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(which was not certified by peer review) is the author/funder.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
