AMP-Activated Kinase Links Serotonergic Signaling
to Glutamate Release for Regulation
of Feeding Behavior in C. elegans
Katherine A. Cunningham,1,2,3Zhaolin Hua,1,4Supriya Srinivasan,5Jason Liu,1,2,3Brian H. Lee,1,2,3Robert H. Edwards,1,4
and Kaveh Ashrafi1,2,3,*
1Department of Physiology
2Cardiovascular Research Institute
4Department of Neurology
University of California, San Francisco, San Francisco, CA 94158-2517, USA
5Department of Chemical Physiology and Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA 92037, USA
studied intensively, both for an understanding of the
basic neurocircuitry of energy balance in various
organisms and as a therapeutic target for human
obesity. However, its underlying molecular mecha-
nisms remain poorly understood. Here, we show
that neural serotonin signaling in C. elegans modu-
lates feeding behavior through inhibition of AMP-
activated kinase (AMPK) in interneurons expressing
the C. elegans counterpart of human SIM1, a tran-
scription factor associated with obesity. In turn, glu-
tamatergic signaling links these interneurons to
pharyngeal neurons implicated in feeding behavior.
We show that AMPK-mediated regulation of gluta-
matergic release is conserved in rat hippocampal
mediators of serotonergic signaling.
In C. elegans, as in mammals, serotonin (5-HT) signaling modu-
are reexposed to food, including foraging, mating, egg-laying,
and changes in metabolism and food-intake behavior (Avery
and Horvitz, 1990; Loer and Kenyon, 1993; Sze et al., 2000;
Waggoner et al., 1998; Srinivasan et al., 2008). Treatment of
food-deprived C. elegans with 5-HT elevates food-intake
behavior even in the absence of food (Horvitz et al., 1982),
whereas tryptophan hydroxylase (tph-1) mutants that lack
serotonin exhibit a reduced feeding rate even in the presence
serotonin signaling in mammals is thought to initiate satiety
(Tecott, 2007), the noted effects of serotonin on C. elegans
are consistent with a role in indicating food availability.
Food intake behavior in C. elegans is assessed by the rate
of pharyngeal pumping, given that increases or decreases in
pumping rate correspond to the amount of ingested nutrients
(Avery and Horvitz, 1990; Avery, 1993) and that pumping rate
is modulated by food availability, food quality, and the experi-
ence of food deprivation (Shtonda and Avery, 2006; You et al.,
2006; Avery and Horvitz, 1990). Here, we report on delineating
the cellular and molecular components of one serotonergic
circuit that links external cues to the modulation of food-intake
behavior. The identified circuit exhibits both molecular and
regulatory parallels to mammalian feeding regulatory circuits,
highlighting regulatory modules and the ancient origins of known
RESULTS AND DISCUSSION
Serotonin from Chemosensory ADF Neurons
Under well-fed, normoxic conditions, tph-1 is expressed in only
a few neurons, most notably NSM, located in the pharyngeal
nervous system; ADF, a chemosensory neuron in the head; and
HSN, a hermaphrodite-specific neuron required for a wild-type
egg-laying rate (Sze et al., 2000). As previously reported (Liang
et al., 2006; Figure S1A available online), we noted that tph-1
levels are reduced off food and are increased as animals return
to food. To determine the neural source of serotonin for the
wild-type feeding rate, we reconstituted tph-1 in only the pair of
ADF or only the pair of NSM neurons using an srh-142 (Sagasti
et al., 1999) or a ceh-2 promoter (Aspo ¨ck et al., 2003), respec-
tively. Reconstitution in the ADF but not in the NSM neurons
restored wild-type pumping rate to tph-1 mutants (Figure 1A).
Whereas animals with tph-1 reconstituted only in the ADF neu-
rons had fast movements, like those of tph-1-deficient animals,
NSM-specific reconstitution of tph-1 restored wild-type move-
ment behavior (Figure S1B). Thus, distinct sources of serotonin
production underlie food intake and movement behaviors.
We next implemented cell-specific RNA interference (RNAi)
to inactivate tph-1 in ADF neurons. As a gauge of RNAi efficacy,
a tph-1 promoter. Selective knockdown of tph-1 in ADF signifi-
cantly reduced pumping (Figures 1A and 1B). By contrast, a pre-
viously established line in which tph-1 is selectively inactivated
Cell Metabolism 16, 113–121, July 3, 2012 ª2012 Elsevier Inc. 113
in NSM (Harris et al., 2011) showed no significant changes in
rating our cell-specific reconstitution studies. However, Li et al.
recently reported that reconstitution of tph-1 in NSM but not
ADF neurons is sufficient to confer a wild-type pumping rate to
tph-1 mutants (Li et al., 2012). The reason for this obvious
discrepancy is not known. It is possible that, depending on the
signaling might be engaged to modulate pharyngeal pumping.
conditions, differentsources ofserotonin
SER-5 Is Required for Serotonergic Regulation
of Feeding from ADF
How serotonin release from ADF neurons modulates pharyngeal
pumping is not immediately apparent from the C. elegans
Figure 1. ADF Serotonin Is Necessary and Sufficient for Wild-Type Feeding
(A) Reconstitution of tph-1 in NSM and ADF by tph-1[Ptph-1::tph-1] or only in ADF by tph-1[Psrh-142::tph-1], but not in NSM tph-1[Pceh-2::tph-1], confers wild-
type pumping rates to tph-1 mutants.
(B) Cell-specific RNAi of tph-1 in ADF results in reduced pharyngeal pumping rates. Efficacy of ADF-specific tph-1 RNAi as monitored by TPH-1::GFP levels.
(E) Animals lacking ser-1, ser-7, or ser-5 fail to elevate pumping rate in the presence of 5-HT.
Data are normalized mean ± SEM. For (A) and (D), ANOVA with Bonferroni correction for multiple comparisons, for (C) and (E), Student’s t test. ***p < 0.001 versus
well-fed animals, unless otherwise indicated. WT, wild-type. See also Figure S1.
Serotonergic Feeding Circuit in C. elegans
114 Cell Metabolism 16, 113–121, July 3, 2012 ª2012 Elsevier Inc.
anatomy. Serotonin produced in ADF can be taken up via the
serotonin reuptake pump MOD-5 into RIH and AIM neurons,
which do not normally synthesize serotonin (Jafari et al., 2011).
Inhibition of MOD-5 by fluoxetine or loss of mod-5 caused
increased pumping in wild-type animals, but not in tph-1-defi-
cient animals (Figures 1C and 1D). Genetic or pharmacological
inactivation of MOD-5 in tph-1 mutants in which tph-1 was
selectively reconstituted in ADF neurons resulted in pumping
rates that were indistinguishable from those of similarly treated
wild-type animals (Figures 1C and 1D). Thus, 5-HT produced
within ADF neurons does not require mod-5-mediated reuptake
into other neurons for feeding regulation, and 5-HT production
in ADF using the srh-142 promoter does not flood the nervous
system with excessive levels of this neuromodulator.
To identify the signaling relays through which ADF-produced
5-HT regulates pumping rate, we first examined two G protein-
coupled receptors (GPCRs), encoded by ser-1 and ser-7, since
loss of either receptor abrogates the increased pharyngeal
pumping rate caused by exogenous 5-HT (Song and Avery,
2012; Carre-Pierrat et al., 2006; Hobson et al., 2006; Srinivasan
et al., 2008; Figure 1E). Loss of neither receptor altered the wild-
type pharyngeal pumping rate of animals in which serotonin
production was restored specifically to ADF neurons (Figure 2A).
We next examined ser-5, which also encodes a putative seroto-
nergic GPCR and plays roles in serotonin-dependent behavioral
processes (Hapiak et al., 2009; Harris et al., 2009; Kullyev et al.,
2010), but has not previously been implicated in feeding
behavior. As in ser-1 andser-7 mutants, ser-5 mutants displayed
wild-type pumping rates under well-fed conditions but failed to
elevate pumping in response to exogenous 5-HT (Figure 1E).
However, loss of ser-5 abrogated the pharyngeal pumping rate
of tph-1 mutants in which tph-1 was selectively reconstituted
in ADF (Figure 2A). Thus, when 5-HT production is limited to
the ADF neurons, the SER-5 receptor plays a nonredundant
function in pumping rate.
SER-5 Activity in SIM1/HLH-34-Expressing Neurons
Regulates Feeding Behavior
To begin dissecting the SER-5 feeding circuit, we generated
transgenic animals expressing a Pser-5::gfp reporter fusion
directed by 5 kb of putative ser-5 promoter sequence (Pser-5).
As previously reported (Carre-Pierrat et al., 2006; Hapiak et al.,
2009), we noted prominent expression in numerous neurons
andalso in thebody-wall muscle, althoughthe body-wall muscle
expression was quite faint in adults.
Next, we examined the roles of several C. elegans counter-
parts of human obesity and fat-regulatory genes in search of
additional gene inactivations that, similar to ser-5 mutants, block
the effects of 5-HT on pumping. This led to the identification of
hlh-34, encoding a basic helix-loop-helix transcription factor,
whose loss blocked the feeding-increasing effects of 5-HT
without altering the basal pumping rate (Figure 2B). hlh-34 is
the closest C. elegans counterpart of the D. melanogaster and
mammalian single-minded 1 (SIM1) genes. Mammalian SIM1 is
a transcription factor expressed in various brain regions and
required for development of the hypothalamic nucleus known
as the PVN (paraventricular nucleus; Michaud et al., 2001), the
site of expression of many of the signaling receptors that play
key roles in energy balance. To determine sites of function of
hlh-34, we generated a transgenic reporter consisting of 2.5 kb
upstream of the predicted hlh-34 start site, fused to mCherry
(Phlh-34::mCherry). This reporter was detected as prominent in
a single pair of interneurons in the head, but nowhere else
(Figures 2C and S2A–S2C). The anatomical characteristics of
the neurons expressing the Phlh-34::mCherry reporter corre-
sponded to either the AVJ or the AVH pair of interneurons,
which have nearly identical anatomical characteristics. To help
identify the hlh-34-expressing neurons and demonstrate that
they are distinct from neurons such as ASH, previously impli-
cated in ser-5-mediated functions (Harris et al., 2009; Harris
et al., 2011), or from the pharyngeal muscle, we crossed reporter
fusions driven by the ocr-2 (Tobin et al., 2002) and myo-2
promoters into the Phlh-34::mCherry line (Figures S2G–S2R).
To help distinguish between AVJ and AVH as the site of the
Phlh-34::mCherry reporter, we relied on the previously reported
expression of eat-4, encoding the vesicular glutamate receptor
(Lee et al., 1999), which is thought to be expressed in AVJ but
not AVH (Lee et al., 1999; http://wormweb.org/neuralnet). As
indicated below, glutamate signaling from the hlh-34 expressing
neurons is required for serotonergic feeding regulation, suggest-
ing that the hlh-34-expressing neurons are the AVJ pair.
We used the 2.5 kb hlh-34 promoter to drive expression of
human SIM1 in hlh-34 mutants. This transgene did not alter
basal pumping rates but conferred responsiveness to exoge-
nous 5-HT (Figure 2B), suggesting that SIM1 is, in fact, the func-
tional counterpart of hlh-34, a remarkable observation given that
C. elegans lacks an anatomically discernable hypothalamus. We
next investigated whether hlh-34-expressing neurons may be
the sitesof serotonin action on SER-5,given thatwesaw overlap
of Phlh-34::mCherry and Pser-5::ser-5::gfp in AVJ (Figure 2C).
Selective targeting of ser-5 to hlh-34-expressing neurons al-
lowed ser-5-deficient animals to once again respond to exoge-
nous 5-HT (Figure 2B). Targeting of the same ser-5 construct
to the pharyngeal muscle or pharyngeal neurons failed to confer
serotonin responsiveness (Figure 2B). To determine whether the
hlh-34-expressing neurons contribute to the pharyngeal pump-
ing rate beyond their role in 5-HT signaling, we genetically
ablated these neurons using the mammalian caspase, inter-
leukin-1-b converting enzyme (ICE). This resulted in a ?10%
reduction in basal pharyngeal pumping rate, as well as unre-
sponsiveness to exogenous 5-HT (Figure 2B). Thus, the AVJ
neurons are essential for both basal and 5-HT-mediated
increased pumping rates, even when all other components of
serotonin signaling are intact.
AMPK Activity in SIM1/HLH-34 Neurons Regulates
A second gene to emerge from our candidate screen was aak-2,
encoding a catalytic subunit of the AMP-activated kinase
(AMPK), a conserved regulator of energy balance (Hardie,
2007). As in mammals, the catalytic subunit of the C. elegans
AMPK is encoded by one of two distinct genes, aak-1 and aak-
2. aak-2 mutants exhibited elevated pumping even in the
absence of exogenous 5-HT (Figure 3A) that did not elevate
further with 5-HT (Figure 3B). This was not simply a reflection
of the upper limit of pumping capacity, as a similarly elevated
basal pumping rate of aak-1 mutants could be elevated further
by 5-HT (Figure S3A). Moreover, treatment with aminoimidazole
Serotonergic Feeding Circuit in C. elegans
Cell Metabolism 16, 113–121, July 3, 2012 ª2012 Elsevier Inc. 115
carboxamide ribonucleotide (AICAR), a pharmacological acti-
vator of AMPK, caused reduced pumping dependent on aak-2
(Figure S3A), highlighting the role of this catalytic subunit in the
regulation of pharyngeal pumping rate.
We next investigated the relationship between ser-5, hlh-34,
and aak-2 mutants. Pharyngeal pumping rates of ser-5;aak-2
double mutants were as elevated as those of aak-2 mutants
(Figure 3A), whereas loss of hlh-34 abrogated the increased
rate of aak-2 mutants (Figure 3A). aak-2 is expressed broadly
in C. elegans (Narbonne and Roy, 2009). To determine where
aak-2 is required to regulate pharyngeal pumping rate in
response to either AICAR or 5-HT, we reconstituted aak-2 within
specific tissues. aak-2 has two predicted isoforms, aak-2a and
aak-2c (WormBase release WS223). We first reconstituted
both isoforms within either the pharyngeal muscle or the nervous
system. Reconstitution within either of these tissues was
sufficient for wild-type pumping but insufficient for responsive-
ness to either 5-HT or AICAR. By contrast, simultaneous
Figure 2. Serotonin from ADF Requires ser-5 in AVJ to Elevate Pumping
(A) Wild-type pumping rate of tph-1 mutants in which tph-1 is reconstituted in ADF alone is dependent on ser-5 (ser-5;tph-1[Psrh-142::tph-1]), but not on ser-1
(tph-1;ser-1[Psrh-142::tph-1]) or ser-7 (tph-1;ser-7[Psrh-142::tph-1]).
(B) ser-5 and hlh-34 are required for elevation of pumping rate in response to 5 mM 5-HT. Reconstitution of ser-5 within hlh-34-expressing neurons, but not
pharyngeal neurons (Pglr-7::ser-5) or pharyngeal muscle (Pmyo-2::ser-5), confers responsiveness to 5-HT. Genetic ablation of hlh-34 neurons (Phlh-34::ICE)
abrogates responsiveness to 5-HT.
(C) Expression of Phlh-34::mcherry and Pser-5::ser-5::gfp overlap in hlh-34 neurons.
Data are normalized mean ±SEM. For (A), ANOVA with Bonferroni correction for multiple comparisons. For (B), Student’s t test. ***p < 0.001 versus well-fed
animals, unless otherwise indicated. For (A) and (B), 2.5 kb upstream of the hlh-34 start site was used to drive expression of mCherry. Images were taken with
a spectral confocal microscope using a Z-stack and compressed into a single file. WT, wild-type. See also Figure S2.
Serotonergic Feeding Circuit in C. elegans
116 Cell Metabolism 16, 113–121, July 3, 2012 ª2012 Elsevier Inc.
reconstitution within the pharyngeal muscle and the nervous
system restored responsiveness to both 5-HT and AICAR
the nervous system for responsiveness to 5-HT and AICAR, we
reconstituted the two aak-2 isoforms within hlh-34-expressing
cells, as well as pharyngeal muscle cells of aak-2-deficient
animals. These animals had wild-type pharyngeal pumping
rates and were responsive to 5-HT and AICAR (Figure 3B,
Figure S3B). By contrast, reconstitution within the two other
Figure 3. Serotonin Regulates AMPK via
(A) Pharyngeal pumping rates of indicated strains.
(B) Reconstitution of aak-2 in hlh-34-expressing
neurons (Phlh-34::aak-2) is sufficient for respon-
siveness to 5-HT. Reconstitution of aak-2 within
RIM and RIC interneurons (Ptdc-1::aak-2) or ADL
et al.,2005)is notsufficient. Alanine substitution at
AMPK Ser244 but not Ser553 blocks the pumping
effect of 5-HT. In all aak-2 reconstitution studies,
aak-2 is expressed in the pharyngeal muscle,
using the myo-2 promoter.
(C) Pharyngeal pumping rates of animals ex-
pressing the constitutively active gsa-1(R182C) in
For (A)–(C), data are normalized mean ± SEM.
ANOVA with Bonferroni correction for multiple
comparisons was used for statistical analysis.
***p < 0.001 versus well-fed animals, unless
otherwise indicated. WT, wild-type. See also
pairs of interneurons, RIM and RIC, pre-
viously implicated in feeding behavior
(Greer et al., 2008), or within a subset of
amphid neurons did not confer respon-
siveness to 5-HT, highlighting the role of
aak-2 within hlh-34-expressing neurons
Serotonin Regulates AMPK via
Protein Kinase A
SER-5 is predicted to be a Gascoupled
GPCR (Harris et al., 2009). To test the
prediction that enhanced cyclic AMP
production in hlh-34 neurons elevates
pumping, we selectively expressed gsa-
1(R182C), a dominant, gain-of-function
mutation of C. elegans Gas (Schade
et al., 2005), in hlh-34 neurons. This
led to an increased pumping rate (Fig-
ure 3C). In mammals, AMPK phosphory-
lation at Ser173 by protein kinase A,
PKA, antagonizes activating phosphory-
lation at Thr172 by the LKB1 kinase
(Djouder et al., 2010). To test this model,
we first examined wild-type animals in
which adenylate cyclase was inactivated
by acy-1 RNAi and found that these
animals failed to elevate pumping rates
in response to 5-HT (Figure S3D). We then substituted analanine
residue at AMPK(S244), the CeAAK-2-residue equivalent of
mammalian Ser173,toprevent PKA-dependent phosphorylation
at this site (Djouder et al., 2010). We reconstituted wild-type iso-
forms of aak-2 within the pharyngeal muscle of aak-2-deficient
animals and the (S244A) versions of AAK-2 in hlh-34-expressing
neurons. These transgenic animals were unresponsive to 5-HT
(Figure 3B), suggesting that PKA-dependent phosphorylation
at this site is required to inactivate AAK-2. As a control, we
Serotonergic Feeding Circuit in C. elegans
Cell Metabolism 16, 113–121, July 3, 2012 ª2012 Elsevier Inc. 117
showedthatchanginganotherPKA phosphorylation siteofAAK-
abrogate responsiveness to 5-HT. Combining the constitutive
allele of gsa-1 with the aak-2(S244A) allele in hlh-34 neurons re-
sulted in animals with essentially wild-type pumping rates (Fig-
ure 3C), indicating that the aak-2 and GSA-1 are probably acting
in the same pathway. Together, these results suggest a model in
which serotonin signaling initiated from ADF neurons acts
directly on SER-5 receptors in hlh-34-expressing neurons to
inactivate AAK-2 via inhibitory phosphorylation by PKA at AAK-
Glutamate Signaling from SIM1/HLH-34 Neurons
Modulates Pumping Rate
To investigate how loss of aak-2 promotes pumping, we took
advantage of the genes previously identified in an RNAi-based
Nile Red screen, a subset of which also shows reduced
pharyngeal pumping (Figure 4A). In most cases, treatment of
aak-2 mutants with these pharyngeal pumping-reducing RNAis
resulted in an intermediate rate, suggesting parallel mecha-
nisms. One exception was glr-7 (C43H6.9), which, when
inactivated, lowered the pumping rate of aak-2 mutants and
wild-type mutants to the same level (Figure 4A). We verified
these RNAi results by examining glr-7;aak-2 double mutants
Given that glr-7 encodes for a non-NMDA-type ionotropic
glutamate receptor expressed in the I1, I2, I3, M1, and NSM
pairs of pharyngeal neurons (Brockie et al., 2001), we asked
whether glutamate signaling could link hlh-34- and glr-7-
expressing sets of neurons. We found that animals deficient in
eat-4, encoding the C. elegans ortholog of the mammalian
BNPI vesicular glutamate transporter (Lee et al., 1999), failed
to elevate their pumping rates on 5-HT, and eat-4;aak-2 double
mutants exhibited the same rates of pumping as eat-4 single
mutants (Figure 4B). However, since the basal pharyngeal
pumping rate of eat-4 mutants is ?25% lower than that of
wild-type, we sought to distinguish between a specific require-
ment for eat-4 within the serotonergic circuit and a general
requirement in pharyngeal pumping. Reconstitution of eat-4
only in the hlh-34-expressing cells of otherwise eat-4-deficient
animals did not alter the reduced basal rate of these animals,
but conferred responsiveness to 5-HT (Figure 4C), revealing
a role for glutamate signaling from hlh-34 neurons in 5-HT-
mediated feeding regulation.
Effect of AMPK on Glutamatergic Release Is Conserved
The mechanisms through which inhibition of AMPK leads to
glutamatergic release from hlh-34 neurons is not currently
known. Nevertheless, we next asked whether a similar regu-
latory relationship may be conserved in mammals. The avail-
ability of experimental reagents prompted us to examine rat
hippocampal neurons, which have been used extensively for
studying glutamatergic synaptic-vesicle recycling (Miesenbo ¨ck
et al., 1998; Voglmaier et al., 2006; Granseth et al., 2006).
VGLUT1 is the vesicular glutamate transporter and is present
in the synaptic vesicles in the majority of excitatory synapses
in the hippocampus. pHluorin, a pH-sensitive form of green
fluorescent protein (GFP), exhibits essentially complete fluo-
rescence quenching at the low pH inside synaptic vesicles
(Miesenbo ¨ck et al., 1998; Sankaranarayanan et al., 2000). The
fluorescence of pHluorin increases with exocytosis and decays
with endocytosis, and alkalinization of the nerve terminal with
a permeant weak base (such as NH4Cl) reveals the total pool
of fluorescent protein. Hippocampal neurons transfected with
VGLUT1-pHluorin were subjected to electric-field stimulation
to elicit synaptic-vesicle exocytosis. To determine the kinetics
and extent of exocytosis without interference from concurrent
endocytosis, bafilomycin was added during the stimulation to
prevent the reacidfication of the newly formed synaptic vesi-
cles. To ask if inhibition of AMPK leads to increased exocytosis,
we added Compound C, an AMPK inhibitor, and measured the
kinetics of exocytosis. The rate of exocytosis was significantly
accelerated in the presence of Compound C (Figures 4D and
4E), without a change in the relative recycling-pool size (Fig-
ure S4D). Endocytosis after synaptic-vesicle exocytosis was
normal, if not faster, in the presence of Compound C (Fig-
ure S4C), indicating that faster exocytosis does not necessarily
result in faster depletion of the recycling pool. Taken together,
these data suggest that inhibition of AMPK can increase the
efficiency of glutamate vesicle release at the synapse.
However, Yang and colleagues recently reported that in a state
of food deprivation, AMPK is activated in the arcuate-nucleus
region of the hypothalamus, resulting in glutamate release
(Yang et al., 2011). Thus, the relationship between AMPK
activity and glutamate release is likely to be dependent on
the specific cell types in which they are studied.
In summary, our findings reveal a circuit that links changes in
environmental cues through two layers of interneurons to the
pharyngeal muscle, the feeding organ of C. elegans. The most
salient feature of our study is identification of common regula-
tory themes in C. elegans and mammalian feeding circuits. In
mammals, anorexic and orexigenic signals regulate feeding
behavior, in part, through modulation of AMPK in the nervous
system (Minokoshi et al., 2004; Seo et al., 2008). Moreover,
the PVN region of the hypothalamus has been identified as
one of the sites in which peripheral signals of food availability
modulate AMPK activity (Blanco Martı ´nez de Morentin et al.,
2011). Here, we found that serotonin, an indicator of food
availability, regulates food-intake behavior through inhibition of
AMPK in neurons that are the site of action of the C. elegans
counterpart of human SIM1. Thus, some of the regulatory
features found in the mammalian hypothalamus predate devel-
opment of this brain region. Finally, we speculate that the link
between serotonin and AMPK is likely to be a common regula-
tory feature in a variety of different cell types in a broad range
of physiological outcomes.
Pharyngeal Pumping Rate Assays
Gravid adults were synchronized using hypochlorite treatment and plated as
L1s. Animals were grown at 20?C, and feeding rate was assayed on gravid
adults. Pharyngeal pumping rates were measured by counting the contraction
of the pharyngeal bulb over a 10 s period using a Zeiss M2-Bio microscope.
For AICAR treatment, animals were picked into liquid media containing
OP50 bacteria and either vehicle (dH20) or drug (2 mM AICAR) and were left
at 20?C for 2 hr. After treatment, animals were returned to nematode growth
media (NGM) plates containing food, and pharyngeal pumping rates were
Serotonergic Feeding Circuit in C. elegans
118 Cell Metabolism 16, 113–121, July 3, 2012 ª2012 Elsevier Inc.
assayed after 15 min. For all measurements, ten animals were counted per
condition for each genotype. Wild-type pumping rates were normalized to
100, and data are presented as the percentage of wild-type treated with
vehicle. Each experiment was repeated at least twice. Error bars represent ±
SEM for all graphs. At least three independent lines were tested for all trans-
Enhanced Slowing Response Locomotion Assay
This assay was performed according to Sawin et al. (2000). In brief, well-fed,
day 1 adult animals were washed twice in S-basal and placed on an unseeded
NGM plate.After5min,bodybends per20swerecountedfortenanimals.The
animals were left on the plate for 30 min before removal to a fresh NGM plate
seeded with HB101 E. coli. After 5 min, body bends of ten animals were
Figure 4. AMPK Links Serotonin to Glutamatergic Signaling in hlh-34 Neurons
(A) Pumping rates of RNAi treated wild-type and aak-2 mutants. Data are normalized mean +/? SEM. Student’s t test was used for statistical analysis. *p < 0.05,
**p < 0.01 versus vector treated animals.
(B) Pumping rates of eat-4;aak-2 relative to eat-4 mutants. Data are normalized mean ± SEM. ANOVA with Bonferroni correction for multiple comparisons was
used for statistical analysis. **p < 0.01.
(C) Reconstitution of eat-4 only in hlh-34-expressing neurons confers 5-HT responsiveness. Data are normalized mean ± SEM. Student’s t test was used for
statistical analysis. ***p < 0.001.
(D) Synaptic-vesicle exocytosis is accelerated in the presence of Compound C.
(E) The time constant for exocytosis (texo) is significantly faster in the presence of Compound C. p = 0.00154; n = 8. 5 coverslips were used containing a total of
300 boutons for each group.
WT, wild-type. See also Figure S4.
Serotonergic Feeding Circuit in C. elegans
Cell Metabolism 16, 113–121, July 3, 2012 ª2012 Elsevier Inc. 119
counted. HB101 plates were incubated at 37?C overnight and cooled to room
temperature prior to the start of the assay.
Hippocampal-Neuron Cell Culture and Molecular Biology
Hippocampal neurons isolated from day 20 rat embryos were transfected with
electroporation (Amaxa) and cultured as previously described (Li et al., 2005).
VGLUT1-pHluorin (Voglmaier et al., 2006) was inserted upstream of an
internal ribosome entry sequence (IRES2, Clontech), driving the translation
Transfected neurons were imaged at 12–14 days in vitro as previously
described (Voglmaier et al., 2006). pHluorin was imaged with 492/18 nm exci-
tation and 535/30 nm emission filters. mCherry was imaged with 580/20 nm
excitation and 630/60 nm emission filters. Images were collected every 3 s.
Neurons were imaged in standard Tyrode’s solution (119 mM NaCl, 2.5 mM
KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose, and 25 mM HEPES; pH
7.4). NH4Cl buffer (69 mM NaCl, 2.5 mM KCl, 2 mM MgCl2, 2 mM CaCl2,
50 mM NH4Cl, 30 mM glucose, and 25 mM HEPES; pH 7.4) was used to reveal
total pHluorin fluorescence. Glutamate-receptor antagonists 6-cyano-7
nitroquinoxaline-2,3-dione (CNQX) (10 mM) and D,L-2-amino-5-phosphonova-
leric acid (APV) (50 mM) were included in the Tyrode’s solution during the
experiments. Bafilomycin (0.6 mM) was diluted from 0.6 mM stock solutions
in dimethyl sulfoxide, and Compound C (20 mM) was preincubated with
neurons at 37?C for 2 hr.
Hippocampal-Neuron Data Analysis
Regions enclosing the entire synaptic bouton were selected using mCherry-
synaptophysin. Arbitrary fluorescence units were normalized to the total
intracellular fluorescence (in NH4Cl), which was determined as FNH4Cl?Finitial.
To determine the kinetics of exo- and endocytosis with 10 Hz stimulation,
the change in fluorescence was normalized to the maximum change in
fluorescence during stimulation (Fpoststim?Fprestim). Endocytosis kinetics
were fit to a single-exponential decay (F = Fplateau+ Fspan3 e?kt). Exocytosis
kinetics were fit to a single-exponential (F = Fmax3 (1?e?kt)). Data indicate
mean ± SEM, and nested analysis of variance (ANOVA) was used to compare
Neuron-specific RNAi transgenes were constructed by fusing neuron-selec-
tive promoters to the 50and 30genomic regions of target genes. Promoter
regions were fused to both the 50and 30ends of the genomic region, with
the 30promoter in the 30-50orientation to create a 50-promoter-30-50-genomic
region-30-30-promoter-50fused PCR product. PCR products were pooled and
injected into the gonads of animals with a coinjection marker.
Student’st testwasused forpairwise comparisons.Formultiple comparisons,
Supplemental Information includes four figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.doi.
K.A.C. was supported by a postdoctoral grant from the National Institutes of
Health (NIH) (HL007731). Additional support was received from the Burroughs
WellcomeFund, to K.A., and the National Institute of Mental Health (MH50712)
and the National Institute on Drug Abuse (DA10154), to R.H.E. Confocal-
microscopy images were acquired at the UCSF Nikon Imaging Center. We
thank the Caenorhabditis Genetics Center, which is funded by the NIH
National Center for Research Resources (NCRR), for nematode strains. We
thank the Komuniecki Lab (University of Toledo) for sharing their Pser-
5::ser-5::gfp and NSM-specific tph-1 RNAi strains, and Mee J. Kim and the
Ahituv Lab (UCSF) for the human SIM1 in pENTR223.1. We thank S. Mitani
and the C. elegans Gene Knockout Consortium for deletion alleles, and
members of the Ashrafi laboratory for helpful discussions.
Received: November 3, 2011
Revised: March 8, 2012
Accepted: May 29, 2012
Published online: July 2, 2012
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