RESEARCH ARTICLE
The Multilayer Connectome of Caenorhabditis
elegans
Barry Bentley
1
, Robyn Branicky
1
, Christopher L. Barnes
1,2
, Yee Lian Chew
1
,
Eviatar Yemini
3
, Edward T. Bullmore
4,5
, Petra E. Ve
´
rtes
4☯
, William R. Schafer
1☯
*
1 Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, 2 HHMI
Janelia Research Campus, Ashburn, VA, United States of America, 3 Department of Biological Sciences,
Columbia University, New York, NY, United States of America, 4 Department of Psychiatry, University of
Cambridge, Cambridge United Kingdom, 5 ImmunoPsychiatry, Alternative Discovery & Development,
GlaxoSmithKline R&D, Cambridge United Kingdom
☯ These authors contributed equally to this work.
*
wschafer@mrc-lmb.cam.ac.uk
Abstract
Connectomics has focused primarily on the mapping of synaptic links in the brain; yet it is
well established that extrasynaptic volume transmission, especially via monoamines and
neuropeptides, is also critical to brain function and occurs primarily outside the synaptic con-
nectome. We have mapped the putative monoamine connections, as well as a subset of
neuropeptide connections, in C. elegans based on new and published gene expression
data. The monoamine and neuropeptide networks exhibit distinct topological properties,
with the monoamine network displaying a highly disassortative star-like structure with a rich-
club of interconnected broadcasting hubs, and the neuropeptide network showing a more
recurrent, highly clustered topology. Despite the low degree of overlap between the extra-
synaptic (or wireless) and synaptic (or wired) connectomes, we find highly significant
multilink motifs of interaction, pinpointing locations in the network where aminergic and neu-
ropeptide signalling modulate synaptic activity. Thus, the C. elegans connectome can be
mapped as a multiplex network with synaptic, gap junction, and neuromodulator layers
representing alternative modes of interaction between neurons. This provides a new topo-
logical plan for understanding how aminergic and peptidergic modulation of behaviour is
achieved by specific motifs and loci of integration between hard-wired synaptic or junctional
circuits and extrasynaptic signals wirelessly broadcast from a small number of modulatory
neurons.
Author Summary
Connectomics represents an effort to map brain structure at the level of individual neu-
rons and their synaptic connections. However, neural circuits also depend on other types
of signalling between neurons, such as extrasynaptic modulation by monoamines and
peptides. Here we present a draft monoamine connectome, along with a partial neuropep-
tide connectome, for the nematode C. elegans, based on new and published expression
PLOS Computational Biology | DOI:10.1371/journal.pcbi.1005283 December 16, 2016 1 / 31
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OPEN ACCESS
Citation: Bentley B, Branicky R, Barnes CL, Chew
YL, Yemini E, Bullmore ET, et al. (2016) The
Multilayer Connectome of Caenorhabditis elegans.
PLoS Comput Biol 12(12): e1005283. doi:10.1371/
journal.pcbi.1005283
Editor: Saad Jbabdi, Oxford University, UNITED
KINGDOM
Received: August 23, 2016
Accepted: December 5, 2016
Published: December 16, 2016
Copyright: © 2016 Bentley et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: We thank the MRC (grant MC-A022-
5PB91 to WRS) and Wellcome Trust (grant
WT103784MA to WRS) for funding. PEV was
supported by a Bioinformatics Research Fellowship
from the Medical Research Council (UK) (MR/
K020706/1). YLC was supported by an EMBO
Long-term Fellowship. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
data for biosynthetic genes and receptors. We describe the structural properties of these
"wireless" networks, including their topological features and modes of interaction with the
wired synaptic and gap junction connectomes. This multilayer connectome of C. elegans
can serve as a prototype for understanding the multiplex networks comprising larger ner-
vous systems, including the human brain.
Introduction
The new field of connectomics seeks to understand the brain by comprehensively mapping the
anatomical and functional links between all its constituent neurons or larger scale brain
regions [
1]. The C. elegans nervous system has served as a prototype for analytical studies of
connectome networks, since the synaptic connections made by each of its 302 neurons have
been completely mapped at the level of electron microscopy [
2, 3]. Through this approach, the
C. elegans nervous system has been found to share a number of topological features in com-
mon with most other real-world networks, from human brain networks through social net-
works to the internet [
1, 4, 5]. One well-known example is the small-world phenomenon,
whereby networks are simultaneously highly clustered (nodes that are connected to each other
are also likely to have many nearest neighbours in common) and highly efficient (the average
path length between a pair of nodes is short) [6, 7]. Another characteristic feature of real-
world networks which has attracted much attention is the existence of hubs or high-degree
nodes, with many more connections to the rest of the network than expected in a random
graph [
8]. As in other networks, these topological features of the C. elegans connectome are
thought to reflect the functional needs of the system [
9, 10]. For example hubs are known to
play a privileged role in coordinating functions across a distributed network [11], while the
short path lengths (often mediated by the hubs) help increase the efficiency of information
transfer across the network [
6].
Although connectomics has primarily focused on mapping the synaptic links between neu-
rons, it is well established that chemical synapses are only one of several modes of interaction
between neurons. For example, gap junctions, which mediate fast, potentially bidirectional
electrical coupling between cells, are widespread in all nervous systems. Likewise, volume
transmission and neurohumoral signalling provide means for local or long-range communica-
tion between neurons unconnected by synapses. As neuromodulators released through these
routes can have profound effects on neural activity and behaviour [
12–14], a full understand-
ing of neural connectivity requires a detailed mapping of these extrasynaptic pathways.
In C. elegans, as in many animals, one important route of neuromodulation is through
monoamine signalling. Monoamines are widespread throughout phyla, with evidence that
they are one of the oldest signalling systems, evolving at least 1 billion years ago [15]. In both
humans and C. elegans, many neurons expressing aminergic receptors are not post-synaptic to
releasing neurons, indicating that a significant amount of monoamine signalling occurs out-
side the wired connectome [
16]. Monoamines are known to be essential for normal brain
function, with abnormal signalling being implicated in numerous neurological and psychiatric
conditions [
17]. In C. elegans, these monoaminergic systems play similarly diverse roles in
regulating locomotion, reproduction, feeding states, sensory adaptation, and learning [
16].
Clearly, if the goal of connectomics is to understand behaviourally relevant communication
within the brain, extrasynaptic monoamine interactions must also be mapped, not just the net-
work of wired chemical synapses and gap junctions.
The Multilayer Connectome of Caenorhabditis elegans
PLOS Computational Biology | DOI:10.1371/journal.pcbi.1005283 December 16, 2016 2 / 31
Competing Interests: ETB is employed half-time by
the University of Cambridge and half-time by
GlaxoSmithKline; he holds stock in GSK. The
authors have declared that no competing interests
exist.
In addition to monoamines, neuropeptides are also widely used as neuromodulators in the
C. elegans nervous system. C. elegans contains over 250 known or predicted neuropeptides syn-
thesized from at least 122 precursor genes, and over 100 putative peptide receptors [
18, 19].
These include homologues of several well-known vertebrate neuropeptide receptors, including
those for oxytocin/vasopressin (NTR-1), neuropeptide Y (NPR-1) and cholecystokinin (CKR-
2) [
19]. As in other animals, neuropeptide signalling is critical for nervous system function,
and frequently involves hormonal or other extrasynaptic mechanisms.
This study describes a draft connectome of extrasynaptic monoamine signalling in C. ele-
gans, as well as a partial network of neuropeptide signalling, based on new and published gene
expression data. We find that the extrasynaptic connectomes exhibit topological properties
distinct from one another as well as from the wired connectome. Overall, the neuronal connec-
tome can be modelled as a multiplex network with structurally distinct synaptic, gap junction,
and extrasynaptic (neuromodulatory) layers representing neuronal interactions with different
dynamics and polarity, and with critical interaction points allowing communication between
layers. This network represents a prototype for understanding how neuromodulators interact
with wired circuitry in larger nervous systems and for understanding the organisational princi-
ples of multiplex networks.
Results
A network of extrasynaptic monoamine signalling
To investigate the extent of extrasynaptic signalling in C. elegans monoamine systems, we sys-
tematically compared the expression patterns of monoamine receptors with the postsynaptic
targets of aminergic neurons. Monoamine-producing cells were identified based on the pub-
lished expression patterns of appropriate biosynthetic enzymes and vesicular transporters (see
Methods). The expression patterns for each of five serotonin receptors (ser-1, ser-4, ser-5, ser-7
and mod-1), three octopamine receptors (octr-1, ser-3 and ser-6), four tyramine receptors (ser-
2, tyra-2, tyra-3 and lgc-55), and four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) were
compiled from published data (see
S1–S7 Tables). Since these receptors are either ion channels
or serpentine receptors predicted to couple to pan-neuronal G-proteins, we therefore assumed
all neurons expressing monoamine receptors are potential monoamine-responding cells.
Three additional genes encode known or candidate monoamine receptors but have missing
or incomplete expression data. Specifically, a ligand-gated chloride channel, lgc-53, has been
shown to be activated by dopamine [
20], but its expression pattern and biological function
have not been characterized. Additional expression profiling using a transgenic lgc-53 reporter
line crossed to a series of known reference strains indicated that lgc-53 is expressed in a small
subset of neurons in the head, body and tail (
Fig 1). Together with the published dop-1, dop-2,
dop-3 and dop-4-expressing cells, these were inferred to make up the domain of dopamine-
responding neurons. In addition, two G-protein coupled receptors, dop-5 and dop-6, have
been hypothesized based on sequence similarity to dop-3 to be dopamine receptors. Using the
same approach used for lgc-53, we identified most of the cells with clear expression of dop-5
and dop-6 reporters (Fig 1). These cells were included in a broader provisional dopamine net-
work, the analysis of which is presented in the supplemental material (S1 Fig, S3 Fig).
Receptor expression patterns suggest that a remarkably high fraction of monoamine signal-
ling must be extrasynaptic. For example, the two tyraminergic neurons, RIML and RIMR, are
presynaptic to a total of 20 neurons. Yet of the 114 neurons that express reporters for one or
more of the four tyramine (TA) receptors, only 7 are postsynaptic to a tyraminergic neuron
(
Fig 2A; Table 1). Thus, approximately 94% of tyramine-responsive neurons must respond
only to extrasynaptic TA. Similar analyses of the other monoamine systems yield comparable
The Multilayer Connectome of Caenorhabditis elegans
PLOS Computational Biology | DOI:10.1371/journal.pcbi.1005283 December 16, 2016 3 / 31
results: 100% of neurons expressing octopamine receptors receive no synaptic input from
octopamine-releasing neurons (Fig 2B), while 82% of neurons expressing dopamine receptors,
and 76% of neurons expressing serotonin receptors receive no synaptic input from neurons
expressing the cognate monoamine ligand (
Table 1). Thus, most neuronal monoamine signal-
ling in C. elegans appears to occur extrasynaptically, outside the wired synaptic connectome.
The prevalence of extrasynaptic monoamine signalling between neurons unconnected by syn-
apses or gap junctions implies the existence of a large wireless component to the functional C.
elegans connectome, the properties of which have not previously been studied.
Using the gene expression data, a directed graph representing a draft aminergic connec-
tome was constructed with edges linking putative monoamine releasing cells (expressing
monoamines, biosynthetic enzymes, or transporters) to those cells expressing a paired receptor
(
Fig 2C; Table 2; S1 Dataset). Since biologically-relevant long-distance signalling (e.g. from
releasing cells in the head to tail motoneurons) has been experimentally demonstrated in C.
elegans for both dopamine and serotonin [
21, 22]–while tyramine and octopamine are each
released from a single neuronal class [
16]–edges were not restricted based on the physical
Fig 1. Expression patterns of the dopamine receptors dop-5, dop-6 & lgc-53. Shown are representative
images showing expression of GFP reporters under the control of indicated receptor promoters in the head (left
panels) or tail/posterior body (right panels). Identified neurons are labelled; procedures for confirmation of cell
identities are described in methods. In all panels, dorsal is up and anterior is to the left. In addition to the neurons
indicated, dopamine receptor reporters were detected in the following neurons: dop-5: BDU (some animals); lgc-53:
CAN (some animals).
doi:10.1371/journal.pcbi.1005283.g001
The Multilayer Connectome of Caenorhabditis elegans
PLOS Computational Biology | DOI:10.1371/journal.pcbi.1005283 December 16, 2016 4 / 31
distance between nodes. For the serotonin network, only those neurons with strong, consistent
expression of serotonin biosynthetic markers such as tryptophan hydroxylase were included
(NSM, HSN and ADF). Additional neurons (AIM, RIH, VC4/5) that appear to take up seroto-
nin but not synthesize it [
23][24] were not included in the network, since they may function
primarily in the homeostatic clearing of serotonin. We also did not include the ASG neurons,
Fig 2. Monoamine signalling in C. elegans is primarily extrasynaptic. (A) RIM tyramine releasing neurons, showing outgoing synaptic edges
(arrows), and neurons expressing one or more of the four tyramine receptors (grey). (B) RIC octopamine releasing neurons, showing outgoing synaptic
edges (arrows), and neurons expressing one or more of the three octopamine receptors (grey). (C) Adjacency matrix showing the monoamine (green),
synaptic (magenta) and gap junction (blue) networks. (D) Multilayer expansion of the synaptic (syn), gap junction (gap), monoamine (MA) and
neuropeptide (NP) signalling networks. Node positions are the same in all layers.
doi:10.1371/journal.pcbi.1005283.g 002
The Multilayer Connectome of Caenorhabditis elegans
PLOS Computational Biology | DOI:10.1371/journal.pcbi.1005283 December 16, 2016 5 / 31