Michael Melkonian7, Vivek Jayaraman3, Martha Constantine-Paton2, Gane Ka-Shu Wong4, Edward S. Boyden1 •
Massachusetts Institute of Technology1, McGovern Institute for Brain Research2, Howard Hughes Medical Institute3, University of Alberta4, Beijing Genomics Institute5, University of Pennsylvania6, University of Cologne7
01 Feb 2014
TL;DR: Chronos and Chrimson as mentioned in this paper have been shown to enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.
Abstract: Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

1,152 citations

Journal Article•DOI•
C. elegans Locomotory Rate Is Modulated by the Environment through a Dopaminergic Pathway and by Experience through a Serotonergic Pathway

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Elizabeth R. Sawin1, Rajesh Ranganathan1, H. Robert Horvitz1•
Massachusetts Institute of Technology1
01 Jun 2000-Neuron
TL;DR: The basal and enhanced slowing responses are distinct and separable neuromodulatory components of a genetically tractable paradigm of behavioral plasticity.
Abstract: Caenorhabditis elegans modulates its locomotory rate in response to its food, bacteria, in two ways. First, well-fed wild-type animals move more slowly in the presence of bacteria than in the absence of bacteria. This basal slowing response is mediated by a dopamine-containing neural circuit that senses a mechanical attribute of bacteria and may be an adaptive mechanism that increases the amount of time animals spend in the presence of food. Second, food-deprived wild-type animals, when transferred to bacteria, display a dramatically enhanced slowing response that ensures that the animals do not leave their newly encountered source of food. This experience-dependent response is mediated by serotonergic neurotransmission and is potentiated by fluoxetine (Prozac). The basal and enhanced slowing responses are distinct and separable neuromodulatory components of a genetically tractable paradigm of behavioral plasticity.

978 citations

"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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Frequently Asked Questions (1)
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.