Serotonergic Neurons Differentially Modulate the Efficacy of Two Motor Neurons Innervating the Same Muscle Fibers in Aplysia

Lyle E. Fox and Philip E. Lloyd

Committee on Neurobiology and Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Fox, Lyle E. and Philip E. Lloyd. Serotonergic neurons differentially modulate the efficacy of two motor neurons innervating the same muscle fibers in Aplysia. J. Neurophysiol. 80: 647-655, 1998. Feeding behavior in Aplysia shows substantial plasticity. An important site for the generation of this plasticity is the modulation of synaptic transmission between motor neurons and the buccal muscles that generate feeding movements. We have been studying this modulation in the anterior portion of intrinsic buccal muscle 3 (I3a), which is innervated by two excitatory motor neurons and identified serotonergic modulatory neurons, the metacerebral cells (MCCs). We have shown previously that serotonin (5-HT) applied selectively to the muscle potently modulates excitatory junction potentials (EJPs) and contractions. All the effects of 5-HT were persistent, lasting many hours after wash out. We examined whether the release of endogenous 5-HT from the MCC could produce effects similar to the application of 5-HT. Stimulation of the MCCs did produce similar short-term effects to the application of 5-HT. MCC stimulation facilitates EJPs, potentiates contractions, and decreases the latency between the onset of a motor neuron burst and the onset of the evoked contraction. The effects of MCC stimulation reached a maximum at quite low firing frequencies, which were in the range of those previously recorded during feeding behavior. The maximal effects were similar to those produced by superfusion with ~0.1 µM 5-HT. Although the effects of MCC stimulation on EJPs were persistent, they were less persistent than the effects of 0.1 µM 5-HT. Mechanisms that may account for differences in the persistence between released and superfused 5-HT are discussed. Thus activity in the MCCs has dramatic short-term effects on the behavioral output of motor neurons, increasing the amplitude and relaxation rate of contractions evoked by both B3 and B38 and shifting the temporal relationship between B38 bursts and evoked contractions.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

A major goal of neurobiology is to understand the mechanisms underlying behavioral plasticity. Much of our current knowledge about the mechanisms and significance of behavioral plasticity has come from studies of simple behaviors in invertebrate preparations. Behavioral plasticity often arises from changes in the output from the central pattern generators. However, in many preparations, it has become increasingly clear that another locus of plasticity is peripheral at neuromuscular junctions and muscles. These are the sites where the central output is transduced to behavior. One of the most extensively studied preparations in which peripheral modulation appears to be well expressed is the buccal muscles that generate feeding movements in Aplysia. These muscles are modulated by neuropeptides intrinsic to the motor neurons and by serotonin (5-HT) released from modulatory neurons that are extrinsic to the motor circuit (Church and Lloyd 1994; Cropper et al. 1987, 1990; Lloyd et al. 1984, 1987; Weiss et al. 1978; Whim and Lloyd 1989, 1990). The preparation we have chosen to study consists of a muscle that participates in the generation of feeding movements termed the intrinsic anterior muscle 3 (I3a). It is innervated by two identified motor neurons, B3 and B38, which likely use glutamate as their fast excitatory transmitter, and B47, which uses acetylcholine as its fast inhibitory transmitter (Church et al. 1993; Fox and Lloyd 1993). All three motor neurons also express modulatory peptide cotransmitters; B3 expresses FMRFamide and an unidentified methionine-containing peptide, B38 expresses the small cardioactive peptides (SCPs), and B47 expresses the myomodulins (Mms) (Church and Lloyd 1991). In addition, I3a is innervated by two giant cerebral neurons termed the metacerebral cells (MCCs). The MCCs are a bilateral pair of large serotonergic neurons that have extensive central and peripheral synaptic outputs (Eisenstadt et al. 1973; Weinreich et al. 1973; Weiss and Kupfermann 1976). The MCCs act centrally to accelerate or trigger bursting activity in motor neurons of the buccal ganglia, act peripherally to modulate muscle contractions, and contribute to an arousal state induced by food stimuli (Weiss et al. 1978). Specifically, they enhance the speed and strength of biting responses. Firing activity of the MCCs recorded in freely behaving animals correlates well with levels of food-induced arousal, and selective lesions of the MCCs affect biting movements but not other aspects of feeding (Kupfermann and Weiss 1982; Rosen et al. 1989).

Excitatory junction potentials (EJPs) and contractions of I3a muscle fibers are modulated by neuropeptide cotransmitters (FMRFamide, SCPs, and Mms) expressed in the motor neurons and 5-HT synthesized by the MCCs (Church et al. 1993; Lotshaw and Lloyd 1990). Application of 5-HT and SCP produce similar short-term effects. They increase the amplitude and relaxation rate of contractions evoked by either B3 or B38, selectively facilitate B38-evoked EJPs, and decrease the latency between the onset of a motor neuron burst and the onset of the resulting contraction much more for B38 than for B3. Although the effects of SCP reversed on wash out, the effects of 5-HT were extremely persistent (Fox and Lloyd 1997).

Although studying the effects from the application of exogenous transmitter are certainly useful, it is also important to determine whether the observed effects occur with the release of transmitter from endogenous sources. Thus we examined the effects of endogenous 5-HT released by stimulation of the MCCs. We were particularly interested in two unusual aspects of the modulation produced by application of 5-HT, the decrease in contraction latency and the persistence of the effects because they would have far ranging behavioral implications. The reduction in the latency of B38-evoked contractions by several seconds compared with B3-evoked contractions might produce a long-lasting change in the relative timing of B3- and B38-evoked contractions. This change in the relative timing of contractions is unusual because it does not require a change in the pattern or frequency of motor neuron bursts. Stimulation of the MCC produced short-term effects that were indeed very similar to those of exogenous 5-HT, and a component of these effects was persistent.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Animals

Aplysia californica (100-300 g) were obtained from Marinus (Long Beach, CA), maintained in circulating artificial seawater (ASW) at 16°C, and fed dried seaweed every 3 days.

Neuron stimulation

Detailed experimental methods have been described previously (Fox and Lloyd 1997). Briefly, animals were immobilized with an injection of isotonic MgCl2 and the dissection carried out in high Ca2+ (33 mM; 3 times normal), high Mg2+ (165 mM; 3 times normal) ASW (termed high Ca, Mg ASW). The buccal mass, buccal ganglia, and cerebral ganglia were removed and the mass bisected along the midline. Buccal nerve 2, which contains the peripheral axons of B3, B38, and the MCCs, and either one or both cerebral-buccal connectives were left intact. The ganglia were desheathed and the buccal ganglia selectively superfused, using tubing with an outlet immediately adjacent to the ganglia, with low Ca2+ (0.5 mM; 0.05 times normal) high Mg2+ (110 mM; 2 times normal) ASW (termed low Ca ASW) to suppress transmitter release because the MCC synapses on many motor neurons in the buccal ganglia and can accelerate or trigger organized bursts (Weiss et al. 1978). In low Ca ASW no central effects of MCC stimulation were observed in buccal motor neurons. Small potentials recorded by electrodes in buccal motor neurons were due to a combination of capacitive coupling and ground electrode polarization from the very large currents used to stimulate the MCC. The cerebral ganglia was selectively superfused with high Ca, Mg ASW to raise the firing thresholds. The buccal and cerebral ganglia were not separated by a barrier, so the remainder of the bath was a mixture of the two ASWs. The bath containing the I3a muscle was superfused with normal ASW and separated by a barrier (except when a perfusion electrode was used; see Measurement of I3a EJPs and contractions) through which ran the intact nerve containing the axons of the motor neurons and MCC (buccal nerve 2). Neurons were normally impaled with two microelectrodes (2-4 MOmega ; filled with 3 M K acetate): one to inject current and one to monitor membrane potential. B3 and B38 were identified by their position, size, and muscle innervation patterns (Church et al. 1993). The MCC was identified in the cerebral ganglia by size and location and was also normally impaled with two electrodes. Individual spikes were driven with long depolarizing pulses (25-40 ms) applied on top of steady-state depolarization to a level just below threshold. The total current (DC and pulses) driving spikes in the MCC was ~1 µA in most preparations. This regimen was developed to improve propagation of action potentials past a presumed region of branch block at a site where the MCC axon branches profusely just proximal to the buccal ganglion (Weiss and Kupfermann 1976). An extracellular suction electrode was placed en passant on buccal nerve 2 to determine whether MCC spikes successfully propagated past this branch point. Unsuccessful propagation of spikes was particularly problematic for the contralateral MCC presumably because its axon branches again at the buccal commissure. In some experiments, we found it impossible to devise stimulation paradigms that would consistently propagate axon spikes into nerve 2 from the contralateral MCC. This may have been due to damage during the dissection as the contralateral MCC axon traverses the length of the surgically desheathed buccal ganglion while the ipsilateral MCC axon branches into nerve 2 before entering the ganglion (Weiss and Kupfermann 1976). Many experiments were carried out by alternatively stimulating bursts in B3 and B38 at 50 s intervals (100 s intervals for each neuron) and stimulating the MCC to fire three bursts of 4 s duration with 6 s interburst intervals between the motor neuron bursts. In other experiments using only one motor neuron, bursts were stimulated in either B3 or B38 at 100 s intervals, and the MCC was stimulated to fire six bursts between the motor neuron bursts. In experiments in which three or more neurons were stimulated, single electrodes in each neuron were used to pass current, and propagated axon spikes were observed using the en passant electrode. MCC stimulation was stopped during motor neuron bursts so the axon spikes of each neuron could be resolved.

Measurement of I3a EJPs and contractions

Individual spikes in motor neurons were driven by brief (10-20 ms) depolarizing current pulses. Experiments were performed at room temperature (~22°C). In all experiments, long interburst intervals (100 s) were used for the motor neurons to minimize release of endogenous peptide cotransmitters and posttetanic potentiation (Church et al. 1993; Lotshaw and Lloyd 1990; Whim and Lloyd 1990). EJPs were recorded with a perfusion electrode (Church et al. 1993; Fox and Lloyd 1997). The perfusion electrode consisted of a small chamber (100 µl) and aperture (~1.5 mm) that was positioned to press firmly down on a portion of the muscle (see Fig. 1 in Church et al. 1993). The inside of this electrode was perfused with normal ASW (containing 5-HT in some experiments) while the rest of the preparation was superfused with low Ca ASW to suppress synaptic transmission and muscle contractions. EJPs were recorded by extracellular electrodes placed inside and just outside the wall of the perfusion apparatus. Signals were amplified using a Grass P15D AC amplifier. This procedure permits us to simultaneously record from a large population of muscle fibers thereby reducing sampling bias. Contraction amplitudes were monitored with an isotonic transducer (Harvard Apparatus), and submaximal contractions were evoked by stimulating B3 or B38. The frequency of action potentials within a burst was usually 16 Hz, but burst durations were adjusted so that EJPs or contractions evoked by B3 and B38 were similar in amplitude.


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FIG. 1. Facilitation of B38-evoked excitatory junction potentials (EJPs) by ipsilateral and contralateral metacerebral cells (MCCs). A: bursts of action potentials driven in B38 at 100 s intervals evoked compound EJPs in the I3a muscle fibers. Stimulation of the ipsilateral MCC (6 bursts at a frequency of 10 Hz and duration of 4 s with 6 s interburst interval repeated 3 times during B38 interburst intervals) facilitated EJPs. Bottom traces: expanded recordings of the compound EJPs. B: identical experiment carried out in the same preparation except the contralateral MCC was stimulated. C: facilitation caused by stimulating the ipsilateral MCC (square ), the 2 MCCs together, and the contralateral MCC (black-square). Stimulation parameters were as described above. In this experiment, action potentials in the MCCs were driven with single electrodes and the neurons' axon spikes monitored with an extracellular en passant electrode on the nerve innervating I3a. Small potentials seen in the B38 recordings during MCC stimulation are due to capacitive coupling and ground electrode polarization.

Dose-response curves were done by increasing the concentration of superfused 5-HT from the lowest (0.001 µM; 1 nM) to the highest concentration (1 µM) at 20 min intervals with no intervening washes. This paradigm was necessary because 5-HT has persistent effects. A similar procedure was used for examining the effects of different frequencies of MCC stimulation. With the use of the bursting paradigm described above, MCC stimulation was increased from the lowest (1 Hz bursts, 0.25 Hz overall firing rate) to the highest frequency (10 Hz bursts, 2.5 Hz overall) at 350 s intervals. This interval was chosen because it allowed us to average the responses to at least two bursts for each motor neuron after the effects of MCC stimulation had stabilized.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Both ipsilateral and contralateral MCCs facilitate B38-evoked EJPs

Motor neurons B3 and B38 send their axons out ipsilateral buccal nerve 2, which is the only one of the three lateral nerves that contains axon branches from both ipsilateral and contralateral MCCs (Weiss and Kupfermann 1976). This raised the possibility that each I3a muscle was innervated by both MCCs. It proved difficult to stimulate the contralateral MCC so it would consistently propagate action potentials past several regions of axonal branching in the buccal ganglia and out contralateral nerve 2. Therefore we only used results from experiments in which en passant extracellular recordings indicated that axon spikes successfully passed the branch points and were propagating toward the I3a muscle (the MCC does not produce junction potentials in I3a muscle fibers). Stimulation of the ipsilateral MCC consistently and reproducibly facilitated B38-evoked EJPs. For example, three sequential 5 min stimulation periods (4 s bursts at 10 Hz with 6 s interburst intervals repeated six times during the B38 interburst intervals), each separated by 30 min increased EJP amplitude 5.59 ± 0.65, 5.22 ± 0.78, and 5.73 ± 0.64 (mean ± SE, n = 5) fold over control. The ipsilateral and contralateral MCCs caused similar facilitation of B38-evoked EJPs (Fig. 1). In three of eight preparations in which the ipsilateral and the contralateral MCCs were stimulated, the contralateral MCC was more effective than the ipsilateral MCC (cf. Fig. 1A with 1B). In four preparations, we stimulated each MCC alone as well as both MCCs together. Stimulation of the ipsilateral MCC facilitated B38-evoked EJPs 5.40 ± 0.95 fold over control. The contralateral MCC produced 82 ± 18%, and both MCCs together produced 123 ± 10% of the facilitation produced by the ipsilateral MCC alone (Fig. 1C). Thus simultaneous stimulation of both MCCs tended to be more effective at facilitating EJPs than stimulation of a single MCC, but the effects were not additive. This result was expected because a twofold increase in 5-HT concentration would be expected to increase EJPs only moderately based on the results from superfusion experiments (see Fig. 5).


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FIG. 5. Pooled results of the effects of MCC stimulation or the application of 5-HT on EJP amplitude (n = 5), contraction amplitude (MCC, n = 6; 5-HT, n = 7), and contraction latency (n = 3). Experiments were carried out as described for Figs. 3 and 4. Data are from experiments in which B3 (square ) and B38 (black-square) were alternately stimulated. C indicates control (no added 5-HT). The amplitude of the 3rd EJPs in a burst were used for these data because they yield a better estimate of facilitation at high concentrations of 5-HT. EJP and contraction experiments are from different preparations. Values are means ± SE.

Effects of MCC stimulation on EJPs and contractions evoked by B3 and B38

We wanted to determine whether MCC stimulation would mimic the effects of exogenously applied 5-HT. These effects include 1) a robust facilitation of B38-evoked EJPs and a much smaller facilitation of B3-evoked EJPs, 2) an increase in the amplitude and relaxation rate of contractions evoked by both B3 and B38, and 3) a decrease in the latency between the onset of a motor neuron burst and the onset of the contraction evoked by both motor neurons, although the effect is more pronounced for B38 (Fox and Lloyd 1997). Stimulation of the ipsilateral MCC was used in these experiments because its axon spike propagated more reliably. Stimulation of the MCC produced a small increase in B3-evoked EJPs and a large increase in B38-evoked EJPs, potentiated contractions evoked by both B3 and B38, and reduced the latency between the onset of a burst and the onset of the evoked contractions more for B38 than B3 (Fig. 2). We also examined the effect of MCC stimulation on the relaxation time constant for B3- and B38-evoked contractions of the I3a muscle. Because the increase in contraction relaxation rates were slow in onset, we used longer stimulation periods similar to the superfusion periods used previously to study this phenomenon (Fox and Lloyd 1997). The relaxation of the contractions was measured in regions in which the amplitude of control and potentiated contractions overlapped and were well fitted to a single exponential. Stimulation of the MCC in 10 Hz bursts for 20 min reduced the relaxation time constant to 77 ± 3% (n = 5) of control for contractions evoked by B3 and to 76 ± 4% for those evoked by B38.


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FIG. 2. Effects of stimulation of the ipsilateral MCC on EJPs and contractions evoked by B3 and B38. Control EJPs and contractions are on the left, those after MCC stimulation are on the right. B3 was stimulated at 100 s intervals. During 3 interburst intervals, the MCC was stimulated (6 bursts at a frequency of 10 Hz and duration of 4 s with 6 s interburst interval). The identical experiment was then carried out using B38. A: MCC stimulation selectively facilitates B38-evoked EJPs. B: MCC stimulation potentiates contractions evoked by both B3 and B38. C: MCC stimulation decreases the latency between the onset of the burst and the onset of the evoked contraction for both B3 and B38, although the effect is more pronounced for B38. Closed pyramids indicate contraction onsets. EJP and contraction experiments are from different preparations.

The patterns and rates of firing of MCCs have been recorded in freely behaving animals (Kupfermann and Weiss 1982; Weiss et al. 1978). In the absence of food stimuli, the MCCs are silent, but they fire regularly when exposed to food and in a bursting pattern when the animal is actually biting (Weiss et al. 1978). Recorded firing rates for the MCC range from 1 to 10 Hz (see DISCUSSION). We wanted to determine the relationship between the rate of MCC stimulation and the magnitude of the effects on EJPs and contractions for the observed firing range (Figs. 3 and 5). We chose to use a pattern similar to the bursting pattern (4 s bursts every 10 s) because MCC axon spikes fatigued less readily with this mode of stimulation. With the use of this pattern, the threshold frequency was ~2 Hz for facilitation of EJPs and ~1 Hz for both the potentiation of contractions and the decrease in latency. All three effects reached a plateau at ~6 Hz with only small increases in response to higher frequency stimulation. Because an intermittent bursting pattern was used (MCC stimulation was stopped during motor neuron bursts so that axon spikes of each neuron could be resolved), 1 Hz bursts correspond to an overall firing rate of 0.25 Hz, whereas 6 Hz bursts correspond to 1.5 Hz. Dose-response curves for 5-HT were carried out in parallel to allow us to estimate the concentrations of superfused 5-HT necessary to cause the same magnitude responses as MCC stimulation (Figs. 4 and 5). For EJPs, contraction amplitude, and contraction latency, we estimate that stimulation of MCC with 6-10 Hz bursts produces effects similar to the application of 0.1-0.3 µM 5-HT.


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FIG. 3. Examples of the effects of stimulating the ipsilateral MCC at different rates on EJPs and contractions. B3 and B38 were alternately stimulated at 50 s intervals. EJPs and contractions evoked by B3 are indicated by dots. For EJPs and contractions, 2 responses to B3 or B38 bursts are shown after the response amplitude had stabilized to the new MCC frequency. A: MCC stimulation selectively facilitated B38-evoked EJPs (MCC was stimulated to fire 3 bursts of 4 s duration with 6 s interburst interval at the indicated frequencies during 7 consecutive periods between motor neuron bursts). B: MCC stimulation potentiates contractions evoked by both B3 and B38, although the effect on B38 was larger. C: MCC stimulation reduces the latency between the onset of the motor neuron burst (vertical line) and the contractions evoked by both B3 and B38, although the effect on B38 was larger. In these experiments, action potentials in the motor neurons were driven with single electrodes and the axon spikes monitored with an extracellular en passant electrode. Closed pyramids indicate contraction onsets. Experiments are from 3 different preparations.


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FIG. 4. Examples of the effects of a series of concentrations of serotonin (5-HT) on EJPs and contractions. B3 and B38 were alternately stimulated at 50-s intervals. EJPs and contractions evoked by B3 are indicated by dots. For EJPs and contractions, 2 responses to B3 or B38 bursts are shown after the response amplitude had stabilized to the new concentration of 5-HT. C indicates control (no added 5-HT). A: 5-HT selectively facilitated B38-evoked EJPs with a threshold below 0.01 µM (10-8 M). B: 5-HT potentiates contractions evoked by both B3 and B38. The threshold was below 0.01 µM for B38. C: 5-HT reduces the latency between the onset of the motor neuron burst (vertical line) and the contractions evoked by both B3 and B38, although the effect on B38 was larger. In these experiments, action potentials in the motor neurons were driven with single electrodes and the axon spikes monitored with an extracellular en passant electrode. Closed pyramids indicate contraction onsets. EJP and contraction experiments are from different preparations.

Does stimulation of the MCC persistently facilitate B38-evoked EJPs?

Although all the effects of 5-HT on the I3a neuromuscular preparation were persistent, we concentrate here on the selective facilitation of B38-evoked EJPs because these provide the most stable recordings (Fox and Lloyd 1997). We have shown previously that 20 min superfusion with 1 µM 5-HT causes persistent facilitation that lasts for hours (Fox and Lloyd 1997), however shorter applications or lower concentrations of 5-HT produced effects that do reverse on wash out (Lotshaw and Lloyd 1990). Because the degree of persistence appeared to be dependent on both the concentration of 5-HT and the duration of application, we tested the effects of 20 min applications of 0.1 and 1 µM 5-HT and a 60 min application of 0.1 µM 5-HT on B38-evoked EJPs. We were most interested in the rates of reversal of facilitation during wash out, so EJPs were normalized to their amplitude at the onset of the ASW wash (time 0 in Figs. 6, 7, and 8). In this mode of normalization, EJP amplitude before 5-HT superfusion or MCC stimulation was set at 0 (no increase), and the EJP amplitude at the beginning of the ASW wash was set at 1.0 (the actual mean percentage increase in EJP amplitude is given in the figure legends). All three of the 5-HT applications produced persistent facilitation. The persistence appears to be a specific effect of 5-HT because the effects of other modulators readily reverse in similar experiments. For example, 1 µM SCP facilitates EJPs much more than 1 µM 5-HT, but the effects fully reverse to control values within 40 min of ASW wash (Fox and Lloyd 1997). EJPs were still markedly elevated compared with control after 60 min wash for all of the 5-HT applications. The level of persistence was indeed dependent on both the concentration of 5-HT and the duration of application. We found that 20 min superfusion with 0.1 µM 5-HT did produce persistent facilitation, although it was much smaller in amplitude than that produced by 1 µM 5-HT. We also determined that 60 min superfusion with 0.1 µM 5-HT produced more persistent facilitation than the 20 min superfusion (Fig. 6). Because MCC stimulation at 10 Hz and application of 0.1 µM 5-HT produced similar short-term effects, we chose to compare the persistence produced by 60 min application of 0.1 µM 5-HT with 60 min MCC stimulation at 10 Hz. Although there was certainly a component of the facilitation elicited by MCC stimulation that was persistent, it was smaller than the persistent component following 5-HT superfusion even though the facilitation produced by MCC stimulation and superfusion with 0.1 µM 5-HT were similar in magnitude in these experiments (Fig. 7). The observation that facilitation elicited by the MCC declined little during the period of stimulation indicates that 5-HT was released at a consistent level throughout the 60 min stimulation period. The duration of application in the superfusion experiments appears to be an important determinant of persistence. To test whether this was also true for MCC stimulation, we reduced the stimulation period to 5 min (Fig. 7) and found that the persistent component was indeed smaller than that observed for the 60 min stimulation period. Finally, the effects of stimulating both MCCs were compared with those of stimulating only the the ipsilateral MCC. Although the degree of facilitation is larger when both MCC are stimulated (see Fig. 1), the persistence is very similar to that observed when only one MCC is stimulated (Fig. 8).


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FIG. 6. Time course of the effects of 5-HT on the amplitude of B38-evoked EJPs. In this and the following 2 figures we used a mode of normalization in which EJP amplitude before 5-HT superfusion or MCC stimulation was set at 0 (no increase) and the EJP amplitude at the beginning of the artificial seawater (ASW) wash was set at 1.0. Percentage increases in EJP amplitude at the onset of ASW wash were 378 ± 80% (mean ± SE, n = 7) for 20 min 1 µM 5-HT, 350 ± 99% (n = 4) for 60 min 0.1 µM 5-HT, and 280 ± 120% (n = 5) for 20 min 0.1 µM 5-HT. Note the persistence was dependent on concentration and the duration of application. The 20 min 1 µM 5-HT data are taken from Fig. 2 in Fox and Lloyd (1997). Bursts in B38 were fired at 100 s intervals. Total EJP amplitude was used in this and the following 2 figures because of the large number of data points.


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FIG. 7. Time course of the effects of 5-HT or stimulation of the ipsilateral MCC on the amplitude of B38-evoked EJPs. Increases in EJP amplitude at the onset of ASW wash were 350 ± 99% (n = 4) for 60 min 0.1 µM 5-HT, 368 ± 58% (n = 5) for 60 min MCC stimulation, and 255 ± 31% (n = 7) for 5 min MCC stimulation. Bursts in B38 were fired at 100-s intervals, and the MCC was stimulated (6 bursts at a frequency of 10 Hz and duration of 4 s with 6 s interburst interval) between the B38 bursts for 5 or 60 min. The 60 min 0.1 µM 5-HT data are the same as shown in Fig. 6. Data acquisition was terminated after 30 min wash for the 5 min MCC experiments.


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FIG. 8. Time course of the effects of stimulation of the ipsilateral MCC or of both MCCs on the amplitude of B38-evoked EJPs. Increases in EJP amplitude at the onset of ASW wash were 368 ± 58% (n = 5) for the ipsilateral MCC, and 573 ± 205% (n = 4) for both MCCs. Bursts in B38 were fired at 100 s intervals, and either one or both MCCs stimulated (6 bursts at a frequency of 10 Hz and duration of 4 s with 6 s interburst interval) between the B38 bursts for 60 min. The 60 min ipsilateral MCC data are the same as shown in Fig. 7.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We were interested in determining whether MCC stimulation would emulate all of the effects of 5-HT superfusion. These include 1) the selective facilitation of B38-evoked EJPs, 2) increases in the amplitude and relaxation rates of contractions evoked by both B3 and B38, and 3) a decrease in the latency between the onset of a burst and the onset of the evoked contraction observed for both motor neurons that was considerably larger for B38 (Fox and Lloyd 1997). In addition, superfusion of 5-HT produced effects that were persistent, lasting several hours after the 5-HT was washed out. We did find that MCC stimulation selectively facilitated B38-evoked EJPs and increased the amplitude and relaxation rates of contractions evoked by both motor neurons. The maximal short-term effects of MCC stimulation were similar to those produced by 0.1-0.3 µM 5-HT. Two unusual aspects of the modulation produced by application of 5-HT were the decrease in contraction latency and the persistence of the effects (Fox and Lloyd 1997). MCC stimulation also produces a substantial decrease in the latency between the onset of a burst in a motor neuron and the onset of the resulting contraction that was more pronounced for B38 than for B3. We found that the MCC also caused persistent facilitation that was dependent on the duration of stimulation. However, the degree of persistence following MCC stimulation was considerably smaller than that observed following superfusion with 0.1 µM 5-HT. This was the case even though the 5-HT application and MCC stimulation were the same duration and the short-term effects of MCC stimulation were similar to the short-term effects produced by superfusion with 0.1 µM 5-HT. There are several possible explanations for why 5-HT released from MCC terminals could be less effective than superfused 5-HT in producing persistent facilitation. One explanation is that superfused 5-HT has access to receptors that 5-HT released from MCC terminals does not. These could be extrasynaptic receptors or receptors that are localized to recognize 5-HT released from other possible serotonergic neurons innervating the I3a muscle. There are no 5-HT-immunoreactive neurons in the buccal ganglia where the motor neurons are located, but there are other unidentified serotonergic neurons in the same cluster as the MCC in the cerebral ganglia that may innervate I3a (Longley and Longley 1986). However, these neurons do not appear to send axons out the cerebral-buccal connective, the most direct pathway for axons of cerebral neurons to innervate the I3a muscle and the pathway used by the MCC axons (Rosen et al. 1991). Extrasynaptic 5-HT receptors could be activated by circulating 5-HT in the hemolymph, which has been reported to reach concentrations as high as 0.045 µM (Hattar et al. 1997), well above threshold for the effects of superfused 5-HT described in this study. The source of 5-HT in the blood is unknown, but there are well over 100 serotonin-immunoreactive neurons in the Aplysia CNS, some of which contain more 5-HT than the MCC (Longley and Longley 1986; Ono and McCaman 1984). Another possible explanation for why MCC stimulation produces less persistence than superfusion with 5-HT is that the MCC may co-release other transmitters that inhibit the expression of persistence. We cannot rule this possibility out, but no transmitter other than 5-HT has yet been identified in the MCC of Aplysia, although other transmitters have been reported to be present in homologous neurons in other species (Gillette et al. 1997; Hanley et al. 1974; Osborne et al. 1982). Finally, it is also possible that superfused 5-HT is taken up by neuronal terminals or the muscle fibers and slowly released. Aplysia ganglia are made up of a neuronal component consisting of neurons and neuropil and a sheath consisting of connective tissue and muscle fibers. Both the neuronal component and the sheath take up 5-HT (Bailey et al. 1983; McCaman et al. 1984). However, there is no increase in the levels of 5-HT in either component at the 5-HT concentrations used in our study (McCaman et al. 1984).

We found that stimulation frequencies of individual MCC that were just threshold for facilitating EJPs or potentiating contractions were very low (1-2 Hz in bursts or 0.25-0.5 Hz overall). These values were obtained using stimulation of one MCC and would presumably be reduced somewhat by stimulating both MCCs. Maximal modulatory effects of MCC were also reached at quite low firing frequencies (6 Hz in bursts or 1.5 Hz overall). These maximal effects likely reflect maximal release of 5-HT because high concentrations of superfused 5-HT produced larger effects. We know that MCC axon spikes were successfully propagating into the nerve leading to I3a at all frequencies tested, but there may be limits on the ability of spikes to fully propagate to MCC terminals. Alternately, there may be limits on the capacity of the release mechanisms in the terminals. Patterns and rates of firing of MCCs have been recorded in freely behaving animals using implanted extracellular electrodes (Kupfermann and Weiss 1982; Weiss et al. 1978). Both MCCs show similar patterns of activity and fire in phase with each other. In the absence of food stimuli, the MCCs are silent but begin to fire regularly at 2-10 Hz when food is placed on the lips. During biting, MCCs fire at lower overall frequencies (1-2 Hz) but in bursts that occur between bites. The frequencies of MCC stimulation we have used lie well within these ranges, suggesting that the modulation we have described is likely to occur during feeding in Aplysia. Thus, in the absence of compensatory changes in motor neuron bursts, I3a contractions evoked by both motor neurons would be larger and relax more rapidly. In addition, B38-evoked EJPs would be facilitated and contraction latencies reduced. This would lead to a change in the relative timing of contractions evoked by B3 and B38, the two major motor neurons innervating I3a.

It is important to emphasize that during vigorous feeding, the motor neurons innervating I3a fire bursts with interburst intervals much shorter than used in this study, which would cause release of their modulatory peptide cotransmitter. These include SCP released from B38, myomodulins from B47, and FMRFamide and an unidentified peptide from B3 (Church et al. 1993). Perhaps most relevant to the present study is the release of SCP because it causes the same short-term effects as 5-HT including a reduction in contraction latency. Indeed, SCP functioning along with 5-HT may produce a more dramatic shift in relative latency between the two excitatory motor neurons because SCP reduces the latency more selectively for B38 compared with B3 than does 5-HT. Indeed, it is striking how the neuropeptide modulators working in concert with 5-HT would dramatically change the input-output relationship of the motor neurons and the I3a muscle. It is also interesting to note that in another buccal muscle in Aplysia, MCC stimulation increases the release of the SCPs from one of the motor neurons that innervates the muscle (Vilim et al. 1996). Finally, the presence of multiple modulatory substances greatly increases the flexibility with which synapses can be regulated (Brezina et al. 1996).

Modulation of neuromuscular synapses also appears to be widespread in invertebrates (Calabrese 1989). Indeed, the effects of 5-HT on B38-evoked EJPs and contractions are similar to those observed previously in crustacean preparations where 5-HT has been shown to facilitate EJPs (Dixon and Atwood 1985; Dudel 1965; Glusman and Kravitz 1982; Kravitz et al. 1980). This effect has been shown to be mediated by an increase in transmitter release, perhaps involving a change in presynaptic calcium metabolism. Furthermore, the effects of 5-HT reversed on wash out in two phases, one rapid and one much slower. 5-HT also has an effect on muscle contractility in lobster (Harris-Warrick and Kravitz 1984). Finally, the actions of 5-HT on crustacean neuromuscular synapses appear to be mediated through increased cAMP levels as well as by other second-messenger systems (Dixon and Atwood 1989a,b; Goy and Kravitz 1989).

Synapses in the central ganglia of Aplysia are also facilitated by 5-HT. Indeed, this heterosynaptic facilitation of sensory to motor neuron synapses is similar to what we observed for B38-evoked EJPs (Byrne and Kandel 1996; Carew et al. 1971; Castellucci et al. 1970; Mackey et al. 1989). In both cases, 5-HT persistently facilitates synaptic potentials (Montarolo et al. 1986). The facilitation of I3a synapses resembles the short and intermediate forms of facilitation of the sensory neuron synapses because it is not suppressed by inhibitors of protein synthesis (Fox and Lloyd 1995; Ghirardi et al. 1995).

The effects of MCC stimulation or application of 5-HT have been investigated in detail in another buccal muscle in Aplysia, the accessory radula closer (ARC). This muscle is innervated by two cholinergic motor neurons and only the ipsilateral MCC. Stimulation of the MCC increases the amplitude and the relaxation rate of contractions evoked by both motor neurons (Weiss et al. 1978, 1992). There are some similarities in the effects of MCC stimulation or 5-HT on the I3a and the ARC. Contractions in both muscles are potentiated by a mechanism that involves a cAMP-mediated increase in the efficiency of excitation-contraction coupling (Lotshaw and Lloyd 1990; Weiss et al. 1979). Extensive recent work has characterized both resting conductances and conductances that are modified by modulators (including 5-HT) in dissociated ARC muscle fibers including a voltage-gated calcium current that is probably responsible for the increase in excitation-contraction coupling (e.g., Brezina et al. 1994). Also there is evidence that phosphorylation by cAMP-dependent protein kinase of a molluscan analogue of twitchin, a large contractile protein, likely underlies the increased rates of relaxation produced by 5-HT and other modulators (Probst et al. 1994). It seems probable that some of these mechanisms are also present in I3a muscle fibers. However, there are also major differences between serotonergic modulation in the ARC and I3a. In the ARCs, both neurons appear to be modulated similarly. There is also little facilitation of EJPs or reduction in contraction latency for either motor neuron. This contrasts with the I3a muscle where the two motor neurons are modulated very differently. Modulatory mechanisms are now beginning to be elucidated at other buccal muscles as well (Evans et al. 1996; Scott et al. 1997). Comparing the similarities and differences in the mechanisms of modulation of these buccal muscles should help us develop a better understanding of the roles of peripheral modulation in shaping behavior.

    ACKNOWLEDGEMENTS

  This work was supported by National Research Service Award 1-F31-MH10656 to L. E. Fox and BNS-9418815 to P. E. Lloyd.

    FOOTNOTES

  Address for reprint requests: P. E. Lloyd, Committee on Neurobiology, University of Chicago, 947 E. 58th St., Chicago, IL 60637.

  Received 23 December 1997; accepted in final form 28 April 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society