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Serotonin regulates repolarization of the C. elegans pharyngeal muscle

Timothy Niacaris^* and Leon Avery

Department of Molecular Biology, The University of Texas Southwestern Medical Center, 6000 Harry Hines, Dallas TX 75390-9148, USA

View larger version (17K): [in a new window]   Fig. 3. (A) Serotonin, gramine and octopamine affect the electropharyngeograms (EPGs) of eat-18 mutants. Serotonin (1 µmoll-1) enhances M3 activity and decreases the action potential duration of eat-18 mutant pharynxes. Gramine (100 µmoll-1) and octopamine (100 µmoll-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 µmoll-1) significantly decreases action potential duration and enhances M3 activity (P<0.005). Gramine (100 µmoll-1) and octopamine (100 µmoll-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 µmoll-1 serotonin.

View larger version (41K): [in a new window]   Fig. 6. (A) Serotonin (1 µmoll-1), gramine (100 µmoll-1) and octopamine (100 µmoll-1) do not affect the action potential duration of eat-18; avr-15 mutants (P>0.5). (B) Serotonin (1 µmoll-1) significantly decreases the action potential duration of avr-15 mutants (P<0.001). This effect is blocked by addition of 100 µmoll-1 gramine (P<0.05), but not by 100 µmoll-1 octopamine (P>0.5). (C) Serotonin (1 µmoll-1), gramine (100 µmoll-1) and octopamine (100 µmoll-1) do not affect the action potential duration of eat-2; avr-15 mutants (P>0.5). (D) Gramine (100 µmoll-1) and octopamine (100 µmoll-1) suppress the M3 activity (P<0.001) and increase the action potential duration of wild-type worms (P<0.05).

View larger version (33K): [in a new window]   Fig. 7. Chronic depletion of endogenous serotonin affects pharyngeal behavior. (A) In the absence of exogenous serotonin (1 µmoll-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 µmoll-1) and octopamine (100 µmoll-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 affect 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 µmoll-1 serotonin. Hatched bars represent significant differences between measurements from eat-18; tph-1 (this figure) and those from eat-18 (Fig. 3B).

View larger version (17K): [in a new window]   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.

View larger version (16K): [in a new window]   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.

View larger version (12K): [in a new window]   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.

View larger version (8K): [in a new window]   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.