Serotonin regulates repolarization of the C. elegans pharyngeal muscle
Department of Molecular Biology, The University of Texas Southwestern Medical Center, 6000 Harry Hines, Dallas TX 75390-9148, USA
* Author for correspondence (e-mail: tim.niacaris{at}utsouthwestern.edu)
Accepted 21 October 2002
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Summary |
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Key words: serotonin, nematode, Caenorhabditis elegans, gramine, octopamine, action potential, motor neuron, feeding, pharynx
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Introduction |
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Proper timing of the pharyngeal contractionrelaxation cycle is
important for efficient feeding (Avery,
1993b). A pair of neurons, the M3s, modulates the timing of
pharyngeal relaxation. When the M3s are ablated with a laser, or decoupled
from the pharyngeal muscle by mutating the M3-post synaptic receptor, the
duration of pharyngeal contraction increases
(Avery, 1993b
;
Dent et al., 1997
;
Raizen and Avery, 1994
). In
addition to regulating pump length, the M3s are important for maintaining
coordinated pharyngeal motions that are required for efficient feeding. When
the M3s are killed along with other pharyngeal neurons, feeding is inefficient
and the pharyngeal lumen becomes clogged with bacteria that are not
efficiently transported to the intestine
(Avery, 1993b
).
Serotonin regulates pharyngeal pumping rate. Exogenous serotonin stimulates
pharyngeal pumping (Avery and Horvitz,
1990; Croll, 1975
;
Horvitz et al., 1982
), while
chronic depletion of endogenous serotonin suppresses pumping
(Duerr et al., 1999
;
Sze et al., 2000
). Gramine, a
competitive serotonin antagonist (Evans
and O'Shea, 1978
), and octopamine decrease pharyngeal pumping rate
(Avery and Horvitz, 1990
;
Horvitz et al., 1982
). In
addition to regulating pharyngeal behavior, serotonin and octopamine also
regulate other behaviors that are influenced by the presence of food, such as
locomotion and egg laying (Horvitz et al.,
1982
; Segalat et al.,
1995
). Serotonin suppresses locomotion and enhances egg laying,
mimicking the behaviors displayed by worms in the presence of abundant food.
Conversely, octopamine enhances locomotion and suppresses egg laying. These
observations suggest that serotonin and octopamine may be important for
signaling the presence and absence, respectively, of food.
To take maximal advantage of abundant food, worms must increase their pharyngeal pumping rate while preserving muscle coordination and efficiency. While it is clear that serotonin and octopamine regulate the rate of pumping, little is known about the effects of these transmitters on pharyngeal physiology. We have used an electrophysiological approach to show that serotonin and octopamine regulate the effective activity of the M3 motor neurons and timing of pharyngeal muscle repolarization. These observations suggest a mechanism for maintaining proper pharyngeal coordination in the context of varied pumping rates.
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Materials and methods |
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Chemicals
Chemicals used were the creatinine sulfate complex of serotonin
(5-hydroxytryptamine) from (Sigma; cat. no. H-7752), gramine
(3-[dimethylaminomethyl]indole) (Sigma; cat. no. G-1647) and
octopamine (1-[p-hydroxyphenyl]-2-aminoethanol) (Research Biochemicals
International; cat. no. O-101).
Preparation of worms for electrophysiological analysis
Eggs of each genotype tested were collected and synchronized as previously
described (Lewis and Fleming,
1995). Approximately 500 synchronized larvae were transferred to
fresh 10 cm agarose plates seeded with HB101 and grown to adulthood. Adult
hermaphrodites were washed from the plate with 5 ml M9 into a 15 ml conical
tube and spun briefly at 800 g. We removed the M9 and washed
the worms twice with 2 ml of fresh M9 to remove any bacteria present in the
M9-worm suspension. For the single-point data (Figs
3,
6 and
7) as well as the gramine and
octopamine titrations (Fig.
5B,C), worms were placed on a rocker at 20°C for 30 min. For
the serotonin titration experiment (Fig.
5A) worms were placed on a rocker at 20°C for 3 h. The longer
starvation period in the serotonin titration experiment was used to promote
further decrease of endogenous serotonin levels.
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Extracellular recordings (electropharyngeograms)
Extracellular recordings were made using methods based on those previously
described (Davis et al., 1995;
Raizen and Avery, 1994
).
Synchronized adult hermaphrodites were transferred into 100 µl Dent's
saline (Avery et al., 1995
) on
a 35 mmx50 mm glass coverslip (Fisher Scientific) and cut just posterior
to the terminal bulb of the pharynx with a #11 surgical blade. Recordings were
done at ambient temperature (21-26°C). Current measurements were done with
an Axopatch-1D amplifier from Axon Instruments (Foster City, CA, USA) in
voltage-clamp mode. The data were low-pass filtered at 1 kHz using a 4-pole
Bessel filter in the amplifier. The electrical signal from the amplifier was
sampled and recorded using an AT-MIO-16X ADC board from National Instruments
(Austin, TX, USA) and Accbin, our own acquisition software written by M. W.
Davis in the National Instruments Labview development environment. The
sampling rate was 2 kHz. Further filtering and analysis were done with Igor
Pro 4.01 from Wave Metrics (Lake Oswego, OR, USA). Each pharynx was assayed
approximately 1 min after dissection and recorded for 3 min.
Single-point drug assays
For single-point drug assays (Figs
3,
6 and
7), the 3 min recordings were
split into two segments. The first 90 s were recorded either in the absence of
drug or in 1 µmol l-1 serotonin, 100 µmol l-1
gramine or 100 µmol l-1 octopamine. The remaining 90 s were
recorded in the presence of 1 µmol l-1 serotonin, 100 µmol
l-1 gramine, 100 µmol l-1 octopamine, 100 µmol
l-1 gramine+1 µmol l-1 serotonin, or 100 µmol
l-1 octopamine+1 µmol l-1 serotonin. The pattern of
drug addition was varied to control for temporal effects. For each strain
tested, two separate synchronized preparations were made. 15 dissected
pharynxes were assayed from each synchronized worm preparation using the drug
exposure pattern shown in Table
1. All recordings were completed less than 2.5 h after
transferring the synchronized worm population to M9.
|
Doseresponse assays
For doseresponse assays (Fig.
5), dissected pharynxes were exposed to a single drug condition
over the entire 3 min recording. For serotonin doseresponse data
(Fig. 5A) at least ten
pharynxes were assayed for each data point. For gramine and octopamine
doseresponse data (Fig.
5B,C) 100 nmol l-1 serotonin was present in addition to
the indicated gramine and octopamine concentrations. Each point in the
serotonin and gramine doseresponse curves represents data from a
minimum of four pharynxes. Data from the final 150 s of each recording were
used to determine the mean action potential duration and M3 activity for the
trial.
Analysis and quantification of electropharyngeograms
Data from each recording were analyzed using Igor Pro 4.01 from Wave
Metrics. We developed an Igor Procedure (available on request) for quantifying
the length and M3 activity of each action potential generated during the
recording. We defined action potential duration as the time between the peak
of the depolarization (E) spike and the peak of the repolarization (R) spike
in the electropharyngeogram (EPG). We fit the EPG to a fourth-order polynomial
equation to correct for baseline drift that was not eliminated by high-pass
filtering. We calculated M3 activity as the mean square deviation about the
drift-corrected baseline during the portion of the plateau phase of the action
potential not affected by current due to the E and R spikes (the interval of
the EPG beginning 10 ms after the E spike and ending 10 ms before the R spike;
Fig. 2, pink box). This value
was corrected for random noise by subtracting the mean variance about the
drift-corrected baseline in regions of the EPG not affected by pharyngeal
currents. M3 activity is reported in units of pA2. The Student's
t-test was used for statistical analysis of both M3 activity and
action potential duration.
|
Pumping rate assays
Unsynchronized adult worms were dissected in 200 µl Dent's saline
containing 1 µmoll-1 levamisole. EPGs were performed as
described above, except that individual trials were separated into three
segments and lasted approximately 8 min. The first segment of the recording
was performed in the absence of serotonin and gramine, the second was
performed in the presence of 1 µmoll-1 serotonin, and the final
segment was performed in the presence of 1 µmoll-1 serotonin and
100 µmoll-1 gramine. Pumping rate was quantified by calculating
the number of pumps (defined by the presence of an R spike in the EPG) during
the last 2 min of each segment of the recording. Data shown are the results of
three independent trials. The Student's t-test was used for
statistical analysis of pumping rate.
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Results |
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M3 is an inhibitory glutamatergic neuron. It fires during the pharyngeal
action potential, releasing glutamate, which acts on a glutamate-gated
chloride channel encoded in part by avr-15. Activation of this
channel causes inhibitory postsynaptic potentials that can end the muscle
action potential (Avery, 1993b;
Dent et al., 1997
;
Li et al., 1997
). M3 thus
decreases action potential duration, promoting rapid relaxation of the
pharyngeal muscle following contraction
(Avery, 1993b
;
Dent et al., 1997
;
Raizen and Avery, 1994
).
Serotonin, gramine and octopamine affect the pharyngeal action
potential
We examined the electrophysiological properties of pharyngeal muscle to
determine whether serotonin affects the pharyngeal action potential. The EPG
allows us to determine the start (Fig.
2, E spike) and end (Fig.
2, R spike) of the pharyngeal action potential. In addition, we
can measure M3 activity (Fig.
2, red portion of trace). To determine whether serotonin can
affect these properties of the pharyngeal action potential we examined EPGs
from eat-18 mutants (Raizen et
al., 1995). In the absence of MC function, pharyngeal pumping rate
decreases to approximately one-fifth the wild-type rate
(Avery and Horvitz, 1989
).
Thus, eat-18 mutants allow us to examine the effects of serotonin
without inducing rapid pumping. In eat-18 mutants, serotonin enhances
the activity of the M3 motor neurons and decreases pharyngeal action potential
duration (Fig. 3A,B). Gramine,
a competitive serotonin antagonist, decreases pumping rate in the presence of
food (Avery and Horvitz, 1990
)
and blocks serotonin-stimulated increases in pumping rate
(Fig. 4). We found that gramine
increases action potential duration and suppresses M3 activity both in the
presence and absence of exogenous serotonin
(Fig. 3A,B). The effects of
gramine in the absence of added serotonin are probably due to antagonism of
endogenous serotonin activity. Serotonin has a residual effect in the presence
of gramine (compare the effects of 1 µmol l-1 serotonin+100
µmol l-1 gramine with 100 µmol l-1 gramine;
Fig. 3), indicating that the
gramine block of serotonin action is incomplete. Octopamine also affects the
pharyngeal action potential in a manner opposite to that of serotonin:
octopamine suppresses M3 activity and increases action potential duration both
in the presence and absence of serotonin
(Fig. 3A,B).
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To determine whether the effects of serotonin, gramine and octopamine on the pharyngeal action potential are dosage sensitive, we measured action potential duration at several drug concentrations. 10 nmol l-1 serotonin decreases action potential duration and enhances M3 activity (Fig. 5A). Higher concentrations of serotonin further decrease action potential duration and enhance M3 activity. Gramine and octopamine block the effects of serotonin in a dosage-sensitive manner (Fig. 5B,C).
The M3 and MC motor neurons mediate regulation of the pharyngeal
action potential by serotonin, gramine and octopamine
The M3s promote repolarization of pharyngeal muscle
(Avery, 1993b;
Dent et al., 1997
). To
determine whether serotonin decreases action potential duration solely by
enhancing the activity of the M3s, we examined avr-15 mutants. AVR-15
is a subunit of the postsynaptic receptor for the M3s and is required for the
pharyngeal muscle to respond to the M3 motor neurons
(Dent et al., 1997
). In the
absence of AVR-15, M3 activity is absent from EPGs
(Fig. 6A-C). We found that
serotonin, gramine and octopamine do not affect action potential duration in
eat-18; avr-15 double mutants
(Fig. 6A). Thus, the regulation
of action potential duration by serotonin, gramine and octopamine in
eat-18 mutants is largely due to regulation of M3 activity. [It is a
formal possibility that AVR-15 regulates action potential duration in response
to serotonin by activity at synapses other than the M3 neuromuscular junction.
Since no other such synapse is known within the pharynx, and since M3 has been
shown previously to affect action potential duration
(Avery, 1993b
;
Dent et al., 1997
), we believe
that the effect of the avr-15 mutation is mostly or entirely caused
by blocking M3 action.] The avr-15 single mutant behaves differently
than the eat-18; avr-15 double mutant: serotonin decreases
and gramine increases the action potential duration of avr-15 mutant
worms (Fig. 6B). These
observations suggest that EAT-18 also mediates the effect of serotonin on
action potential duration.
eat-18 mutants have MC motor neurons that are functionally
decoupled from pharyngeal muscle (Raizen
et al., 1995). To determine whether EAT-18 mediates serotonergic
regulation of the action potential solely by affecting MC function, we
examined eat-2; avr-15 mutants. EAT-2 is a non-alpha
nicotinic acetylcholine receptor subunit that localizes to the MC-pharyngeal
muscle synapses and is required for MC transmission to pharyngeal muscle (J.
McKay, personal communication). Thus, it is likely that absence of EAT-2
specifically affects MC function. Similar to eat-18; avr-15
mutants, eat-2; avr-15 mutant pharynxes do not modulate
their action potential duration in response to serotonin, gramine or
octopamine (Fig. 6C). These
data suggest that EAT-18 and EAT-2 mediate the effects of serotonin, gramine
and octopamine by an MC-dependent mechanism.
In wild-type worms that contain intact MC and M3 motor neurons, exogenous serotonin does not significantly enhance M3 activity or decrease action potential duration (Fig. 6D). This is likely to be a consequence of the high levels of M3 activity and short action potentials we observe in the absence of added serotonin. However, gramine and octopamine can suppress M3 activity and increase the action potential duration of wild-type worms. This suggests that the serotonergic and octopaminergic regulatory mechanisms are involved in regulating the action potential of wild-type worms (Fig. 6D).
Chronic depletion of endogenous serotonin affects the pharyngeal
action potential
To determine whether chronic depletion of endogenous serotonin affects
action potential duration and M3 activity we examined eat-18;
tph-1 double mutants. TPH-1 is the sole C. elegans ortholog
of tryptophan hydroxylase. Tryptophan hydroxylases are required for converting
tryptophan to the immediate precursor of serotonin, 5-hydroxytryptophan, but
are not involved in the synthesis of other bioamines. Worms lacking TPH-1 have
several defects consistent with reduced levels of endogenous serotonin
(Sze et al., 2000). However,
tph-1 mutants retain some serotonin immunoreactivity, so may contain
residual serotonin (C. Loer, personal communication).
eat-18; tph-1 double mutants have altered responses to serotonin, gramine and octopamine relative to eat-18 single mutants (Fig. 7A,B). In the absence of exogenous serotonin, eat-18; tph-1 mutants have reduced M3 activity and fail to respond to gramine and octopamine. The level of M3 activity in eat-18; tph-1 in the absence of added drugs is not different from that of eat-18 in the presence of gramine. Therefore, it is likely that chronic depletion of serotonin and acute blocking of the serotonin signal affect M3 activity similarly. However, action potential duration in the absence of added drug is not significantly different between eat-18; tph-1 and eat-18, suggesting there is a serotonin and M3-independent mechanism for shortening the action potential.
Exogenous serotonin increases M3 activity and decreases action potential duration in eat-18; tph-1 mutants. Serotonin also restores the ability of gramine to regulate action potential duration and M3 activity in eat-18; tph-1 mutants. Indeed, gramine can suppress the M3 activity of serotonin-stimulated eat-18; tph-1 worms to the same extent as in eat-18 worms. Octopamine lengthens the serotonin-stimulated action potential duration of eat-18; tph-1 mutants, but does not significantly suppress serotonin-stimulated M3 activity.
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Discussion |
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Two classes of pharyngeal motor neurons, MC and M3, are required for serotonergic regulation of the action potential. In the absence of MC and M3 function, serotonin does not affect action potential duration. However, when either MC or M3 activity is restored, serotonin decreases the length of the action potential. Thus, both MC and M3 dependent-mechanisms are required for normal regulation of the action potential by serotonin. Since the M3s act to promote pharyngeal muscle repolarization, it is clear that serotonergic enhancement of M3 function can directly influence action potential duration. However, it is less clear how serotonin affects action potential duration in an MC-dependent fashion. Since MC acts to depolarize pharyngeal muscle, the effect of MC on action potential duration is likely to be indirect. One possibility is that serotonin enhances MC-dependent stimulation of pharyngeal muscle causing elevation of intracellular calcium, which could activate calcium-sensitive repolarization mechanisms.
The MC and M3-dependent effects of serotonin could be the result of direct
stimulation of the activity of the MC and M3 motor neurons or, alternatively,
serotonin may regulate the action potential by modulating postsynaptic
receptors for MC and M3 that are located in the pharyngeal muscle. Consistent
with the latter model, we have identified a serotonin receptor, SER-1, that is
expressed in pharyngeal muscle, but have not identified any candidate
serotonin receptors within the C. elegans genomic sequence that
express in MC or M3 (Hamdan et al.,
1999; T. Niacaris and L. Avery, unpublished observations). While
this suggests that serotonin acts within the pharyngeal muscle to regulate
action potential duration, it is possible that serotonin affects the activity
of MC and M3 by an unidentified serotonin receptor.
Chronic depletion of endogenous serotonin suppresses M3 activity, similar to the acute effects of the serotonin antagonist gramine. However, the chronic absence of serotonin does not significantly affect action potential duration, despite the suppression of M3 activity. This suggests the presence of a serotonin and M3-independent mechanism for shortening the action potential. Since long action potentials limit the rate of pharyngeal pumping and slow pumping inhibits growth, it is likely that worms have an adaptive mechanism for shortening the action potential under these conditions. Our observation that eat-18; tph-1 worms have significantly shorter action potentials in the presence of gramine than those of eat-18 worms further supports the existence of a serotonin-independent adaptive mechanism that regulates pump length.
We have recently shown that GPB-2, a G-protein ß5 subunit,
also affects M3 activity and action potential duration
(Robatzek et al., 2001). In
the absence of GPB-2 function, M3 activity is greatly reduced; however, action
potential duration is not significantly affected. This is the same phenotype
that we observe in worms chronically deprived of serotonin. Since chronic
deprivation of serotonin mimics loss of GPB-2 function, GPB-2 may be required
for pharyngeal muscle to respond to serotonin. In addition, the GPB-2
phenotype provides further evidence that there is an adaptive mechanism for
shortening the action potential when M3 is chronically suppressed.
Reciprocal regulation of pharyngeal behavior by serotonin and octopamine
provides a mechanism for adapting to a range of food availability. In the
presence of abundant food, serotonin acts to increase the speed and efficiency
at which worms feed. When the food source is exhausted, falling levels of
serotonin and, possibly, rising levels of octopamine switch the pharynx to an
inactive state characterized by slow pumping. Thus, serotonin and octopamine
may function to adapt pharyngeal behavior to the presence or absence,
respectively, of food. Since reciprocal chronotropic effects of serotonin and
octopamine have been identified for several rhythmically contracting organs
(Collins and Miller, 1977;
Zornik et al., 1999
), it will
be interesting to determine whether serotonin and octopamine also modify
action potential duration in these systems similarly to their effects in
C. elegans.
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Acknowledgments |
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