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The Journal of Neuroscience, August 15, 2001, 21(16):5871-5884
Mutations in the Caenorhabditis elegans Serotonin
Reuptake Transporter MOD-5 Reveal Serotonin-Dependent and -Independent
Activities of Fluoxetine
Rajesh
Ranganathan,
Elizabeth R.
Sawin,
Carol
Trent, and
H.
Robert
Horvitz
Howard Hughes Medical Institute, Department of Biology,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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ABSTRACT |
We isolated two mutants defective in the uptake of exogenous
serotonin (5-HT) into the neurosecretory motor neurons of
Caenorhabditis elegans. These mutants were
hypersensitive to exogenous 5-HT and hyper-responsive in the
experience-dependent enhanced slowing response to food modulated by
5-HT. The two allelic mutations defined the gene mod-5
(modulation of locomotion defective), which encodes the only serotonin
reuptake transporter (SERT) in C. elegans. The selective
serotonin reuptake inhibitor fluoxetine (Prozac) potentiated the
enhanced slowing response, and this potentiation required
mod-5 function, establishing a 5-HT- and SERT-dependent behavioral effect of fluoxetine in C. elegans. By
contrast, other responses of C. elegans to fluoxetine
were independent of MOD-5 SERT and 5-HT. Further analysis of the
MOD-5-independent behavioral effects of fluoxetine could lead to the
identification of novel targets of fluoxetine and could facilitate the
development of more specific human pharmaceuticals.
Key words:
C. elegans; SERT; fluoxetine; serotonin; reuptake; modulation of behavior; SSRI
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INTRODUCTION |
The activity of serotonin (5-HT), a
key neuromodulator, is mediated postsynaptically through metabotropic
(Martin et al., 1998
) and ionotropic (Maricq et al., 1991; Ranganathan
et al., 2000) 5-HT receptors and their downstream signaling components (Hille, 1992). 5-HT modulates several behaviors of the nematode Caenorhabditis elegans, including egg laying and locomotion
(Horvitz et al., 1982; Trent et al., 1983; Avery and Horvitz, 1990;
Schafer and Kenyon, 1995). 5-HT also mediates the enhanced slowing
response exhibited by food-deprived nematodes after they encounter
bacteria (Sawin et al., 2000). 5-HT neurotransmission can be regulated by the removal of 5-HT from the synaptic cleft by a serotonin reuptake
transporter (SERT; Cooper et al., 1996).
Na+/Cl
-dependent
SERTs were cloned first from rats (Blakely et al., 1991; Hoffman et
al., 1991) and subsequently from other species (Mortensen et al., 1999,
and references therein), including humans and Drosophila
melanogaster. SERT antagonists, such as the selective serotonin
reuptake inhibitors (SSRIs) fluoxetine (Prozac), paroxetine (Paxil),
and sertraline (Zoloft), are broadly used in the treatment of
psychiatric disorders (Schloss and Williams, 1998). Therefore, in
addition to the basic question of SERT function and regulation, it is
of particular clinical importance to understand SERT function in
vivo. SERT-deficient mice do not show gross developmental defects but have reduced 5-HT levels in the brain (Bengel et al., 1998), are
insensitive to 3,4-methylenedioxymethamphetamine (ecstasy)-induced hyperactivity (Bengel et al., 1998), and show a brain region- and
gender-specific reduction in the density and expression of 5-HT1a receptors (Li et al., 2000). However, no
obvious behavioral defects or abnormal responses to SSRIs in these
SERT-deficient mice have been reported.
In this article, we report the isolation of two C. elegans SERT-deficient mutants and describe studies of these
mutants revealing that the C. elegans SERT is required for
the experience-dependent enhanced slowing response and that the SSRI
fluoxetine can act on both 5-HT- and SERT-dependent and -independent targets.
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MATERIALS AND METHODS |
mod-5 mapping, cloning, and cDNA. Nematodes
were grown at 20°C as described previously (Brenner, 1974), except
that Escherichia coli strain HB101 rather than OP50 was used
as the food source (Sawin et al., 2000). Wild-type animals were
C. elegans strain N2. mod-5(n822) and
mod-5(n823) were isolated from a genetic screen in which
clonal populations of F3 animals descended from
P0 animals mutagenized with ethyl
methanesulfonate (Brenner, 1974) were pretreated with 5-HT (15 min
incubation in 500 µl of 13 mM 5-HT followed by
two washes with M9) (Wood et al., 1988) and then examined for the presence of formaldehyde-induced fluorescence (FIF) (Sulston et
al., 1975) in the neurosecretory motor neurons (NSMs).
mod-5(n3314) was isolated from a library of animals
mutagenized with UV illumination and trimethylpsoralen (Jansen et al.,
1997). The deletion library was constructed essentially as described
previously (Jansen et al., 1997; Liu et al., 1999; P. Reddien, R. Ranganathan, and H. R. Horvitz, unpublished results).
mod-5(n3314) was outcrossed to the wild type six times
before behavioral assays. mod-5(n823) was mapped to linkage
group I (LG I) on the basis of two-factor linkage to dpy-5
unc-75 I. The following three-factor data were obtained:
mod-5 (47/47) dpy-5 (0/47)
unc-75, mod-5 (35/35) unc-73 (0/35)
lin-44 dpy-5, lin-6 (27/27) lin-17
(0/27) mod-5, lin-17 (13/13) fog-1
(0/13) mod-5, and fog-1 (3/26) mod-5
(23/26) unc-11. All mapping experiments were performed by
mating hermaphrodites homozygous for the recombinant chromosome with
mod-5(n823) males and scoring the F1 cross progeny for 5-HT
hypersensitivity at 5 min in 10 mM 5-HT. Germ
line transformation experiments (Mello et al., 1991) were performed by
injecting various constructs with 80 µg/ml pl15EK (which contains the
wild-type lin-15 gene) into a mod-5(n823);
lin-15(n765ts) strain and scoring 5-HT sensitivity in transgenic
lines that produced non-Lin progeny at 22.5°C. Long-range PCR was
performed using the Advantage cDNA PCR kit (Clontech, Cambridge, UK).
DNA sequences were determined using an automated ABI 373A DNA sequencer
(Applied Biosystems, Foster City, CA). RT-PCR was performed with
primers corresponding to exons predicted by Genefinder (C.
elegans Sequencing Consortium, 1998). The 5' and 3' ends of the
mod-5 cDNA were determined using 5'- and 3'-rapid amplification of cDNA ends (RACE) kits (Life Technologies,
Gaithersburg, MD), respectively. To construct the mod-5
minigene, we used PCR and primers that contained restriction enzyme
sites at their ends to amplify 2.7 kb of the mod-5 promoter
region. A PstI-BamHI fragment of this PCR
product was ligated into the pPD49.26 vector (A. Fire, Carnegie
Institute of Washington, Baltimore, MD) digested with PstI and BamHI. This mod-5 promoter
construct was then digested with NcoI and SacI
and ligated to an NcoI-SacI fragment of the mod-5 coding region that was PCR-amplified in a manner
similar to that used for the mod-5 promoter region.
Laser microsurgery. Neurons were ablated during the second
larval stage using a laser microbeam, as described previously (Avery and Horvitz, 1987; Bargmann and Horvitz, 1991). Behavioral assays of
young adult animals were performed 2 d later. Mock-ablated animals
were animals transferred to agar pads and anesthetized in parallel to
the animals that underwent laser ablation. Sawin et al. (2000)
described details concerning how ablated animals were assayed
sequentially in each of the different behavioral conditions.
Neurotransmitter and drug pretreatment and behavioral
assays. The locomotory rate was assayed and 5-HT-containing plates
were prepared as described previously (Sawin et al., 2000). Fluoxetine (HCl salt; Sigma, St. Louis, MO) was dissolved in water, and 400 µl
of a 25× stock solution were added to each 5 cm plate, containing ~10 ml of agar, to obtain the various final concentrations of fluoxetine. The plates were allowed to dry at room temperature with
their lids removed for >2 hr.
To assay 5-HT hypersensitivity, we placed 20 animals in 200 µl of
5-HT solution (creatinine sulfate salt; Sigma; dissolved in M9 buffer;
Wood et al., 1988) in 96-well microtiter wells and scored the swimming
behavior of the animals as either active or immobile at 5 min; an
animal was scored as immobile if it did not exhibit any swimming motion
for 5 sec. Fluoxetine-induced paralysis was scored in a similar manner
at 10 min.
Egg-laying assays were performed as described previously (Trent et al.,
1983). Briefly, 1-d-old adult animals (staged by picking late fourth
larval stage animals 36 hr before the assay) were placed in
wells of microtiter dishes containing 100 µl of 12.5 mM
5-HT or 500 µg/ml fluoxetine, and the number of eggs laid was counted
after 90 min.
5-HT-uptake assays in vivo. FIF assays were performed as
described previously (Sulston et al., 1975). For the anti-5-HT antisera experiments shown in Figure 1B, mod-5(n823);
cat-4 and cat-4; lin-15(n765ts) double mutants were
grown at 20°C, and animals of both genotypes were incubated
separately on plates containing 2 mM 5-HT and
bacteria (for details concerning how plates were prepared, see Sawin et
al., 2000) for 2 h and then incubated on plates with bacteria but
without 5-HT for 30 min. Controls without exogenous 5-HT were similarly
treated in parallel. Before fixation, mod-5(n823); cat-4 and
cat-4; lin-15 double mutants preincubated on 5-HT-containing
plates were combined, and mod-5(n823); cat-4 and
cat-4; lin-15 double mutants preincubated on control plates were combined. 5-HT staining was performed as described previously (Desai et al., 1988) using affinity-purified rabbit polyclonal anti-5-HT antisera (H. Steinbusch, Maastrict University, Maastrict, the
Netherlands). The cat-4; lin-15 mutants were not defective in the uptake of 5-HT (data not shown) and served as internal controls
for each staining reaction. These animals could be distinguished from
the test animals by the Multivulva phenotype caused by
lin-15. Neurons with bright staining in cell bodies, axonal
processes, and varicosities were termed "bright," and neurons with
weak staining in just the cell bodies and axonal processes were termed
"weak." For the results in Table 1, the procedure was essentially
the same, except that the animals experienced an additional 1 hr
incubation on control or fluoxetine-containing plates before the 2 hr
incubation with 5-HT but did not experience the 30 min incubation on
plates without drug after the 5-HT preincubation (for details, see
Table 1 legend). lin-15 adult animals grown at 22.5°C were
added to all plates at the first incubation step, and these animals
served as internal controls for the staining reaction.
MOD-5-mediated uptake in mammalian cells. We obtained a
modified version of the MSCVpac vector (Hawley et al., 1994) in which the pac gene had been replaced with the gfp gene
[green fluorescent protein (GFP) vector; a generous gift from J. Schwartz, Massachusetts Institute of Technology, Cambridge,
MA]. We further modified the GFP vector as follows. The ends of
a BglII-Mfe I fragment containing the entire
mod-5 cDNA were blunted using the Klenow fragment of DNA
polymerase I and then ligated to the GFP vector digested with HpaI, placing mod-5 under the control of the
retroviral long terminal repeat promoter (GFPMOD-5). The Phoenix
packaging cell line (American Type Culture Collection, Manassas, VA)
was used to generate a virus containing either GFPMOD-5 or GFP vector.
Human embryonic kidney 293 (HEK293) cells were infected with these
viral stocks in the presence of 4 mg/ml polybrene, and clones
expressing high levels of GFP were isolated using a
fluorescence-activated cell sorter (FACstar or FACSvantage; Becton
Dickinson, Mountain View, CA). The GFPMOD-5 clones were then screened
for MOD-5 C. elegans SERT (CeSERT)-mediated
[3H]5-HT uptake activity, and one clone
was chosen for use in all further uptake experiments. Cells were plated
at 106 cells per well of a six-well dish
and allowed to grow overnight before being assayed. Cells were
incubated in prewarmed wash buffer (120 mM NaCl,
10 mM HEPES, pH 7.4, 4.7 mM
KCl, 2.2 mM CaCl2, 1.2 mM
KH2PO4, 1.2 mM MgSO4, 1.8 mg/ml
glucose, 100 µM pargyline, and 100 µM ascorbic acid) for 10 min at 37°C, and the
buffer was then replaced with prewarmed wash buffer plus substrate. In
Figure 4B, the NaCl was substituted with an
equivalent amount of sodium gluconate or choline chloride. Except for
the trials shown in Figure 4, C and D, 50 nM [3H]5-HT
(specific activity, 24 Ci/mmol; New England Nuclear, Boston, MA) was
used as a substrate (there was no dilution with nonradioactive substrate in these experiments). For the trials shown in Figure 4,
C and D, radiolabeled substrates were diluted
with nonradioactive substrate to maintain a specific activity of 0.1 Ci/mmol. Uptake was allowed to proceed at 37°C for varying times for
the time course and for 10 min in all other experiments. Cells were
then washed three times with ice-cold wash buffer and solubilized in 1% SDS, and the radioactivity retained in the cells was determined by
liquid scintillation. Cell numbers, quantified in parallel wells taken
through all steps of the assay, were used to convert counts per minute
to nmoles per cell per minute. The specific uptake of each substrate
for each condition was obtained by subtracting the average value
obtained from at least three trials with the GFP vector cell line from
the average value obtained from at least six trials with the GFPMOD-5
cell line. Inhibitor Ki values were determined from concentration-versus-uptake profiles after adjustment for substrate concentrations (Cheng and Prusoff, 1973). Statistical significance was evaluated using Student's t test (Statview).
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RESULTS |
Isolation of mutants defective in 5-HT uptake
FIF histochemistry indicates that the C. elegans NSMs,
located in the pharynx, contain 5-HT in their cell bodies and axonal processes (Horvitz et al., 1982). FIF in the NSMs was more readily observed when animals were preincubated with exogenous 5-HT before the
staining protocol (Horvitz et al., 1982; our unpublished results). This
observation suggested that the NSMs possess an active uptake system
that can concentrate 5-HT from the extracellular environment.
We performed a genetic screen for mutants lacking FIF in the NSMs after
preincubation with exogenous 5-HT (see Materials and Methods). Two
mutations that failed to complement each other, n822 and
n823, were isolated. n822 and n823
mutants lacked FIF in the NSM cell bodies after 5-HT preincubation but
retained FIF in the NSM axonal processes (Fig.
1A). Nomarski optics
(Ellis and Horvitz, 1986) revealed that these mutants had NSM cell
bodies in their usual positions (data not shown).
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Figure 1.
The NSMs of mod-5 mutants
are defective in 5-HT uptake. A, Wild-type and
mod-5(n822) animals preincubated with exogenous 5-HT
were stained using FIF to visualize 5-HT (Sulston et al., 1975).
Arrow, NSM cell body; arrowheads, NSM
axonal processes. No NSM cell bodies were FIF-positive in
mod-5(n822) mutants. The fluorescence seen in the axonal
processes may be the consequence of partial 5-HT reuptake activity (see
B). B, NSMs in mod-5(n822)
and mod-5(n823) mutants show reduced uptake of exogenous
5-HT when assayed using anti-5-HT antibodies. The mutation
cat-4(e1141) was included to reduce the levels of
endogenous 5-HT, which would otherwise obscure the detection of 5-HT
uptake. At least 100 animals were tested for each genotype in each
condition. For details, see Materials and Methods. Error bars indicate
SEM.
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5-HT can also be detected in C. elegans using anti-5-HT
antisera, which have proven to be more sensitive than FIF and have allowed the reliable detection of endogenous 5-HT in the NSMs and other
neurons without requiring preincubation with exogenous 5-HT (Desai et
al., 1988; Loer and Kenyon, 1993; Sawin et al., 2000). We used
anti-5-HT antisera to evaluate 5-HT reuptake in n822 and
n823 mutants. Because endogenous 5-HT masks 5-HT reuptake in
NSMs visualized using anti-5-HT antisera, we used a
cat-4(e1141) (catecholamine-defective) genetic background to
reduce endogenous 5-HT levels (Desai et al., 1988; Weinshenker et al.,
1995). cat-4 encodes GTP cyclohydrolase I (C. Loer, personal
communication), which is required for the synthesis of a
biopterin cofactor needed for dopamine and 5-HT biosynthesis (Kapatos
et al., 1999).
We counted the number of immunoreactive NSMs in cat-4 single
and n822; cat-4 and n823; cat-4 double mutants
with or without exogenous 5-HT preincubation. Although NSMs in the
cat-4 mutants were capable of 5-HT uptake, the NSMs in the
n822; cat-4 and n823; cat-4 double mutants were
partially defective in 5-HT uptake (Fig. 1B),
confirming our FIF observations. For example, we observed no brightly
fluorescing NSMs in n823; cat-4 mutants preincubated with
5-HT, whereas 43% of the NSMs in cat-4 mutants had bright immunofluorescence (Fig. 1B, black bars).
That a slight increase in NSM immunoreactivity was observed in
n822; cat-4 and n823; cat-4 mutants after 5-HT
preincubation suggests that the n822 and n823
mutations do not lead to a complete loss of 5-HT uptake activity.
Although previous studies did not detect anti-5-HT immunoreactivity in
cat-4 mutants (Desai et al., 1988; Loer and Kenyon, 1993;
Weinshenker et al., 1995), we observed, using the same staining
protocol as in the previous studies, a small percentage of weakly
anti-5-HT-immunoreactive NSMs in cat-4 mutants in the
absence of 5-HT preincubation (Fig. 1B). This
staining is 5-HT, because this antibody did not result in any
immunoreactivity in tph-1 mutants (see below), which lack the 5-HT biosynthetic enzyme tryptophan hydroxylase and are
specifically deficient in 5-HT (Sze et al., 2000). Our data suggest
that the cat-4 mutation does not lead to a complete loss of
5-HT, consistent with the conclusions of Desai et al. (1988) and Avery
and Horvitz (1990).
5-HT-uptake mutants exhibit a hyperenhanced slowing response
Because n822 and n823 mutants were defective
in 5-HT uptake, we sought to determine whether these mutants were
abnormal in their responses to endogenous 5-HT release. We tested the
5-HT-dependent enhanced slowing response (Sawin et al., 2000) of these
mutants. Whereas well-fed wild-type animals slow their locomotory rate slightly in response to bacteria (the basal slowing response), food-deprived wild-type animals display a greater degree of slowing of
locomotory rate in response to bacteria (the enhanced slowing response;
Sawin et al., 2000) (Fig.
2A). Strikingly,
n822 and n823 mutants exhibited a hyperenhanced
slowing response: on Petri plates with bacteria, the locomotory rates
of food-deprived n822 and n823 mutants slowed
significantly more than did those of food-deprived wild-type animals
(Fig. 2A, gray bars). On Petri plates
without bacteria, the locomotory rates of food-deprived n822
and n823 mutants were not significantly different from those
of food-deprived wild-type animals (Fig. 2A,
gray bars). Well-fed n822 and n823 mutants exhibited no defect in the 5-HT-independent dopamine-dependent basal slowing response to bacteria (Fig. 2A,
black bars) (Sawin et al., 2000). Genes involved in the
enhanced slowing response are called mod (modulation of
locomotion defective; Sawin et al., 2000), and we named the gene
defined by the allelic mutations n822 and n823
mod-5. The hyperenhanced slowing responses of these mod-5 mutants were presumably a consequence of a defect in
the clearing of 5-HT from the relevant synapses by uptake into
serotonergic neurons, thereby leading to increased 5-HT signaling and a
greater inhibition of locomotion.
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Figure 2.
Phenotypic characterization of
mod-5. A, mod-5 mutants
exhibited a hyperenhanced slowing response. Well-fed (black
bars) and food-deprived (gray bars)
animals were transferred to assay plates with or without a bacterial
lawn, and the locomotory rate of each animal was recorded after 5 min;
food-deprived animals were transferred to plates without bacteria 30 min before the transfer to locomotory assay plates (for details, see
Sawin et al., 2000). At least 10 trials were performed for each
genotype for each condition. For this and all subsequent panels, each
trial involved testing at least five animals for each of the
conditions; a given animal was tested in only one condition.
p values were calculated by comparing the combined data
for the mutants from all of the separate trials under one set of
conditions to the combined data for the wild-type animals assayed in
parallel under the same conditions. B, The hyperenhanced
slowing response exhibited by mod-5(n823) mutants was
suppressed by ablation of the NSMs. Number of animals tested: three of
each ablation state when well-fed; seven mock-ablated, food-deprived;
and 12 NSM-ablated, food-deprived. C, A decrease in
endogenous 5-HT partially suppressed the mod-5
phenotype. mod-5(n823); cat-4 double mutants displayed
an enhanced slowing response intermediate to that of
mod-5(n823) (see A) and
cat-4 mutants. D, mod-5
mutants were hypersensitive to exogenous 5-HT. 5-HT
dose-response curves for wild-type and mod-5 mutant
animals were generated from averages of five trials with 20 animals of
each genotype at each concentration in which animals were scored for
movement after 5 min. Error bars indicate SEM. ***p < 0.0001, Student's t test.
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Ablation of the serotonergic NSMs or decrease in endogenous 5-HT
suppresses mod-5 mutations
Ablation of the serotonergic NSMs with a laser microbeam leads to
a defect in the enhanced slowing response (Sawin et al., 2000). Because
the NSMs were defective in 5-HT uptake in mod-5 mutants (see
above), we tested whether ablation of the NSMs affected the
hyperenhanced slowing response of mod-5 mutants. On Petri plates with bacteria, food-deprived NSM-ablated mod-5(n823)
mutants exhibited an enhanced slowing response that was significantly reduced in comparison with that of food-deprived, mock-ablated mod-5(n823) mutants (Fig. 2B, gray
bars). Well-fed NSM-ablated mod-5(n823) mutants were
not significantly affected in their basal slowing response to bacteria
(Fig. 2B, black bars). Ablation of the I5
neuron, another pharyngeal neuron, had no effect on the enhanced
slowing response of mod-5 mutants (data not shown),
indicating that the effect of the NSM ablations was not a consequence
of the ablation protocol per se.
We reasoned that the ablation of the NSMs probably led to a loss of
5-HT needed for the hyperenhanced slowing exhibited by mod-5
mutants. To test this hypothesis, we determined whether the
cat-4 mutation, which decreases 5-HT levels (see above),
could suppress the mod-5 phenotype. cat-4 mutants
are defective in the enhanced slowing response (Sawin et al., 2000)
(Fig. 2C), and the reduced 5-HT in these mutants is the
cause of this defect (Sawin et al., 2000).
On Petri plates with bacteria, the locomotory rate of
food-deprived mod-5(n823); cat-4 double mutants was
significantly faster than that of food-deprived mod-5(n823)
mutants (Fig. 2, compare C, A, gray
bars) but similar to that of NSM-ablated mod-5(n823) mutants (Fig. 2B), suggesting that for the enhanced
slowing response the ablation of the NSMs is equivalent to a reduction
in 5-HT levels in the animal. That the locomotory rate of food-deprived mod-5(n823); cat-4 double mutants was significantly slower
than that of cat-4 mutants (Fig. 2C) is likely a
consequence of the effect of residual 5-HT in cat-4 mutants
(Fig. 1B).
We propose that when food-deprived mod-5 mutants encounter
bacteria, the NSMs release 5-HT, and this 5-HT is inefficiently cleared, thus causing the hyperenhanced slowing response.
mod-5 mutants are hypersensitive to exogenous 5-HT
Exogenous 5-HT inhibits wild-type C. elegans locomotion
(Horvitz et al., 1982). To determine whether mod-5(n822) and
mod-5(n823) mutants were abnormal in their response to
exogenous 5-HT, we used a liquid swimming assay (Ranganathan et al.,
2000). In this assay, mod-5(n822) and mod-5(n823)
mutants were hypersensitive to exogenously added 5-HT (Fig.
2D), presumably because this 5-HT was not efficiently
cleared from the relevant synapses.
MOD-5 is similar to SERTs
We used the 5-HT hypersensitivity of mod-5 mutants in
the liquid swimming assay to map and clone the gene. We mapped
mod-5 to a ~2.0 map unit interval on chromosome I, between
fog-1 and unc-11 (Fig.
3A) (see Materials and
Methods). We noted an open reading frame, Y54E10BR.b (GenBank accession
number AC024812), in this region predicted to encode a protein with
similarity to serotonin reuptake transporters (SERTs). Because loss of
SERT function would provide a simple explanation for both the defective 5-HT uptake and 5-HT hypersensitivity of mod-5 mutants, we
decided to determine whether Y54E10BR.b was mod-5.
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Figure 3.
mod-5 encodes a protein similar to the human
and Drosophila 5-HT reuptake transporters.
A, Top, Genetic map of the
mod-5 region of LG I. Bottom,
Intron-exon structure of mod-5 (Y54E10BR.b), inferred
from cDNA sequences. Open boxes, Coding regions;
lines, untranslated regions, arrow,
direction of transcription; SL1, SL1
trans-spliced leader. The mod-5 open
reading frame is 2016 bp within a 2594 bp cDNA. The extent of the 1688 bp n3314 deletion is depicted. B, Amino
acid sequence alignment of MOD-5 CeSERT with hSERT and dSERT. The 12 predicted transmembrane regions are underlined. Amino
acids conserved between MOD-5 and at least one of the two other
proteins are shown in black boxes, and the two
mod-5 point mutations are indicated.
mod-5(n822) is a Thr-to-Ala transversion mutation
resulting in a C225opal nonsense substitution, and
mod-5(n823) is a Cys-to-Thr transition mutation
resulting in a P569S missense substitution. Triangles,
Potential PKA or PKC phosphorylation sites. Such sites have been
implicated in SERT membrane distribution (Qian et al., 1997;
Ramamoorthy et al., 1998a) and the rate of 5-HT transport (Miller and
Hoffman, 1994). *Potential N-linked glycosylation sites (consensus
NXS/T). Such sites have been implicated in proper folding and insertion
of SERTs into the membrane, protection from degradation, or both (Tate
and Blakely, 1994; Ramamoorthy et al., 1998b). Diamond,
The aspartate residue conserved in SERTs, norepinephrine transporters,
and dopamine transporters.
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We generated an 8 kb PCR product from the genomic region spanning the
first eight exons of Y54E10BR.b (Fig. 3A) and encoding the
first 507 amino acids of the corresponding predicted protein. This
construct rescued the 5-HT hypersensitivity phenotype of mod-5(n823) mutants in 16 of 20 transgenic lines tested
[0-50% animals immobilized after 5 min in 10 mM 5-HT, contrasted with 100%
mod-5(n823) mutants immobilized under the same conditions; data not shown; Fig. 2D], suggesting that not all
671 amino acids of MOD-5 are essential for at least some aspects of
SERT function.
We obtained a partial cDNA clone of Y54E10ABR.b using RT-PCR and
determined the 5' and 3' ends of the cDNA using 5'- and 3'-RACE, respectively. The 5' end of the cDNA contained an SL1
trans-spliced leader, which is found at the 5' ends of many
C. elegans transcripts (Krause and Hirsh, 1987). The 3' end
contained a poly(A) stretch, indicating that we had determined the
complete Y54E10BR.b transcriptional unit (Fig. 3A). This
cDNA was capable of rescuing the 5-HT hypersensitivity of
mod-5 mutants (see below).
Protein sequence comparisons revealed that the predicted protein
encoded by our full-length cDNA is 44% identical to human SERT (hSERT;
Ramamoorthy et al., 1993) and 45% identical to the other known
invertebrate SERT, from D. melanogaster (dSERT; Corey et
al., 1994), which is itself 51% identical to hSERT (Fig.
3B). We identified single-base mutations in the Y54E10BR.b
coding sequence in mod-5(n822) and mod-5(n823)
mutants (Fig. 3B). The mutation in mod-5(n822) is
predicted to change cysteine 225 (codon TGT) to an opal stop codon
(TGA). The mutation in mod-5(n823) is predicted to change
proline 569 (CCG) to serine (TCG) within a transmembrane region. We
concluded that Y54E10BR.b is mod-5.
Like SERTs from other species (Barker and Blakely, 1998, and references
therein), the MOD-5 protein is predicted to contain 12 putative
transmembrane regions (Fig. 3B). Much of the sequence conservation is clustered in or around these transmembrane regions, suggesting that the membrane topology of the SERTs is important for
their function. At position 119 (Fig. 3B,
diamond) within the first predicted transmembrane domain,
MOD-5 has an aspartate residue that is conserved in 5-HT, dopamine, and
norepinephrine (NE) reuptake transporters but not in 
-aminobutyric
acid (GABA) reuptake transporters (Barker and Blakely, 1998). This
aspartate may be involved in binding to the amino group in 5-HT,
dopamine, and NE (Kitayama et al., 1992; Barker et al., 1999). At
position 116, MOD-5 has a phenylalanine residue that is conserved in
hSERT and participates in ligand binding (Adkins et al., 2001). MOD-5 also has other similarities to hSERT and dSERT (Fig. 3B, legend).
mod-5(n3314) is a null allele
To determine the phenotypic consequence of completely eliminating
mod-5 function, we screened libraries of mutagenized animals using PCR to identify large deletions (Jansen et al., 1997) in the
mod-5 genomic locus. We isolated a deletion allele,
n3314, that contains a 1688-bp deletion in the
mod-5 genomic locus (Fig. 3A). The altered open
reading frame (ORF) is predicted to encode the first 42 amino acids of
MOD-5 and, if the end of exon 2 splices onto the next available splice
acceptor site at the start of exon 8, an additional 18 out-of-frame
amino acids before ending at a premature stop codon.
n3314 mutants displayed both the hyperenhanced slowing
response and 5-HT hypersensitivity in the liquid swimming assay (Fig. 2A,D), and the n3314 mutation failed to
complement the mod-5(n822) and mod-5(n823)
alleles for both of these behaviors (data not shown), confirming that
n3314 is an allele of mod-5.
mod-5(n3314) mutants were more hypersensitive to 5-HT than
were mod-5(n822) and mod-5(n823) mutants (Fig.
2D). mod-5(n3314) mutants also exhibited a
more severe hyperenhanced slowing response than did the nonsense mod-5(n822) and the missense mod-5(n823) mutants
(Fig. 2A). On Petri plates without bacteria, the
locomotory rate of mod-5(n3314) mutants was not different
from that of the wild type (Fig. 2A). Well-fed
mod-5(n3314) mutants showed no defect in their basal slowing
response to bacteria (Fig. 2A). Given the stronger
behavioral defects of mod-5(n3314) mutants and the molecular
nature of the n3314 deletion, we think that n3314
is a null allele of mod-5 and that both
mod-5(n822) and mod-5(n823) are partial
loss-of-function alleles. mod-5(n822), which is predicted to
encode only the first 224 amino acids of MOD-5, is not a null allele,
based on comparisons of the phenotypes of mod-5(n822),
mod-5(n3314), and mod-5(n822)/mod-5(n3314) trans-heterozygous animals (Fig. 2A,D; data not
shown). This activity of mod-5(n822) might be a consequence
of the presence of functional mod-5 transcripts produced by
alternative splicing or read-through of the stop codon. Alternatively,
it is conceivable that the first 225 amino acids of MOD-5 retain
partial SERT function. This latter possibility is consistent with our
rescue of the 5-HT hypersensitivity of mod-5(n823) mutants
with a construct that encodes only the first 507 amino acids of
MOD-5.
Because we had isolated the mod-5 cDNA using RT-PCR and
RACE, we sought to confirm that the protein encoded by this cDNA could function in vivo. We constructed a mini-gene in which the
mod-5 cDNA was placed under the control of 2.7 kb of genomic
DNA upstream to the first predicted methionine of mod-5.
mod-5(n3314) animals transgenic for extrachromosomal arrays
consisting of this minigene construct were no longer
5-HT-hypersensitive (data not shown), confirming that we had defined a
functional mod-5 gene and that the mod-5 cDNA
could encode a functional SERT and was suitable for 5-HT-uptake assays
in a heterologous system (see below).
MOD-5 functions as a SERT in mammalian cells
Using retroviral-mediated gene transfer (see Materials and
Methods), we generated HEK293 cell lines that stably expressed MOD-5.
Using these cell lines, we performed uptake assays similar to those
previously done for other SERTs (Ramamoorthy et al., 1993; Demchyshyn
et al., 1994). The uptake of [3H]5-HT by
MOD-5-expressing cell lines was saturable, indicating that the
accumulation of [3H]5-HT in the cells
was facilitated by MOD-5 (Fig.
4A). MOD-5-mediated [3H]5-HT transport was strictly
dependent on Na+ ions (Fig.
4B), as has been observed for 5-HT transport by hSERT (Ramamoorthy et al., 1993), rat SERT (rSERT; Blakely et al., 1991; Hoffman et al., 1991), and dSERT (Demchyshyn et al., 1994). By contrast, MOD-5 did not display a strict dependence on
Cl ions (Fig. 4B),
whereas both hSERT and rSERT, but not dSERT, did display such strict
dependence (Blakely et al., 1991; Hoffman et al., 1991; Ramamoorthy et
al., 1993; Demchyshyn et al., 1994).
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Figure 4.
Physiological characterization of MOD-5
CeSERT. Experiments were performed using HEK293 cell lines stably
transfected with either a mod-5:: gfp
(GFPMOD-5) or a gfp control (GFP vector) construct (for
details, see Materials and Methods). Results indicate mean ± SEM
from at least six replicate trials with a MOD-5 CeSERT-expressing cell
line after subtracting the nonspecific 5-HT uptake from at least three
replicate trials with a cell line expressing GFP from the parent vector
lacking mod-5. Except in A, all assays
were performed for 10 min. In A, B, 50 nM
[3H]5-HT was used. In C, D, the
radiolabeled neurotransmitters were present at 0.1 Ci/mmol specific
activity. Error bars indicate SEM. A, Time dependence of
MOD-5 CeSERT-mediated [3H]5-HT transport.
Circles, GFPMOD-5; squares, GFP vector.
B, Dependence of MOD-5 CeSERT-mediated
[3H]5-HT transport on Na+ and
Cl ions in the external buffer. Results are shown
as a percentage of the normalized 5-HT uptake in standard
NaCl-containing buffer. Transport is strictly dependent on
Na+ but only partially dependent on
Cl. C, MOD-5 CeSERT-mediated
[3H]5-HT transport as a function of 5-HT
concentration. [3H]5-HT uptake was measured at
various 5-HT concentrations. Inset, Eadie-Hoftsee
transformation (Stryer, 1995) of the data.
Km, 150 ± 8 nM;
Vmax, 8.31 × 109
nmol · cell1 · min1.
This Vmax value cannot be compared in
a meaningful way with values from other studies in the absence of
information about relative SERT levels on the cell membrane.
D, MOD-5 CeSERT-mediated transport was specific for
[3H]5-HT. Assays were performed with 1 µM [3H]5-HT and a 50 µM concentration of each of the other radiolabeled
neurotransmitters, and the results are presented as a percentage of
normalized 1 µM [3H]5-HT
uptake. There was no detectable transport when neurotransmitters other
than 5-HT were added at 1 µM (data not shown).
E, Antagonism of MOD-5 CeSERT-mediated 5-HT uptake.
Inhibition curves for SSRIs and other transporter inhibitors are shown.
Assays were performed for 10 min with 50 nM
[3H]5-HT in the presence of varying concentrations
of each of the compounds. The extent of [3H]5-HT
uptake is plotted as the percentage of [3H]5-HT
uptake observed in the absence of antagonists versus log[inhibitor].
See Results for Ki values
calculated from these data.
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MOD-5-mediated [3H]5-HT transport
occurred in a concentration-dependent and saturable manner (Fig.
4C), with a Km of 150 ± 8 nM, a value similar to those reported for other
SERTs (Km range, 280-630
nM; Blakely et al., 1991; Hoffman et al., 1991;
Ramamoorthy et al., 1993; Corey et al., 1994; Demchyshyn et al., 1994;
Chang et al., 1996; Padbury et al., 1997; Chen et al., 1998; Mortensen et al., 1999).
We tested the specificity of MOD-5 by assaying the ability of MOD-5 to
transport various radiolabeled neurotransmitters besides 5-HT.
MOD-5-mediated uptake was highly specific for
[3H]5-HT and inefficient at
translocating radiolabeled GABA, glutamate, glycine, NE, histamine, and
dopamine (Fig. 4D). We also tested the ability of
these neurotransmitters to inhibit
[3H]5-HT uptake via MOD-5. None of the
six neurotransmitters tested, even when present at 100 µM, substantially inhibited the uptake of 50 nM 5-HT. Specifically, the extent of
[3H]5-HT uptake in the presence of a 100 µM concentration of each neurotransmitter was
as follows: GABA, 104 ± 17% of control; glutamate, 92 ± 12%; glycine, 107 ± 18%; NE, 122 ± 15%; histamine,
107 ± 15%; and dopamine, 118 ± 15%. We also tested
whether octopamine or tyramine, two invertebrate-specific
neurotransmitters, could inhibit
[3H]5-HT transport; we could not test
MOD-5-mediated uptake of these neurotransmitters, because radiolabeled
octopamine and tyramine are not available. Tyramine (100 µM) partially inhibited (54 ± 10% of
control) the transport of [3H]5-HT (50 nM) by MOD-5; octopamine (100 µM) did not inhibit (86 ± 12% of
control) MOD-5-mediated [3H]5-HT (50 nM) transport. By comparison, dSERT-mediated
transport of 100 nM
[3H]5-HT was reduced to 95 ± 15%
of control by 200 µM tyramine and to 82 ± 15% of control by 200 µM octopamine (Corey et
al., 1994). These data suggest that there are subtle differences in the
properties of MOD-5 and dSERT.
We tested whether MOD-5-mediated
[3H]5-HT transport was inhibited by
tricyclic antidepressants, SSRIs, and nonspecific monoamine transporter
inhibitors (Blakely et al., 1991; Ramamoorthy et al., 1993; Demchyshyn
et al., 1994). The rank order of potency for inhibition of
MOD-5-mediated [3H]5-HT transport was
imipramine (Ki, 89 ± 58 nM) ~ fluoxetine (Ki, 133 ± 90 nM) ~ paroxetine
(Ki, 179 ± 64 nM) > desipramine (Ki, 334 ± 115 nM) > citalopram
(Ki, 994 ± 298 nM) 
cocaine (Ki, 4076 ± 349 nM) (Fig. 4E).
This rank order is different from that of other SERTs (for example, the
rank order of potency for inhibition of hSERT-mediated
[3H]5-HT transport is paroxetine > fluoxetine > imipramine ~ citalopram cocaine;
Ramamoorthy et al., 1993), and for some of the inhibitors the
Ki values were higher than those reported
for the other SERTs (Blakely et al., 1991; Hoffman et al., 1991;
Ramamoorthy et al., 1993; Corey et al., 1994; Demchyshyn et al., 1994;
Chang et al., 1996; Padbury et al., 1997; Chen et al., 1998; Mortensen
et al., 1999).
Taken together, the specificity of MOD-5-mediated transport for 5-HT,
the dependence of such transport on Na+
and Cl ions, and the inhibition of 5-HT
transport by SSRIs establish that MOD-5 is a SERT from C. elegans, CeSERT.
MOD-5 is likely the only SERT in C. elegans
To determine whether MOD-5 is the only SERT in C. elegans, we analyzed the C. elegans genomic sequence
for other potential SERTs and performed in vivo assays of
5-HT uptake in mod-5 mutants. We found 15 Na+/Cl-dependent
neurotransmitter transporter-like predicted ORFs in the completed
C. elegans genomic sequence (C. elegans
Sequencing Consortium, 1998). Only two of these ORFs, T23G5.5 and
T03F7.1, are nearly as similar (43 and 41% identity, respectively) to
hSERT, as is MOD-5 CeSERT, and only MOD-5 CeSERT and T23G5.5 have an aspartate corresponding to aspartate 119 in MOD-5 CeSERT, a conserved residue likely to be functionally important for amine transport (see
above). T23G5.5 is a dopamine reuptake transporter and is very
inefficient at transporting 5-HT (Jayanthi et al., 1998). Hence, from
sequence analysis, it appeared likely that MOD-5 CeSERT is the only
SERT in C. elegans. This finding is consistent with the
observation that to date only one SERT gene per species has been identified.
If there existed a second SERT in C. elegans, serotonergic
neurons in mod-5(n3314) mutants might be able to take up
exogenously added 5-HT. It is also conceivable that nonserotonergic
cells possess SERT activity. We tested both these possibilities using anti-5-HT antisera to detect the uptake of 5-HT. To eliminate endogenous 5-HT, we used the tph-1(mg280) mutant, which
contains a deletion in the tryptophan hydroxylase gene and is hence
defective in an enzyme essential for 5-HT biosynthesis (Sze et al.,
2000). tph-1 mutants completely lack anti-5-HT
immunofluorescence (Sze et al., 2000). We have confirmed these findings
(Table 1) using the same anti-5-HT antibodies used in Figure
1B. tph-1 mutants are unlikely to be
perturbed in the levels of other biogenic amines, because tryptophan
hydroxylase functions in only 5-HT biosynthesis (Cooper et al., 1996;
Sze et al., 2000).
To identify cells capable of 5-HT uptake, we examined the head, ventral
cord, gut, and tail of tph-1 mutants pretreated with 5-HT
and observed 5-HT immunofluorescence in only the serotonergic neurons
(see below; data not shown). To examine more carefully the requirement
of MOD-5 CeSERT for 5-HT uptake by serotonergic neurons, we scored the
NSMs for 5-HT uptake, because these neurons are the most brightly
staining serotonergic neurons in the animal after incubation with
exogenous 5-HT. Without 5-HT pretreatment, both tph-1 single
and mod-5(n3314); tph-1 double mutants had no NSMs that were
5-HT-positive (Table 1). By contrast, when pretreated with 5-HT,
tph-1 mutants displayed robust 5-HT staining in the NSMs,
whereas mod-5(n3314); tph-1 double mutants showed none
(Table 1). We observed similar results for the serotonergic ADF
neurons in the head and for the hermaphrodite-specific neurons (HSNs) in the midbody (data not shown). Thus, no other transporter appeared to
transport 5-HT into serotonergic neurons in the absence of the MOD-5
CeSERT. We also examined the head, ventral cord, gut, and tail of
mod-5(n3314); tph-1 double mutants pretreated with 5-HT and
observed no 5-HT immunofluorescence anywhere in the animal (data not
shown), indicating that no other cells display 5-HT uptake activity in
the absence of MOD-5 CeSERT.
These 5-HT uptake experiments, taken together with the analysis of the
C. elegans genomic sequence, suggest that MOD-5 is the only
SERT in C. elegans.
mod-5 interacts genetically with mod-1
and goa-1
Mutants defective in the 5-HT-mediated enhanced slowing response
defined several mod genes (Sawin et al., 2000). One of these genes, mod-1, encodes a novel ionotropic 5-HT receptor, a
5-HT-gated chloride channel (Ranganathan et al., 2000). On Petri plates
with bacteria, the locomotory rate of food-deprived mod-1
mutants is substantially faster than that of the wild type (Ranganathan
et al., 2000; Sawin et al., 2000) (Fig.
5A, gray bars). By
contrast, mod-5 mutants exhibit a hyperenhanced slowing
response (Fig. 5A).
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Figure 5.
mod-5 genetic interactions and 5-HT and
MOD-5 CeSERT dependence of the potentiating effect of fluoxetine.
A, Gray bars, Enhanced slowing response
of mod-1, goa-1, mod-5
goa-1, goa-1; mod-1, mod-5;
mod-1, and mod-5 goa-1; mod-1 mutants. At least
five trials were performed for each genotype. Hatched
bars, Effect of fluoxetine on the enhanced slowing response of
wild-type animals (data reproduced from Sawin et al., 2000) and
mod-1 and mod-5; mod-1 mutants (at least
10 trials for each genotype). B, Rescue by 5-HT
preincubation (see Materials and Methods) of the resistance of
bas-1; cat-4 mutants to the potentiating effect of
fluoxetine on the enhanced slowing response. At least five trials were
performed with each genotype. C,
mod-5(n3314) mutants retained normal sensitivity to
fluoxetine-mediated paralysis. Five trials with 20 animals of each
genotype at each concentration were conducted, and the animals were
scored for paralysis after 10 min. Error bars indicate SEM.
***p < 0.0001, Student's t
test.
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To define the genetic pathway in which mod-1 and
mod-5 act in the enhanced slowing response, we characterized
mod-5(n3314); mod-1(ok103) double mutants.
mod-1(ok103) is a null allele by genetic and molecular
criteria (Ranganathan et al., 2000). If the function of the MOD-1 5-HT
receptor were essential for the effects of 5-HT not cleared from
synapses in mod-5 mutants, then eliminating mod-5
function should have had no effect in a mutant that lacked
mod-1 function; i.e., mod-5(n3314); mod-1(ok103)
double mutants should exhibit the same phenotype as
mod-1(ok103) single mutants. However, the enhanced slowing
response of mod-5(n3314); mod-1(ok103) double mutants was
intermediate to the responses of mod-1(ok103) and
mod-5(n3314) single mutants (Fig. 5A, gray bars). This observation suggests that the 5-HT signaling triggered by bacteria in the enhanced slowing response acts via at least two
parallel 5-HT signaling pathways, a MOD-1-dependent pathway and a
MOD-1-independent pathway. This observation is also consistent with the
observation that mod-1(ok103) single mutants were not completely defective in the enhanced slowing response (Fig.
5A).
Animals carrying mutations in the G-protein gene goa-1
(G
o, G0 protein, subunit; Mendel et al., 1995; Segalat et al., 1995) are also defective
in the enhanced slowing response (Sawin et al., 2000) (Fig.
5A). Because GOA-1 animals are resistant to 5-HT in assays
of locomotion (Segalat et al., 1995; our unpublished observations),
pharyngeal pumping (Segalat et al., 1995), and egg laying (Mendel et
al., 1995; Segalat et al., 1995), we tested whether the
MOD-1-independent pathway might involve goa-1. As with
mod-5; mod-1 double mutants, the enhanced slowing response of mod-5(n3314) goa-1(n1134) double mutants was intermediate
to the responses of mod-5(n3314) and goa-1(n1134)
single mutants (Fig. 5A, gray bars), indicating
that 5-HT signaling triggered by bacteria in the enhanced slowing
response does not act solely through goa-1. By contrast,
food-deprived mod-5(n3314) goa-1(n1134); mod-1(ok103) triple
mutants exhibited very little slowing in response to bacteria (Fig.
5A). These observations suggested that MOD-1 and GOA-1 act
in two parallel pathways that together mediate the response to the
excess 5-HT signaling in mod-5(n3314) mutants.
Fluoxetine blocks 5-HT uptake in vivo
Because fluoxetine blocked [3H]5-HT
transport in mammalian cells expressing MOD-5 CeSERT (see above), we
tested whether fluoxetine could block 5-HT uptake in vivo in
C. elegans (Table 1). We pretreated tph-1 mutants
with fluoxetine, incubated the animals with 5-HT, and scored the number
of 5-HT-positive NSMs. We observed, for example, few 5-HT-positive NSMs
when tph-1 mutants were pretreated with 0.22 mM fluoxetine (Table 1), a concentration
sufficient to potentiate the enhanced slowing response (see below).
Furthermore, tph-1 mutants pretreated with as little as 0.44 mM fluoxetine, a concentration lower than that
required for all the MOD-5 CeSERT-independent effects of fluoxetine
(see below), were as defective in 5-HT uptake as were untreated
mod-5(n3314); tph-1 double mutants (Table 1). These
observations suggested that fluoxetine can block 5-HT uptake in
C. elegans in vivo and does so by inhibiting MOD-5 CeSERT.
The potentiation of the enhanced slowing response by fluoxetine
requires MOD-5 CeSERT and 5-HT
When wild-type animals that have been food-deprived in the
presence of 0.22 mM fluoxetine encounter bacteria, they
slow their locomotory rate more than if they had been food-deprived in
the absence of fluoxetine (Sawin et al., 2000) (Fig. 5A).
This fluoxetine-mediated potentiation of the enhanced slowing response
resembles the hyperenhanced slowing response exhibited by
mod-5(n3314) mutants (Fig. 5A), suggesting that
fluoxetine causes this potentiation by blocking MOD-5 CeSERT function.
If so, mod-5(n3314) mutants should be resistant to the
potentiating effect of fluoxetine on the enhanced slowing response.
Because food-deprived mod-5(n3314) mutants exhibit an extreme hyperenhanced slowing response that cannot be further potentiated by fluoxetine treatment (Fig. 5A; data not
shown), we used mod-5(n3314); mod-1(ok103) double mutants to
test this hypothesis. These double mutants are partially suppressed for the hyperenhanced slowing response exhibited by mod-5(n3314)
animals (Fig. 5A, gray bars); therefore, a
potentiation of the enhanced slowing response could be observed.
The enhanced slowing response of mod-1(ok103) mutants was
potentiated by fluoxetine (Fig. 5A, hatched
bars), indicating that fluoxetine can potentiate the enhanced
slowing response in the absence of MOD-1 5-HT receptor function. This
observation was consistent with the phenotype of mod-5(n3314);
mod-1(ok103) double mutants in this assay, which suggested that
there are MOD-1-independent 5-HT pathways through which the enhanced
slowing response is effected. By contrast, mod-5(n3314);
mod-1(ok103) double mutants were completely resistant to the
potentiating effect of fluoxetine on the enhanced slowing response
(Fig. 5A, hatched bars). Therefore, the MOD-5 CeSERT is likely the only in vivo target in C. elegans on which fluoxetine acts to potentiate the enhanced
slowing response.
Because fluoxetine-mediated potentiation of the enhanced slowing
response is MOD-5 CeSERT-dependent, it is also likely to be
5-HT-dependent, as we previously suggested (Sawin et al., 2000) on the
basis of the observation that the enhanced slowing response of
bas-1(ad446); cat-4 double mutants (bas, biogenic
amine synthesis-defective) is resistant to such potentiation. However,
our studies of egg laying by tph-1 mutants suggest that the
resistance of cat-4 animals to the effects of high
concentrations of fluoxetine is likely not to be caused by a deficiency
in 5-HT in these animals (see below). Because tph-1 mutants
display sluggish locomotion (data not shown), they could not be assayed
for resistance to the fluoxetine-mediated potentiation of the enhanced
slowing response.
We sought to determine whether it is the 5-HT-deficiency of
bas-1; cat-4 double mutants that renders these animals
resistant to the potentiating effect of fluoxetine. The defect in the
enhanced slowing response of bas-1; cat-4 double mutants in
the absence of fluoxetine treatment can be rescued by preincubating the
animals on Petri plates containing 2 mM 5-HT
(Sawin et al., 2000) (Fig. 5B), a pretreatment sufficient
for the detection of 5-HT in the NSMs of cat-4 (Fig.
1B) and tph-1 (Table 1) mutants. When
bas-1; cat-4 mutants were preincubated with 5-HT and then
food-deprived in the presence of fluoxetine, they exhibited a
potentiated enhanced slowing response (Fig. 5B). Therefore,
restoration of 5-HT to bas-1; cat-4 mutants is sufficient
for fluoxetine to potentiate the enhanced slowing response of these
mutants. We conclude that the effect of fluoxetine on the enhanced
slowing response is dependent not only on MOD-5 CeSERT but also
on 5-HT.
Fluoxetine induces nose contraction and paralysis in
mod-5 and tph-1 mutants
Treatment of C. elegans with high concentrations
(0.25-1 mg/ml, 0.7-2.9 mM) of fluoxetine leads
to paralysis (Choy and Thomas, 1999), contraction of nose muscles (Choy
and Thomas, 1999), and stimulation of egg laying (Weinshenker et al.,
1995). The concentrations of fluoxetine required for these effects are
at least 2.5-fold higher than that required to detect a block of 5-HT
uptake in vivo (see above) and for the potentiation of the
enhanced slowing response (Fig. 5B) (Sawin et al., 2000). If
the only in vivo target for fluoxetine were MOD-5 CeSERT,
mod-5 mutants would be paralyzed, have contracted noses, and
display excessive egg laying. mod-5 mutants exhibit none of
these characteristics (data not shown), suggesting that either MOD-5
CeSERT is not the sole target of fluoxetine in C. elegans or
that an altered developmental program compensates for the animals
growing up without a SERT. In the latter case, mod-5(n3314)
mutants would be expected to be resistant to the effects of fluoxetine.
Therefore, we tested mod-5(n3314) mutants for their
responses to high concentrations of fluoxetine.
mod-5(n3314) mutants retained wild-type sensitivity to
fluoxetine in assays of paralysis induced by fluoxetine treatment (Fig. 5C). There was no difference in the time course of paralysis
at any of the concentrations tested (data not shown). These
observations suggest that fluoxetine-induced paralysis in C. elegans is not caused by the lack of 5-HT uptake from synapses.
Fluoxetine-treated wild-type and mod-5(n3314) mutant animals
assumed a rigid body posture (data not shown), as observed by others
(Choy and Thomas, 1999). By contrast, 5-HT-treated animals assumed a
relaxed and flaccid body posture (data not shown), suggesting that the
mechanisms of locomotory inhibition by 5-HT and fluoxetine are
distinct. 5-HT has been proposed to decrease excitatory input to the
locomotory muscles (Nurrish et al., 1999). Given the two distinct body
postures, we suggest that fluoxetine may directly or indirectly
increase excitatory input or decrease inhibitory input to the
locomotory muscles.
When treated with fluoxetine for 20 min, a similar proportion of
wild-type and mod-5(n3314) mutant animals had contracted noses (100% at 2.9 mM and ~25% at 1.5 mM). Thus, the effect of fluoxetine on nose
contraction also appears to act via a MOD-5 CeSERT-independent pathway.
This conclusion is consistent with the conclusion by Choy and Thomas
(1999) that fluoxetine-mediated nose contraction is 5-HT-independent,
although their hypothesis was based on studies of
cat-1(e1111) mutants, which are defective in signaling by
several biogenic amines (Duerr et al., 1999), and cat-4
mutants, which we found to not completely lack 5-HT (Fig.
1B).
That the effects of high concentrations of fluoxetine on nose
contraction and paralysis were independent of MOD-5 CeSERT suggested that fluoxetine acts either on another SERT or on a distinct non-SERT target(s). As discussed above, we think that MOD-5 is the only SERT in
C. elegans, making it likely that a non-SERT target(s) of
fluoxetine mediates the MOD-5 CeSERT-independent effects. Such non-SERT
targets may or may not be part of a serotonergic signaling pathway. We
decided to explore the requirement for 5-HT by testing whether
fluoxetine can act in animals that lack 5-HT. The 5-HT-deficient mutants that have been used in numerous previous studies (Weinshenker et al., 1995; Choy and Thomas, 1999; Sawin et al., 2000), such as
cat-1, cat-4, and bas-1 mutants, all
affect multiple biogenic amines. None has been shown to cause a
complete loss of 5-HT function. We therefore tested tph-1
mutants, which appear to completely lack 5-HT (see above), for their
response to fluoxetine.
One hundred percent of tph-1 animals displayed contracted
noses after treatment with 2.9 mM fluoxetine for
20 min. These data confirm the hypothesis of Choy and Thomas (1999)
that this effect of fluoxetine is 5-HT-independent. We found that
fluoxetine treatment paralyzed tph-1 mutants to a similar
extent as wild-type animals (data not shown), suggesting that paralysis
by high concentrations of fluoxetine is also a 5-HT-independent process.
Fluoxetine stimulates egg laying in mod-5 and
tph-1 mutants
In 1.5 mM fluoxetine, mod-5(n3314) mutants
were stimulated to lay eggs to nearly the same extent as was the wild
type (Fig. 6A,B,
black bars), suggesting that fluoxetine can stimulate egg laying via one or more MOD-5 CeSERT-independent pathways. Nevertheless, mod-5(n3314) mutants were hypersensitive to exogenous 5-HT
in assays of egg laying (Fig.
7A,B), suggesting that MOD-5
CeSERT can affect serotonergic synapses that regulate egg laying.
mod-5(n3314) mutants and wild-type animals contain similar
numbers of eggs (25.7 ± 2.8 and 26.3 ± 2.2, respectively),
indicating that this hypersensitivity to exogenous 5-HT was not a
consequence of differences in basal egg-laying rates between wild-type
animals and mod-5 mutants but rather a result of excess 5-HT
signaling in mod-5(n3314) mutants.
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Figure 6.
5-HT and MOD-5 CeSERT independence of
fluoxetine-induced egg laying. Fluoxetine-induced egg laying:
n = 50 for each genotype. A,
Wild-type animals. B, mod-5(n3314)
mutants. C, tph-1 mutants.
D, cat-4 mutants. E,
egl-1 mutants.
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Figure 7.
mod-5(n3314) mutants, but not
tph-1 mutants, are hypersensitive to stimulation of egg
laying by 5-HT. 5-HT-induced stimulation of egg laying:
n = 50 for each genotype. A,
Wild-type animals. B, mod-5(n3314)
mutants. C, tph-1 mutants.
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Given that the stimulation of egg laying by fluoxetine did not require
the MOD-5 CeSERT, we wondered whether this stimulation required 5-HT.
tph-1 mutants were partially resistant to the stimulation of
egg laying by fluoxetine (Fig. 6A,C), indicating that
5-HT mediated some but not all of the egg-laying response to
fluoxetine. By contrast, cat-4 mutants were completely
resistant to fluoxetine-induced egg laying (Fig.
6A,D), as reported by Weinshenker et al. (1995).
The reduction in egg laying by tph-1 mutants in response to
fluoxetine (Fig. 6C) is unlikely to be caused by a lower
number of eggs within tph-1 mutants or the inability of
egg-laying muscles in tph-1 mutants to respond to
stimulatory input: tph-1 mutants contain more eggs than do
wild-type animals (Sze et al., 2000; and data not shown), and
tph-1 mutants laid at least as many eggs in response to
exogenous 5-HT as did wild-type animals (Fig. 7C). That
mod-5(n3314) and tph-1 mutants laid significant
numbers of eggs in response to fluoxetine argues that the mechanism(s)
through which fluoxetine stimulates egg laying in C. elegans
is not only MOD-5 CeSERT-independent but also, in part,
5-HT-independent.
The serotonergic HSN motor neurons innervate the egg-laying muscles and
drive egg laying (Trent et al., 1983; Desai et al., 1988).
egl-1(n1084) mutants, which lack the HSNs (Desai et al., 1988), released some eggs in the absence of fluoxetine (Fig.
6E, gray bars), presumably because these
animals were severely bloated with eggs. However, treatment with
fluoxetine had no effect on egg laying in egl-1 mutants
(Fig. 6E, black bars). These observations indicate that the HSNs are required for the stimulation of egg laying
by fluoxetine.
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DISCUSSION |
The C. elegans gene mod-5 encodes a homolog
of human SERT, a protein of major importance in human neurobiology and
psychiatric disease. MOD-5 CeSERT acts in a pathway that includes both
a novel type of 5-HT receptor, the 5-HT-gated chloride channel MOD-1, and the G-protein GOA-1. We have identified a C. elegans
modulatory behavior (the potentiation of the enhanced slowing response)
in which fluoxetine (Prozac) affects 5-HT signaling by antagonizing MOD-5 CeSERT. We have also identified C. elegans behaviors
in which fluoxetine acts independently of both SERT and 5-HT. The analysis of these behaviors could define novel targets of fluoxetine action. Choy and Thomas (1999) initiated such studies, screening for
mutants that fail to show the nose contraction response to fluoxetine
treatment, and in this way they defined two genes required for this response.
MOD-5 probably acts upstream of MOD-1 and GOA-1
mod-5 mutants, which exhibit a hyperenhanced slowing
response, are opposite in phenotype to all other mod
mutants, which were isolated on the basis of their failures to exhibit
the enhanced slowing response (Sawin et al., 2000). These opposite
phenotypes allow genetic epistasis experiments to be performed to help
define a genetic pathway for this behavior.
We performed such epistasis analysis with the genes mod-1,
goa-1, and mod-5 (Fig. 5A). The
phenotypes of mod-5 goa-1 and mod-5; mod-1 double
mutants were intermediate between those of either mod-5 or
goa-1 or mod-5 or mod-1 single
mutants, respectively; mod-5 goa-1; mod-1 triple mutants
were almost completely defective in the enhanced slowing response. The
intermediate phenotypes of these double mutants with mod-5
indicate that mod-1 and goa-1 act in parallel
pathways for the enhanced slowing response (Fig. 8A). mod-1
and goa-1 probably both act downstream of mod-5
(Fig. 8A), because in the absence of both
mod-1 and goa-1, the presence or absence of
mod-5 has little effect.
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Figure 8.
Models for the effects of fluoxetine on C.
elegans behaviors. A, MOD-5 CeSERT acts upstream
of the MOD-1 5-HT-gated chloride channel and the GOA-1 G-protein.
B, The potentiating effect of low concentrations of
fluoxetine on the enhanced slowing response is 5-HT- and MOD-5
CeSERT-dependent. The stimulus of food in a food-deprived animal
results in the release of 5-HT. This 5-HT acts through the MOD-1
5-HT-gated chloride channel and through a GOA-1-dependent 5-HT
signaling pathway, which likely acts in parallel to the MOD-1 pathway.
Together, these two pathways result in the slowing of the locomotory
rate. Mutation in mod-5 or application of fluoxetine
leads to inefficient clearing of 5-HT and thus a hyperenhanced slowing
response. 5-HT receptor, A metabotropic 5-HT receptor
that GOA-1 might couple to. Triangles, 5-HT;
circles, chloride ions. C, The effects of
high concentrations of fluoxetine on egg laying, nose contraction, and
paralysis are MOD-5 CeSERT-independent. High concentrations of
fluoxetine act on non-SERT targets and on a non-5-HT pathway to
paralyze C. elegans and to lead to the contraction of
nose muscles. By contrast, stimulation of egg laying by fluoxetine is
MOD-5 CeSERT-independent but still partially dependent on 5-HT (for
details, see Discussion).
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Because mod-5 encodes a SERT and mod-5 mutants
are defective in the loading of 5-HT into serotonergic neurons, it is
likely that MOD-5 CeSERT functions in neurons that synthesize and
release 5-HT. That mod-1 probably acts downstream of
mod-5 and encodes a 5-HT receptor (Ranganathan et al., 2000)
suggests that MOD-1 acts in cells that are postsynaptic to the
serotonergic neurons in which MOD-5 functions (Fig.
8B). Alternatively, MOD-1 could be a presynaptic 5-HT
receptor, acting in the same serotonergic cells as MOD-5. We consider
this latter possibility unlikely, given that a
mod-1:: GFP reporter that rescues the
mod-1 mutant phenotype (Ranganathan et al., 2000) is not
expressed in the NSM, HSN, and ADF serotonergic neurons (R. Ranganathan
and H. R. Horvitz, unpublished data). Moreover, expression of this
mod-1:: GFP reporter was observed in certain
nonserotonergic neurons (data not shown) that are likely to be direct
postsynaptic partners of some of the serotonergic neurons (White et
al., 1986
TOP
ABSTRACT
INTRODUCTION
