Serotonin and the small cardioactive peptides differentially modulate two motor neurons that innervate the same muscle fibers in Aplysia

Abstract

The anterior portion of intrinsic buccal muscle 3 (I3a) is innervated by two motor neurons, B3 and B38, which appear to use glutamate as their fast excitatory transmitter. B3 and B38 express the neuropeptides FMRFamide and the small cardioactive peptides (SCPs), respectively. We have shown previously that stimulation of B38 causes release of the SCPs from terminals in the muscle. The I3a muscle also receives input from neurons that use 5HT as a modulatory transmitter. The SCPs and 5HT potently facilitated B38-evoked excitatory junction potentials (EJPs) but had only a small effect on B3-evoked EJPs; however, both the SCPs and 5HT strongly potentiated contractions evoked by both B3 and B38, indicating that the two substances must also act on excitation-contraction coupling. The selective facilitation of B38-evoked EJPs, however, did manifest itself in other parameters. Decreases in the firing frequencies and burst durations that were threshold to evoke contractions and decreases in the latency between the onset of a burst and the onset of the evoked contraction were all much larger for B38 than for B3. Indeed, B38 bursts recorded during feeding-like behavior would be subthreshold for evoking contractions in the absence of this modulation. All of the effects of the SCPs reversed during washout, whereas those of 5HT were persistent, lasting many hours after washout. Thus, the SCPs and 5HT dramatically change the behavioral output of these motor neurons, increasing the amplitude of contractions evoked by both B3 and B38, and shifting the temporal relationship between bursts in B38 and its evoked contractions.

Fig. 1.

Effects of 1 μm SCP_A or 1 μm 5HT on EJPs recorded intracellularly in I3a muscle fibers. A, Selective effects of SCPs and 5HT on EJPs evoked by B3 and B38. Note that B38-evoked EJPs were facilitated markedly by both the SCPs and 5HT, whereas these substances had little effect on the amplitude of B3-evoked EJPs. B, Time courses of the effects of the SCPs and 5HT on B38-evoked EJPs. Records for 5HT or the SCPs are each from a single muscle fiber. Application of the SCPs caused a 4 mV hyperpolarization from a rest potential of −72 mV; 5HT caused an 8 mV hyperpolarization from a rest of −78 mV. Note that the effects of the SCPs reversed during washout, whereas those of 5HT persisted for at least 3 hr.

Fig. 2.

Effects of 1 μm SCP_A or 1 μm 5HT on EJPs recorded extracellularly in I3a muscle using a perfusion electrode. A, Effects of SCPs and 5HT on EJPs evoked by stimulating B38. Note that the effects of the SCPs reversed during washout, whereas those of 5HT persisted for at least 3 hr. Recordings are from different experiments. B, Cumulative results for the SCPs and 5HT on B38-evoked EJPs (n = 11). To pool results from different neuromuscular preparations, EJPs were normalized to the maximum EJP amplitude (set at 1.0). 5HT or the SCPs were applied from −20 to 0 min. Note that the effects of the SCPs reverse during washout, whereas those of 5HT persisted for at least 1 hr of washout. The small upward inflection of EJP amplitude observed at the end of 5HT perfusion suggests that 5HT may also have a small inhibitory effect that reverses during washout.

Fig. 4.

Summary of the effects of the SCPs and 5HT on extracellularly recorded EJPs and contractions. Top, The effect of 1 μm SCPs or 1 μm 5HT on EJPs evoked by the stimulation of B3 or B38. The mean amplitude of the third EJP in the bursts was used because the first EJP was often too small to measure reliably (e.g., Fig. 2). The B38-evoked EJPs were dramatically facilitated by both the SCPs (n = 12;p ≤ 0.01; t test) and 5HT (n = 11; p ≤ 0.01), whereas the B3-evoked EJPs were only slightly facilitated by the SCPs (n = 12; p ≤ 0.01) or 5HT (n = 10; not significant). The SCPs were more effective at increasing EJPs than 5HT (B3, p ≤ 0.05; paired t test; B38, p ≤ 0.01). Bottom, The effect of 1 μm SCPs or 1 μm 5HT on contractions evoked by the stimulation of B3 or B38. Both the SCPs and 5HT increased the amplitude of contractions evoked by both motor neurons (n = 4;p ≤ 0.01). Note that 5HT was more effective than the SCPs at potentiating contractions (p ≤ 0.01) and that the SCPs were more effective at potentiating contractions evoked by B38 than those evoked by B3 (p ≤ 0.05). The values for the effects of the SCPs on the amplitude of B3-evoked EJPs and contractions are very similar to those reported previously (Church et al., 1993).

Fig. 3.

Effects of the SCPs and 5HT on I3a muscle contractions. A, Effects of 1 μmSCP_A on contractions evoked by stimulating B3 or B38. Bursts were alternately fired in B3 or B38 (16 Hz for 1.6 sec at interburst interval of 100 sec). The first contraction is in ASW, the second is after 20 min superfusion with the SCPs, and the third is after 1 hr wash in ASW. Note that the effects of the SCPs reverse during washout. B, Effects of 1 μm 5HT on contractions evoked by alternately stimulating B3 or B38. Bursts were alternately fired in B3 (16 Hz for 1.3 sec at interburst interval of 100 sec) or B38 (16 Hz for 1.6 sec at interburst interval of 100 sec). Note that the effects of 5HT persisted for at least 3 hr. Recordings are from different experiments.

Fig. 5.

Cumulative results for the effects of the SCPs and 5HT on motor neuron-evoked contractions. Top, Time course of the effects of SCPs and 5HT on B38-evoked contractions (n = 7). Bottom, Time course of the effects of SCPs and 5HT on B3-evoked contractions (n = 8). To pool results from different preparations, contraction amplitudes were normalized to the maximum contraction amplitude (set at 1.0). SCP_A (1 μm) or 5HT (1 μm) were applied from −20 to 0 min. Note that the effects of the SCPs reversed during washout, whereas those of 5HT persisted for at least 1 hr of washout.

Fig. 6.

Cumulative results for the effects of SCPs and 5HT on relaxation time constants of motor neuron-evoked contractions.Top, Time course of the effects of SCPs and 5HT on the relaxation time constants of B38-evoked contractions (n = 3). Bottom, Time course of the effects of SCPs and 5HT on the relaxation time constants of B3-evoked contractions (SCPs, n = 4; 5HT,n = 3) To pool results from different neuromuscular preparations, relaxation time constants were normalized to control (set at 1.0; decreasing time constants indicate more rapid relaxation rates). SCP_A (1 μm) or 5HT (1 μm) were applied from −20 to 0 min (indicated byblack bars). Note that the effects of the SCPs reverse during washout, whereas those of 5HT continued to increase throughout the washout. It is difficult to interpret the relaxation rate results in light of the large changes in contraction amplitude, but it is clear that the effects of 5HT on amplitude and relaxation rate were persistent.

Fig. 7.

Effects of the SCPs and 5HT on I3a muscle contractions evoked by bolus applications of glutamate.A, Superfusion with 0.1 μm 5HT or 0.1 μm SCP_A increased the contractions evoked by boluses of 100 nmol glutamate (upward arrows). The first contraction is in ASW, the second is after 15 min superfusion with the SCPs or 5HT. B, Superfusion of higher concentrations (1 μm) of the SCPs or 5HT (horizontal bar) caused I3a muscles to contract rhythmically.

Fig. 8.

Effects of the SCPs and 5HT on the latency between the onset of a motor neuron burst and the onset of the resulting I3a contraction. A, Muscle contractions were evoked by stimulation of B3 or B38. Bursts of spikes were fired alternatively in B3 or B38 (16 Hz for 1.6 sec at interburst interval of 100 sec) for the application of the SCPs. B3 bursts were increased to 1.7 sec before application of 5HT so that the contractions evoked by B3 and B38 would be of similar amplitude. The timing of the onset of muscle contractions is emphasized by recording at a fast time base and high gain. As a result, most of the contractions are off scale. Superfusion with either 1 μm SCP_A or 1 μm 5HT increased the amplitude of muscle contractions evoked by both B3 and B38 (visible as an increased rate of rise in the contraction traces; also see Fig.3); however, the reduction in latency between the onset of a motor neuron burst and the onset of the resulting contraction was reduced much more dramatically for contractions evoked by B38 than it was for contractions evoked by B3. B, The effects of 5HT are persistent, whereas those of the SCPs reverse during washout. The first contraction is in ASW, and the second is after 20 min superfusion with either 1 μm 5HT or 1 μm SCP_A. Subsequent contractions are after washout in ASW for the indicated periods. All recordings were from a single experiment. Black arrowheads indicate the onset of the contraction.

Fig. 11.

Summary of the effects of 1 μm SCPs and 1 μm 5HT on (1) the threshold firing frequency required to elicit contractions, (2) the latency between the onset of the burst and onset of the evoked contraction, and (3) the threshold burst duration required to elicit contractions. A, The threshold firing frequency to elicit B38-evoked contractions was reduced by the SCPs and 5HT (n = 3;p ≤ 0.01; t test). The threshold firing frequency for B3 was reduced by 5HT (n = 3;p ≤ 0.01), whereas the effect of the SCPs was not significant (n = 3). B, The latency between the onset of the burst and onset of the B38-evoked contraction was reduced by the SCPs and 5HT (n = 5;p ≤ 0.01). This latency for B3 was also reduced by 5HT (n = 6; p ≤ 0.01), whereas the effect of the SCPs was not significant (n = 6). C, The threshold burst duration to elicit contractions was reduced by the SCPs and 5HT for B38-evoked contractions (n = 5;p ≤ 0.01). Note that the SCPs and 5HT were more effective at reducing contraction latency and threshold burst duration for B38-evoked contractions than for B3-evoked contractions (p ≤ 0.01; paired ttest)

Fig. 9.

Effects of firing frequency and 5HT on the latency between the onset of a B38-evoked burst and the onset of the resulting I3a contraction. Contractions were evoked by alternately stimulating B3 or B38 (4 sec bursts at interburst interval of 100 sec). Only recordings from B38 are shown. Stimulation frequency was increased 1 spike/sec (Hz) during the interburst intervals and is indicated next to the motor neuron burst. Superfusion with 1 μm 5HT dramatically decreased the latency between the onset of the B38-evoked burst and the onset of the contraction. Note that 5HT reduced the latency to an extent similar to that produced by ∼4 Hz increase in stimulation frequency (compare 12 Hz in ASW with 8 Hz in 5HT). The first contraction at each frequency is in ASW, and the second is after 10 min superfusion with 5HT, so the effects are nearly maximal (see Fig. 5). Black arrowheads indicate the onset of the contraction.

Fig. 10.

Effects of the SCPs and 5HT on the latency of contractions evoked by firing the motor neuron at different frequencies. Contractions were evoked by alternately stimulating B3 or B38 (4 sec bursts at interburst interval of 100 sec) at frequencies between 8 and 13 Hz. Stimulation frequency was increased 1 spike/sec (Hz) during the interburst intervals preceding the B38-evoked bursts. It was not possible to present pooled results because the threshold frequencies varied widely between preparations, so representative examples are shown.A, SCP_A (1 μm) had little effect on the relationship between frequency and latency for B3-evoked contractions. B, 5HT (1 μm) shifted this relationship 1–2 Hz lower for B3. C, SCP_A(1 μm) shifted this relationship between frequency and latency ∼1 Hz for B38-evoked contractions. D, 5HT (1 μm) shifted this relationship ∼4 Hz lower for B38. Note that the SCPs and 5HT did not change the slope of the relationship between frequency and latency, suggesting that they do not modulate frequency-dependent facilitation within a burst.