Dissecting the Serotonergic Food Signal Stimulating
Sensory-Mediated Aversive Behavior in C. elegans
Gareth Harris, Amanda Korchnak, Philip Summers, Vera Hapiak, Wen Jing Law, Andrew M. Stein, Patricia
Komuniecki, Richard Komuniecki*
Department of Biological Sciences, University of Toledo, Toledo, Ohio, United States of America
Nutritional state often modulates olfaction and in Caenorhabditis elegans food stimulates aversive responses mediated by
the nociceptive ASH sensory neurons. In the present study, we have characterized the role of key serotonergic neurons that
differentially modulate aversive behavior in response to changing nutritional status. The serotonergic NSM and ADF
neurons play antagonistic roles in food stimulation. NSM 5-HT activates SER-5 on the ASHs and SER-1 on the RIA
interneurons and stimulates aversive responses, suggesting that food-dependent serotonergic stimulation involves local
changes in 5-HT levels mediated by extrasynaptic 5-HT receptors. In contrast, ADF 5-HT activates SER-1 on the
octopaminergic RIC interneurons to inhibit food–stimulation, suggesting neuron-specific stimulatory and inhibitory roles for
SER-1 signaling. Both the NSMs and ADFs express INS-1, an insulin-like peptide, that appears to cell autonomously inhibit
serotonergic signaling. Food also modulates directional decisions after reversal is complete, through the same serotonergic
neurons and receptors involved in the initiation of reversal, and the decision to continue forward or change direction after
reversal is dictated entirely by nutritional state. These results highlight the complexity of the ‘‘food signal’’ and serotonergic
signaling in the modulation of sensory-mediated aversive behaviors.
Citation: Harris G, Korchnak A, Summers P, Hapiak V, Law WJ, et al. (2011) Dissecting the Serotonergic Food Signal Stimulating Sensory-Mediated Aversive
Behavior in C. elegans. PLoS ONE 6(7): e21897. doi:10.1371/journal.pone.0021897
Editor: Hiroaki Matsunami, Duke University, United States of America
Received April 11, 2011; Accepted June 8, 2011; Published July 21, 2011
Copyright: ? 2011 Harris 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.
Funding: This work was supported by the National Institutes of Health (NIH) grant AI-145147 awarded to RWK and funds from the Joan L. and Julius H. Jacobson
Biomedical Professorship. The present study is supported by the NIH National Center for Research Resources. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Olfactory acuity is modulated by nutritional status, with
behaviors initiated by attractive stimuli generally enhanced by
starvation and those initiated by repulsive (noxious) stimuli
enhanced by satiety. Both monoamines, such as serotonin (5-HT),
and neuropeptides translate nutritional status into olfactory
modulation. Serotonergic signalingincreases odor-evoked responses
in moths and reduces sensory input and odor processing in mice,
apparently coordinating sensory gain in the olfactory bulb with
behavioral state [1,2]. Similarly, insulin and leptin differentially
modulate spontaneous and odor-evoked activity in rat olfactory
neurons . Serotonergic neurons often co-release classical
neurotransmitters, such as glutamate, and neuropeptides, but our
understanding of the nutritional modulation of serotonergic/
peptidergic interactions is limited, given the complexity of the
mammalian nervous system [4,5]. To better understand serotoner-
gic modulation of olfactory signaling, we have examined the food-
dependent modulation of sensory-mediated locomotory behaviors
in the model, Caenorhabditis elegans. In C. elegans, food dramatically
alters monoaminergic signaling and modulates nutritionally-depen-
dent locomotory transitions [6,7]. Recent work suggests that 5-HT
provides a balance of both excitatory and inhibitory input into key
behaviors, through five different 5-HT receptors, most with
orthologues in mammals [8,9].
In C. elegans, food increases aversive responses mediated by
the ASH sensory neurons through three 5-HT receptors,
operating within the ASH-mediated locomotory circuit, but
the relationship between food availability and serotonergic
transmission or the source of the 5-HT activating these
receptors has not been identified [10–12]. C. elegans contains
at least nine serotonergic neurons; two NSMs, ADFs and HSNs
that synthesize 5-HT directly and two AIMs and the RIH that
do not express tph-1, required for serotonin synthesis, and
instead accumulate secreted 5-HT through a 5-HT transporter,
MOD-5 . Interactions between NSM and ADF signaling are
complex, with both neuron-specific and cooperative interactions
described, although few studies have functionally localized the
5-HT receptors involved in NSM and/or ADF-dependent
In the present study, we have dissected the food signal that
modulates ASH-mediated aversive behaviors and demonstrated
that 5-HT released from the NSMs and ADFs functions
antagonistically, with NSM 5-HT stimulating aversive responses
and ADF 5-HT inhibiting food-stimulation. 5-HT from the NSMs
and ADFs activates distinct subsets of 5-HT receptors, suggesting
that food-dependent serotonergic signaling is characterized by
changes in local 5-HT levels, involving primarily extrasynaptic 5-
HT receptors. Finally, ins-1 appears to act cell autonomously in
both the NSMs and ADFs to inhibit serotonergic signaling.
Together, these results highlight the complexity of serotonergic
modulation and the obligate interactions among the multiple
ligands released from the serotonergic neurons in the food-
associated modulation of aversive behavior.
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Materials and Methods
All reagents were purchased from Sigma Aldrich (St. Louis,
MO). Neurochemicals were purchased from Sigma Aldrich (St.
Louis, MO), restriction enzymes from New England Biolabs
(Beverly, MA) and Promega (Madison, WI) and oligonucleotide
primers from Integrated DNA Technologies (Coralville, IA). A C.
elegans cDNA pool was purchased from OriGene Technologies
(Rockville, MD), and additional cDNA pools were constructed
from mixed stage mRNA using standard techniques. Green
fluorescent protein (GFP) expression vectors were obtained from
Andy Fire (Stanford School of Medicine).
Cultures and maintenance of strains
The N2 Bristol WT isolate of C. elegans was used for all studies.
All animals were raised at 20uC under uncrowded conditions .
The following mutant alleles were used in this study: ins-
1(tm1888)IV, mod-5(n822)I, ser-5(tm2647)I, ser-5(tm2654)I, ser-
4(ok512)III, mod-1(ok103)V, ser-7(tm1325)X, and ser-1(ok345)X. All
strains were obtained from the Caenorhabditis Genetics Center
7(tm1325)X, ser-5(tm2647)I, ser-5(tm2654)I, which were obtained
from the National Bio-Resources Project (Tokyo Women’s
Medical University, Tokyo, Japan). Animals containing combina-
tions of null or gf alleles were constructed using standard genetic
techniques and confirmed by PCR. All mutant animals were
backcrossed with the N2 Bristol strain at least 56before use.
Assay plates (5 cm NGM plates) were prepared daily and
serotonin (4 mM) was added to NGM liquid media just prior to
pouring. Dilute 1-octanol was prepared daily using 100% ethanol
(vol/vol) [10,20]. Synchronized fourth-stage larvae (L4) were
picked 24 hrs pre-assay and assays were performed at 23–25uC.
All assays were performed with blinded samples to remove
experimental bias. Octanol avoidance was measured, as described
by . Briefly, the blunt end of a hair (Loew-Cornell 9000
Kolinsky 8 paintbrush), was taped to a toothpick, dipped in 30%
1-octanol and placed in front of an animal exhibiting forward
sinusoidal locomotion. Time to reverse was recorded and assays
were terminated after 20 sec, to minimize spontaneous reversals,
which are effected by food availability [10,21–23]. For assays in
the absence of food or 5-HT, well-fed young adults (three to five
per plate) were transferred to intermediate non-seeded plates and
left for 1 min to prevent bacteria/media carry over, then
transferred to NGM plates and assayed after 10 min. For assays
in the presence of food (E. coli OP50) or 5-HT, animals were
transferred to plates containing OP50 or 4 mM 5-HT and assayed
after 20 and 30 min, respectively.
Post-initiation assays were performed as follows: synchronized
fourth-stage larvae (L4) were picked 24 hrs pre-assay. Octanol
avoidance was measured as above, except that animals were
examined for duration of reversal by 1) counting the number of
head swings per reversal and 3) determining the angle from the
initial trajectory once reversal was complete (Figure 1). Twenty
animals/strain/condition were assayed. Data are presented as
a mean 6 SE and analyzed by two-tailed Student’s t test.
Rescue constructs and RNAi
All rescue constructs were created by overlap fusion PCR or by
cloning into pPD95.75 [24,25]. The following neuron-selective
promoters were used: srh-142p (ADF), ceh-2p (I3, M4, NSM, M3),
tdc-1p (RIC, RIM, spermetheca). For overlap PCR, constructs
were pooled from at least 3 reactions and were co-injected with
myo-3p::gfp, F25B3.3p::gfp or rol-6 and carrier DNA (to 100 ng) into
gonads of wild-type and null mutant animals by standard
techniques [24,26]. At least three lines were examined and all
constructs in pPD95.75 were confirmed by sequencing.
Neuron selective/specific RNAi transgenes were constructed as
described by  by fusing neuron-selective promoters to exon
rich regions of target genes. Exon-rich regions were amplified
using forward and reverse primers to create template A from either
cDNA or genomic DNA by PCR-fusion, as previously described
by . Neuron-selective promoters were amplified using a
forward primer with reverse (sense) or reverse (antisense) primers
to create templates B and C, respectively . Templates A and B
were fused using a forward internal promoter primer and a reverse
internal target gene primer to create the sense construct (Product
D). Templates A and C were fused using the forward internal
promoter primer and the forward internal target gene primer to
Figure 1. The serotonergic NSM and ADF neurons modulate the food-dependent stimulation of aversive responses by the ASH
sensory neurons. Wild-type, mutant, transgenic and RNAi expressing animals were examined for aversive responses to dilute 1-octanol (30%) in the
presence (A) or absence (B) of food, as described in Methods. NSM and ADF selective promoters: (ceh-2, I3, M4, NSM, M3) and (srh-142, ADF),
respectively [51,52]. Data are presented as a mean 6 SE and analyzed by two-tailed Student’s t test. ‘‘*’’ P,0.001, significantly different from wild-
type animals incubated in the presence of food.
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create the antisense construct (Product E). At least three products
were pooled and sense and antisense transgenes were microinject-
ed at 25–100 ng/ml. At least 3 transgenic lines were examined for
each RNAi. All strains are listed in Strain List S1. Primer
sequences used for RNAi and rescue construct generation are
shown in Table S1 and S2.
Serotonergic signaling from the NSMs, but not the ADFs,
is required for the food-stimulation of aversive responses
mediated by the ASH sensory neurons
Food or 5-HT stimulate ASH-mediated aversive responses to
dilute (30%) 1-octanol and tph-1 null animals that lack tryptophan
hydroxylase, the rate-limiting enzyme for 5-HT biosynthesis, do
not increase aversive responses on food [10–12]. As noted above,
C. elegans contains at least nine serotonergic neurons and 5-HT
from the NSMs and ADFs, the major 5-HT synthesizing neurons
surrounding the nerve ring, appear to function independently to
modulate behavior [14,15,17]. Indeed, both the NSMs and the
ADFs have been previously implicated in a number of serotonin-
dependent, sensory-mediated, behaviors [14–18].
Therefore, to better understand the serotonergic ‘‘food signal,’’
in the modulation of ASH-mediated aversive responses, the
individual roles of the major 5-HT synthesizing neurons in the
nerve ring, the NSMs and ADFs, in food-stimulation were
dissected, using neuron-specific rescue and RNAi. Since Esposito
first described neuron-specific RNAi knockdown in individual
pairs of sensory neurons in 2007, many labs, in addition to our
own, have used this technique effectively [12,27–30]. In fact, we
have found that the major problem with this RNAi technique is
not whether it effectively knocks down expression (it usually does),
but whether the RNAi will spread to additional tissues, especially if
it is more broadly expressed (i.e., muscle or motorneurons),
significantly complicating the interpretation of any neuron-specific
results (Komuniecki, unpublished). However, it is important to
note that as this technique is more broadly applied, each neuron
has the potential to respond differently to RNAi, emphasizing the
need for independent corroboration of any RNAi result. In
examining the roles of NSM and ADF signaling below, we have
confirmed any neuron specific RNAi result by the neuron-specific
rescue of null mutants, the RNAi knockdown of two different
genes predicted to decrease serotonergic signaling, and most
importantly, the neuron-specific RNAi knockdown of genes
predicted to increase serotonergic signaling (mod-5) that yield
phenotypes opposite to those observed for the inhibition of
serotonergic signaling. In addition, to control for any potential
RNAi spreading, we have also used NSM and ADF specific RNAi
to knockdown the expression of ser-5, a receptor essential for food
stimulation but not expressed in the NSMs or ADFs. As predicted,
ser-5RNAi knockdown in the ASHs abolished food-stimulation,
but ser-5RNAi knockdown in the NSMs or ADFs had no effect
(Komuniecki, data not shown) .
The food-dependent stimulation of aversive responses in tph-1
null animals was rescued by the expression of tph-1 in the NSMs,
but not the ADFs ((ceh2p::tph-1(+) vs srh-142p::tph-1(+); Figure 1).
Similarly, food-stimulation was abolished by the tph-1RNAi
knockdown in the NSMs, but not in the ADFs, of wild-type
animals (Figure 1). To confirm a role for the NSMs in food-
stimulation, RNAi was also used to selectively knockdown unc-86
in the NSMs and osm-9 in the ADFs, on the observation that
UNC-86 (POU domain transcription factor) and OSM-9 (TRPV
channel subunit) were essential for 5-HT synthesis in the NSMs
and ADFs, respectively [15,31] (Figure 1). As predicted, the
Fp::osm-9RNAi had no effect on aversive responses (Figure 1).
Thus, using three different approaches, we have demonstrated that
that 5-HT signaling from the NSMs, but not the ADFs, is required
for stimulation of ASH-mediated aversive responses by food.
Conversely, as predicted none of these treatments has any effect off
food (Figure 1B).
Serotonergic signaling from the ADFs abolishes the
stimulation of ASH-mediated aversive responses by food
To further define the roles of serotonergic signaling from the
NSMs and ADFs, mod-5, which encodes a 5-HT reuptake
transporter, was selectively knocked down in the NSMs or ADFs,
using RNAi [27,32]. These studies are based on the assumption
that the neuron-specific knockdown of mod-5 would selectively
modulate signaling from these neurons, but more global effects on
other 5-HT pools cannot be ruled out. Reassuringly, the RNAi
knockdown of mod-5 in either the NSMs or ADFs had different
effects on octanol sensitivity. As predicted, mod-5 null animals and
wild-type animals with mod-5 knocked down in the NSMs
exhibited increased aversive responses both off and on food when
compared to wild-type animals, presumably driven by the
accumulation of 5-HT released from the NSMs (Figure 2).
Similarly, NSMp::tph-1 overexpression in wild type animals also
increased aversive responses on food (Figure 1A). Interestingly,
NSMp::tph-1 overexpression had no effect off food, presumably
because food-stimulation of the NSMs was required for 5-HT
release (Figure 1B). In contrast, ADFp::mod-5RNAi abolished
food-stimulation, suggesting that 5-HT released from the ADFs
inhibited food-stimulation, but not basal ASH signaling (data not
shown, Figure 2). Indeed, ADFp::tph-1 overexpression in wild-type
animals also abolished food-stimulation (Figure 1A). These results
confirm a stimulatory role for NSM 5-HT and suggest that ADF 5-
HT inhibits the stimulation of ASH-mediated aversive responses
INS-1 from the NSMs and ADFs inhibits serotonergic
The insulin-like peptide, INS-1 is expressed in the both NSMs
and ADFs and ins-1 null animals exhibited more rapid aversive
Figure 2. The neuron-selective increase in NSM or ADF
serotonergic signaling by RNAi knockdown of mod-5 that
encodes a 5-HT reuptake transporter differentially modulates
aversive responses. Wild-type, mutant, transgenic and RNAi express-
ing animals were examined for aversive responses to dilute 1-octanol
(30%) in the presence or absence of food and/or 5-HT (4 mM), as
described in Methods. Data are presented as a mean 6 SE and analyzed
by two-tailed Student’s t test. ‘‘*’’ P,0.001, significantly different from
wild-type animals incubated in the presence of food.
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responses than wild-type animals off food, suggesting that INS-1
inhibited ASH-mediated aversive behavior [33,34] (Figure 3). To
determine if this ins-1 phenotype was mediated by INS-1 from the
NSMs and/or ADFs, ins-1 was selectively knocked down using
RNAi. NSMp::ins-1RNAi stimulated aversive responses both on
and off food (Figure 3). In contrast, NSM ins-1 overexpression in
wild-type animals abolished food-dependent increases in aversive
responses (Figure 3). Importantly, animals with both tph-1 and ins-
1 knocked down in the NSMs did not exhibit the more rapid
aversive responses off food observed after the NSM knockdown of
ins-1 alone, suggesting that NSM INS-1 inhibited serotonergic
signaling from the NSMs (Figure 3). In contrast, ADFp::ins-1RNAi
abolished food-stimulation, suggesting that ins-1 knockdown in the
ADFs might stimulate ADF serotonergic signaling and inhibit
aversive responses, in agreement with observations described
above for the ADF knockdown of mod-5 or overexpression of tph-1
(Figure 3). Indeed, animals with both ins-1 and tph-1 knocked
down in the ADFs exhibited wild-type responses that were
stimulated by food (Figure 3). These results suggest that ADF
INS-1 may have an autocrine function in the ADFs and inhibits
ADF serotonergic signaling. However, the possibility that INS-1 in
acting in parallel to or downstream of serotonergic signaling
cannot be ruled out.
Distinct 5-HT receptors are involved in the NSM
stimulation and ADF inhibition of aversive responses
As noted above, NSM 5-HT is essential for the stimulation of
aversive responses by food, while ADF 5-HT abolishes food-
stimulation, suggesting that food and/or ASH-mediated signaling
may inhibit serotonergic signaling from the ADFs. To identify the
specific 5-HT receptors mediating the effects of NSM or ADF 5-
HT, aversive responses were examined in animals with null alleles
for ser-1, ser-5 or mod-1, the three 5-HT receptors previously
identified as playing a role in 5-HT-dependent aversive responses,
after RNAi knockdown of either mod-5 or ins-1 in the NSMs or
ADFs, on the assumption that knockdown should specifically
increase serotonergic signaling from either neuron pair .
NSMp::ins-1RNAi or NSMp::mod-5RNAi increased aversive
responses both on and off food in both wild-type and mod-1
animals, but had no effect on aversive responses in ser-1 or ser-5
animals, suggesting that NSM serotonergic signaling required both
Figure 3. Insulin signaling modulates 5-HT release from the
ADFs and NSMs. Wild-type, mutant, transgenic and RNAi expressing
animals were examined for aversive responses to dilute 1-octanol (30%)
in the presence or absence of food, as described in Methods. Data are
presented as a mean 6 SE and analyzed by two-tailed Student’s t test.
‘‘*’’ P,0.001, significantly different from wild-type animals incubated in
the presence or absence of food.
Figure 4. Serotonergic signaling from the NSMs or ADFs
requires unique subsets of 5-HT receptors in the modulation
of ASH-mediated aversive responses. Wild-type and 5-HT receptor
null mutants expressing neuron selective mod-5 or ins-1 RNAi
transgenes (Top: NSMp::RNAi, Bottom: ADFp::RNAi) were examined for
aversive responses to dilute 1-octanol (30%) in the presence or absence
of food, as described in Methods.
Figure 5. 5-HT from the HSNs increases aversive responses on
and off food. Wild-type and 5-HT receptor null animals expressing
neuron selective mod-5RNAi were examined in the presence or absence
of food for aversive responses to dilute 1-octanol (30%). Data are
presented as a mean 6 SE and analyzed by two-tailed Student’s t test.
‘‘*’’ P,0.001, significantly different from wild-type animals incubated in
the presence or absence of food.
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Figure 6. NSM 5-HT activates SER-1 in the RIA interneurons to stimulate aversive responses and ADF 5-HT activates SER-1 in the
octopaminergic RIC interneurons to inhibit food-stimulation. Wild-type and transgenic animals were assayed for aversive responses to dilute
1-octanol (30%) in either the presence or absence of food. Neuron selective promoters for RIA and RIC neurons: (glr-3, RIA) and (tdc-1, RIC, RIM,
spermetheca), respectively [53,54]. Data are presented as a mean 6 SE and analyzed by two-tailed Student’s t test. ‘‘*’’ P,0.001, significantly
different from animals incubated in the presence or absence of food.
Figure 7. Food and 5-HT modulate locomotory behavior after reversal is initiated. A. Diagram outlining the measurement of post-
initiation behaviors. B. Wild-type animals were examined for post-initiation responses in the presence or absence of food or 5-HT (4 mM), as
described in Methods. After reversal was complete, the angle at which the animals resumed forward locomotion, relative to their initial trajectory, was
recorded, i.e., the angle of animals proceeding along the same path was 0, and for animals proceeding in the opposite direction was 180. In addition,
animals were examined for reversal duration (number of heads swings). C. Locomotory trajectory after the completion of reversal was assayed in
mutant animals in the presence and absence of food. Data are presented as a mean 6 SE and analyzed by two-tailed Student’s t test. ‘‘*’’ P,0.001,
significantly different from animals incubated in the presence or absence of food.
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SER-1 and SER-5 (Figure 4, data not shown). MOD-1 is also
essential for food or 5-HT-dependent stimulation, so that the
ability of NSM 5-HT to stimulate aversive responses in mod-1 null
animals was surprising . Interestingly, mod-5RNAi knock-
down in the third pair of 5-HT biosynthetic neurons, the HSNs,
using the egl-47 promoter, also increased basal and 5-HT-
stimulated aversive responses in both wild-type and mod-1 null
animals, suggesting either that MOD-1 is not required for 5-HT
stimulation from any of the biosynthetic neurons or that the levels
of 5-HT released after mod-5 knockdown are sufficient to
overcome the need for MOD-1 (Figure 5). In contrast,
ADFp::ins-1RNAi or ADFp::mod-5RNAi inhibited food stimula-
tion in wild-type, ser-5, and mod-1 null animals, but had no effect in
ser-1 null animals, suggesting that the activation of SER-1 by ADF
5-HT inhibited food-stimulation (Figure 4). This result was also
surprising, as previous work demonstrated that the expression of
ser-1 in the RIA interneurons, major downstream synaptic partners
of the ADFs, was essential for food or 5-HT stimulation and
suggested that ADF SER-1 signaling antagonized NSM SER-1
signaling in the RIAs .
To identify the neuron(s) involved in NSM and ADF SER-1
signaling, neuron-specific RNAi was used to create transgenic
NSMp::ins-1, NSMp::mod-5, ADFp::ins-1, and ADFp::mod-
5RNAi. The more rapid aversive responses observed after
NSMp::ins-1RNAi or NSMp::mod-5RNAi were absent when ser-
1 was also knocked down in the RIAs, supporting our hypothesis
that food-stimulation and increased serotonergic signaling from
the NSMs required SER-1 in the RIAs (Figure 6) .
Importantly, NSMp::ins-1or NSMp::mod-5RNAi in ser-1 mutant
animals phenocopies their double RNAi, supporting the effective-
ness of this double RNAi approach (compare Figures 4 and 6). In
contrast, although the inhibition of food-stimulation observed after
ADFp::ins-1 or ADFp::mod-5RNAi knockdown was absent in ser-
1 null animals, it was unaffected by the simultaneous knockdown
of ser-1 in the RIAs, suggesting that the ADF SER-1 inhibition of
food-stimulation involved other neurons (Figure 6).
Interestingly, ADFp::mod-5RNAi or ADFp::ins-1RNAi did not
inhibit food-stimulation in tdc-1 or tbh-1 null animals that lack
octopaminergic signaling, suggesting that the ADF-dependent
inhibition of food-stimulation might require OA (Figure 6). ser-
1p::gfp is robustly expressed in both the RIAs and the
octopaminergic RICs . As predicted, aversive responses in
animals with ADFp::mod-5/RICp::ser-1 or ADFp:ins-1/RICp::-
ser-1 knocked down by RNAi were still stimulated by food
(Figure 6). Together, these results suggest that NSM 5-HT
activates SER-1 in the RIAs to stimulate aversive responses and
ADF 5-HT activates SER-1 in the RICs to stimulate OA release
and inhibit food-stimulation.
NSM 5-HT also modulates locomotory behavior after
reversal is initiated
In addition to stimulating the initiation of reversal (time to
reverse after exposure to stimulus), food also decreased reversal
length (head swings/reversal) and directional decisions after
reversal was complete (Figure 7A, B). For example, on food
reversals were short (,1 head swing/reversal) and after reversal
was complete most animals continued forward along their previous
path (,45u from initial trajectory). In contrast, off food animals
backed up more extensively and turned significantly away from
their previous trajectory (.45u from initial trajectory). Surprising-
ly, these post-initiation phenotypes were independent of the
intensity of the initiating stimulus, i.e., even though animals
initiated reversal much more rapidly to 100% than 30% 1-octanol,
food had identical effects on post-initiation responses, suggesting
nutritional state and not intensity of the noxious stimulus dictated
these aversive responses (Figure 7B). Exogenous 5-HT mimicked
food in these post-initiation assays and modulated the length and
directionality of reversal, and, as predicted, post-initiation
responses in tph-1 null animals on food were similar to those of
animals off food (Figure 7). SER-1, SER-5 and MOD-1, the 5-HT
receptors essential for food stimulation of aversive responses, were
also essential in the food-dependent modulation of post-initiation
responses (Figure 7C).
To identify the source of 5-HT responsible for food-dependent
modulation of post-initiation behaviors, animals with altered NSM
and ADF signaling were examined on food (Figure 8). Food-
dependent alterations in post-initiation behaviors were abolished
by NSMp::tph-1RNAi, but were unaffected by ADFp::tph-1RNAi,
suggesting 5-HT from the NSMs, but not the ADFs was required
for modulation of post-initiation responses on food. As predicted,
increasing NSM serotonergic signaling by NSMp::mod-5RNAi
suppressed turns and reversal duration off food (Figure 8). These
results suggest that NSM 5-HT not only increased responsiveness
to ASH-mediated odorants, but also markedly altered post-
initiation behaviors, regardless of the intensity of the initiating
aversive stimulus. This result suggests that NSM signaling may
provide differential input into potentially overlapping locomotory
Figure 8. Food-dependent modulation of post-initiation
behaviors requires NSM, but not ADF 5-HT. Wild-type animals
and animals expressing neuron-selective RNAi were examined for post-
initiation behaviors in the presence or absence of food. A. After reversal
was complete and the angle at which the animals resumed forward
locomotion, relative to their initial trajectory, was recorded, i.e., the
angle of animals proceeding along the same path was 0, and for
animals proceeding in the opposite direction was 180. B. Head swings
per reversal, as described in Methods. Data are presented as a mean 6
SE and analyzed by two-tailed Student’s t test. ‘‘*’’ P,0.001, significantly
different from wild-type animals incubated in the presence or absence
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circuits to modulate both acute aversive responses and post-
initiation locomotory behaviors.
Food availability and nutritional status modulate olfactory
acuity and behavior in most organisms [35–39]. In C. elegans, food
stimulates ASH-mediated aversive responses and the present
studies highlight the complexity of the serotonergic signal defining
food availability (Figure 9). 5-HT released from the serotonergic
NSMs and ADFs appears to have antagonistic effects on octanol-
stimulated aversive behavior, with NSM 5-HT stimulating
aversive responses and ADF 5-HT inhibiting food-stimulation.
For example, increasing serotonergic signaling from the NSMs by
knocking down NSM mod-5 or ins-1 or overexpressing tph-1
stimulates basal aversive responses to levels observed on food or
exogenous 5-HT. Conversely, increasing serotonergic signaling
from the ADFs by knocking down ADF mod-5 or ins-1 or
overexpressing tph-1 abolishes the stimulation of aversive responses
by food or exogenous 5-HT. These genetic manipulations have
been interpreted as having acute effects on serotonergic signaling,
but it is worth noting that altered serotonergic signaling can
potentially also have effects on neuronal development in C. elegans
. These results highlight the complexity of serotonergic
modulation and the potential limitations of exogenous ligands in
characterizing serotonergic signaling.
Interactions between NSM and ADF signaling are complex,
with both neuron-specific and cooperative interactions described
previously. The branched NSMs make synapses onto the
pharyngeal basement membrane and muscle, but also secrete 5-
HT and neuropeptides directly into the pseudocoelomic fluid from
vesicle-filled varicosities [13,41]. Many mammalian monoaminer-
gic neurons are also ‘‘asynaptic’’ . In contrast, the ADFs are
innervated by the ASHs directly and synapse onto interneurons
modulating key locomotory transitions . The NSMs are
required for the enhanced slowing response and both the NSMs
and ADFs are required for dispersal during starvation [16,18].
However, these studies often have relied on NSM ablation, so
whether NSM 5-HT, glutamate and/or neuropeptides were
involved is unclear. In contrast, the food-sensitization of hyperoxia
avoidance and aversive learning were rescued in tph-1 animals by
tph-1 expression in the ADFs, but not the NSMs, supporting a role
for ADF 5-HT in food-sensitization [14,15]. Whether the ADFs
sense food directly or respond to signals from other neurons in
response to food availability/quality is unclear. However,
ADFp::tph-1 expression is elevated in daf-7 animals . daf-7
encodes a TGF-b homologue and the ASI expression of DAF-7
decreases during stress/starvation, suggesting that starvation
[14,43,44]. Serotonergic signaling from ADFs requires MOD-1
for avoidance to pathogenic bacteria . Bacteria should
stimulate NSM 5-HT and activate feeding, but pathogenic
bacteria may also stimulate ADF 5-HT to antagonize NSM
signaling, with products from the pathogens either activating the
ADFs directly or indirectly through other sensory neurons. In the
present studies, SER-1, not MOD-1, was essential for the ADF 5-
HT inhibition of food-stimulation and it is unclear if SER-1 was
required for aversive learning to pathogenic bacteria, highlighting
the potential complexity of ADF serotonergic signaling.
Food and 5-HT stimulate ASH-mediated aversive responses
through at least three different 5-HT receptors, operating within
the ASH-mediated circuit [10,11,12]. 5-HT from the NSMs,
ADFs and potentially other serotonergic neurons differentially
interacts with subsets of these 5-HT receptors and serotonergic
signaling appears to involve precisely modulated changes in local
5-HT levels, mediated primarily by extra-synaptic 5-HT receptors
food stimulatesNSM 5-HT
Figure 9. Model for the food dependent modulation of the ASH-mediated aversive behavior. On food, the release of 5-HT from the NSMs
stimulates ASH-mediated aversive responses. In contrast, 5-HT from the ADFs and OA from the RICs inhibits 5-HT sensitization. ASH, sensory neuron
necessary and sufficient for aversive responses to dilute octanol; AVA, backward command interneuron; AVB, forward command interneuron; INS-1,
insulin-like peptide; OA, octopamine; OCTR-1, a-adrenergic-like OA receptor; SER-1, 5-HT2-like receptor; SER-5; 5-HT6-like receptor; MOD-1; 5-HT-gated
chloride channel; Green (NSM and ADF serotonergic neurons expressing TPH-1); MOD-5, 5-HT reuptake transporter; Yellow (RIC octopaminergic
neurons expressing TDC-1 and TBH-1); Blue (RIM tyraminergic neurons expressing TDC-1).
Food Increases Octanol Avoidance
PLoS ONE | www.plosone.org7 July 2011 | Volume 6 | Issue 7 | e21897
(Figure 9). In fact, C. elegans 5-HT receptors are expressed on many
neurons that are not directly innervated by serotonergic neurons,
suggesting that most serotonergic signaling is humoral and extra-
synaptic [22,32,45]. Our data suggest that stimulation of aversive
responses by NSM 5-HT requires SER-5 and SER-1, and confirm
the role of the RIAs in NSM 5-HT stimulation . In contrast,
MOD-1 is not required, although it is essential for the sensitization
of aversive responses by food or exogenous 5-HT, suggesting that
additional sources of 5-HT may contribute to food-sensitization or
that the levels of 5-HT released from the NSMs in these
experiments overcome the need for MOD-1. The inhibition of
food-stimulation by ADF 5-HT requires only SER-1, not in the
RIAs but in the RICs. Presumably the Gaq-coupled SER-1
stimulates OA release from the RICs that, in turn, inhibits ASH
signaling through the Gao-coupled OCTR-1, as we have
demonstrated previously [12,46]. Together, these results demon-
strate that SER-1 is required for both ADF inhibition and
NSM stimulation, highlighting the complexity of serotonergic
Nutritional status modulates multiple aspects of the ASH-
mediated aversive response, including reversal duration and
trajectory after reversal is complete. For example, off food,
animals initiate reversal more slowly, back up further and are
more likely to turn from their initial trajectory, often initiating an
omega turn. These post-initiation responses are independent of the
intensity of the initiating stimulus (30 vs 100% 1-octanol),
suggesting that the decision to continue forward or change
direction after contact with an aversive stimulus is dictated largely
by nutritional state. The 5-HT receptors modulating the initiation
of reversal also modulate post-initiation behaviors, suggesting that
nutritional status is defined by an extrasynaptic ‘‘serotonergic
circuit.’’ The presence of food is perceived by a subset of sensory
neurons and modulates many sensory-mediated behaviors. For
example, the AWCs, ASKs, and ASIs are involved in locomotory
changes associated with removal from food . The AWCs are
required for turning and reversals , the ASKs and AWCs for
long reversals and omega turns, and the ASIs for dispersal during
starvation [7,16,37]. Nutritional status also modulates odor
conditioning in the AWCs and salt conditioning in the ASEs
[47–50]. Interestingly, the ablation of sensory input has no
apparent effect on reversal frequency on food, but dramatically
alters reversal/turning off food, i.e., off food animals with
compromised sensory input behave as if they are on food [6,17].
Since, tph-1 null animals on food behave as if they are off food, the
‘‘serotonergic circuit’’ identified in the present study may also
mediate other food-dependent locomotory transitions.
The present study has demonstrated that food availability is
translated by complex changes in both monoaminergic and
peptidergic signaling to modulate aversive responses mediated by
the ASHs and highlights the advantages of this model system for
dissecting nutritional modulation, as these same excitatory and
inhibitory food signals most certainly also modulate other
nutritionally-dependent behaviors, including attraction, feeding,
locomotion, and egg-laying. Given the advantages of the C. elegans
model, it should now be possible to fully dissect these nutritionally-
sensitive signaling pathways in the modulation of individual
neurons or circuits.
genes. Table includes all sense and antisense primers used for the
generation of full-length rescue, neurons specific rescue or
overexpression constructs, generated by PCR fusion .
Creation of rescue and overexpression trans-
knockdown transgenes. Table represents all sense and
antisense primers used for the generation of neuron specific/
selective RNAi constructs, using PCR fusion [25,27].
Creation of Neuron specific/selective RNAi
Strain List S1
and examined in present study. The list includes all rescue,
overexpressor and neuron specific/selective RNAi expressing
animals that were generated for examination in octanol avoidance
or post-initiation assays.
The strain list represents all strains made
We would like to thank Dr. Robert Steven for critical discussion of the
manuscript, Dr. S. Mitani at the National Bioresources Project and the
Caenorhabditis elegans Genetics Center for strains. tph-1(mg280)II
animals were kindly provided by Dr. Mark Alkema (UMass, Boston,
MA), ceh-2p::tph-1(+) and srh-142p::tph1(+) rescue animals from Dr. Cori
Bargmann (Rockefeller University, New York, NY, ). ins-1 over-
expressors were kindly provided by Dr. Yuichi Iino (The University of
Tokyo, Tokyo, Japan).
Conceived and designed the experiments: RK GH. Performed the
experiments: GH AK VH PS WJL AS. Analyzed the data: RK GH PK.
Wrote the paper: RK GH. Critical reading of the manuscript: VH PK.
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