Serotonin regulates repolarization of the C. elegans pharyngeal muscle

Abstract

Caenorhabditis elegans feeds by rhythmically contracting its pharynx to ingest bacteria. The rate of pharyngeal contraction is increased by serotonin and suppressed by octopamine. Using an electrophysiological assay, we show that serotonin and octopamine regulate two additional aspects of pharyngeal behavior. Serotonin decreases the duration of the pharyngeal action potential and enhances activity of the pharyngeal M3 motor neurons. Gramine, a competitive serotonin antagonist, and octopamine have effects opposite to those of serotonin: gramine and octopamine increase action potential duration and suppress M3 activity. The effects of serotonin, gramine and octopamine on action potential duration are dependent on the pharyngeal motor neurons MC and M3. When the MC and M3 motor neurons are functionally defective, serotonin and octopamine do not regulate the action potential. Our data suggest that serotonin alters pharyngeal physiology to allow for rapid contraction-relaxation cycles. Reciprocal regulation of pharyngeal behavior by serotonin and octopamine provides a mechanism for adapting to the presence and absence of food, respectively.

Fig. 1

Motor neurons MC and M3 control the timing of pharyngeal muscle action potentials. MC is an excitatory cholinergic neuron (Raizen et al., 1995; T. Niacaris and L. Avery, unpublished observations). Its firing triggers a pharyngeal muscle action potential via the release of acetylcholine, which acts on a muscle nicotinic receptor (Raizen et al., 1995; J. McKay, personal communication). MC thus controls the frequency of pharyngeal pumping. M3 is an inhibitory glutamatergic neuron. It fires during the action potential, releasing glutamate, which causes inhibitory postsynaptic potentials that can end the muscle action potential (Avery, 1993b; Dent et al., 1997; Li et al., 1997). M3 thus acts to decrease action potential duration. Serotonin acts to increase the activity or effect of both of these neurons on pharyngeal muscle, resulting in decreased action potential duration. Note that MC and M3 are actually located within the pharynx although they are shown here as external for clarity.

Fig. 2

The electropharyngeogram (EPG) reflects current movement across the pharyngeal muscle membrane. In the EPG, the large positive transient (E, blue) corresponds to depolarization of the pharyngeal muscle and onset of contraction. The large negative transient (R, green) corresponds to repolarization of the pharyngeal muscle and the end of the action potential. We defined action potential duration as the time difference between the peaks of the E and R spikes. The interval between the E and R spikes represents the plateau phase of the action potential (red portion of the trace). The negative transients during the plateau phase are pharyngeal muscle inhibitory postsynaptic potentials (IPSPs) caused by firing of the M3 motor neurons. We calculated the activity of the M3 motor neurons as the mean-square deviation about the baseline during the portion of the action potential affected only by M3-induced currents (pink box). Regions of the EPG trace not affected by pharyngeal currents (regions outside the gray and pink boxes) were used to determine the portion of baseline deviation due to random noise. This value is subtracted from the preliminary measurement of M3 activity to determine a noise-corrected value, which we report as M3 activity.

Fig. 3

(A) Serotonin, gramine and octopamine affect the electropharyngeograms (EPGs) of eat-18 mutants. Serotonin (1 μmol l⁻¹) enhances M3 activity and decreases the action potential duration of eat-18 mutant pharynxes. Gramine (100 μmol l⁻¹) and octopamine (100 μmol l⁻¹) increase action potential duration and suppress M3 activity in the presence and absence of exogenous serotonin. EPGs shown for each drug condition are statistically representative of the average action potential duration and M3 activity of all recordings. (B) Quantitative analysis of M3 activity and action potential duration in eat-18 mutants. Serotonin (1 μmol l⁻¹) significantly decreases action potential duration and enhances M3 activity (P<0.005). Gramine (100 μmol l⁻¹) and octopamine (100 μmol l⁻¹) block the serotonin-stimulated decrease in action potential duration and enhancement of M3 activity (P<0.001). Gramine and octopamine also increase action potential duration and suppress M3 activity in the absence of exogenous serotonin (P<0.05). Values are means + s.e.m. Significant differences between the indicated measurement and data recorded * in the absence of drug or ^† in the presence of 1 μmol l⁻¹ serotonin.

Fig. 4

Gramine suppresses serotonin-stimulated pumping in wild-type worms. 1 μmol l⁻¹ serotonin induces rapid pharyngeal pumping (P<0.0001). Addition of 100 μmol l⁻¹ gramine suppresses serotonin-stimulated pumping (P<0.0001). However, serotonin still has residual effects on pumping rate in the presence of 100 μmol l⁻¹ gramine (P<0.005). Values are means + s.e.m. Significant differences between the indicated measurement and data recorded * in the absence of drug or ^† in the presence of 1 μmol l⁻¹ serotonin.

Fig. 5

Serotonin, gramine and octopamine affect the M3 activity (red lines) and action potential duration (blue lines) of eat-18 mutant pharynxes in a dosage-sensitive manner. (A) Serotonin enhances M3 activity and decreases action potential duration with an EC₅₀ of approximately 30 nmol l⁻¹. In the presence of 100 nmol l⁻¹ exogenous serotonin, gramine (B) and octopamine (C) suppress M3 activity and increase action potential duration in a dosage-sensitive manner. Values are means ± s.e.m.

Fig. 6

(A) Serotonin (1 μmol l⁻¹), gramine (100 μmol l⁻¹) and octopamine (100 μmol l⁻¹) do not affect the action potential duration of eat-18; avr-15 mutants (P>0.5). (B) Serotonin (1 μmol l⁻¹) significantly decreases the action potential duration of avr-15 mutants (P<0.001). This effect is blocked by addition of 100 μmol l⁻¹ gramine (P<0.05), but not by 100 μmol l⁻¹ octopamine (P>0.5). (C) Serotonin (1 μmol l⁻¹), gramine (100 μmol l⁻¹) and octopamine (100 μmol l⁻¹) do not affect the action potential duration of eat-2; avr-15 mutants (P>0.5). (D) Gramine (100 μmol l⁻¹) and octopamine (100 μmol l⁻¹) suppress the M3 activity (P<0.001) and increase the action potential duration of wild-type worms (P<0.05).

Fig. 7

Chronic depletion of endogenous serotonin affects pharyngeal behavior. (A) In the absence of exogenous serotonin (1 μmol l⁻¹), eat-18; tph-1 worms have decreased M3 activity relative to eat-18 worms (Fig. 3B) (P<0.05), and are unresponsive to gramine (100 μmol l⁻¹) and octopamine (100 μmol l⁻¹) (P>0.2). Exogenous serotonin enhances M3 activity to levels comparable to serotonin-stimulated eat-18 worms (P<0.05). Gramine can suppress the serotonin-stimulated enhancement of M3 activity in eat-18; tph-1 worms (P<0.05). (B) In the absence of exogenous serotonin, eat-18; tph-1 worms have action potentials similar in length to those of eat-18 worms (P>0.05). Gramine and octopamine have no effect on action potential duration in the absence of added serotonin (P>0.6). Exogenous serotonin shortens the action potential of eat-18; tph-1 worms (P<0.05). This effect is blocked by gramine and octopamine (P<0.05). Values are means + S.E.M. Significant differences between the indicated measurement and data recorded * in the absence of drug or ^† in the presence of 1 μmol l⁻¹ serotonin. Hatched bars represent significant differences between measurements from eat-18; tph-1 (this figure) and those from eat-18 (Fig. 3B).