Differential Modulation of Motor Neurons That Innervate the Same
Muscle but Use Different Excitatory Transmitters in Aplysia
CHRISTOPHER KEATING AND PHILIP E. LLOYD
Committee on Neurobiology and Department of Neurobiology, Pharmacology, and Physiology, University of Chicago,
Chicago, Illinois 60637
INTRODUCTION
The coexistence of multiple transmitters in neurons has
proven to be an important means by which neuronal circuits
can influence synaptic plasticity. Typically, a neuron contains
a single conventional neurotransmitter, such as acetylcholine
or glutamate, and one or more families of peptide cotransmitters that can act as modulators of synaptic transmission. Neuromuscular synapses have been used extensively for the study
of synaptic physiology. Whereas the role of conventional transmitters in synaptic transmission is well established at neuromuscular synapses, less is known about the precise physiological effects of peptide cotransmitters at these synapses.
Modulation of neuromuscular synapses has been extensively
studied, especially in invertebrate preparations (Calabrese
1989; Cropper et al. 1987; Jorge-Rivera et al. 1998; Kravitz et
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al. 1980; Lu et al. 1993; O’Shea and Schaffer 1985; Worden
1998), and we are continuing to investigate these processes
using the synapses between motor neurons and their target
muscles in Aplysia. Specifically, we are studying the motor
neurons in the buccal ganglia that generate the cyclic motor
output that drives biting and swallowing movements of muscles of the buccal mass (Kupfermann 1974). This system has
been used extensively to investigate the neural mechanisms
underlying synaptic plasticity (Brezina et al. 1996; Weiss et al.
1992, Whim et al. 1993).
The buccal ganglia of Aplysia contain many large identifiable motor neurons that innervate discrete muscle fields in the
buccal mass and have been shown to express a variety of
peptide cotransmitters (Church and Lloyd 1991; Lotshaw and
Lloyd 1990). This information allowed us to choose a preparation amenable for studying the role of these transmitters. This
neuromuscular preparation consists of the medial region of
intrinsic muscle 3 (I3m) that is innervated by six identified
motor neurons, all of which express peptide cotransmitters.
Three of them express SCP, and three express FMRFamide
(Church and Lloyd 1991). In the present study, we focus on
B3, which likely uses glutamate as its conventional transmitter
and expresses FMRFamide and a second methionine-containing peptide that has yet to be identified, and B9, which is
cholinergic and expresses SCP (Church and Lloyd 1991; Fox
and Lloyd 1999; Lloyd and Church 1994). These neurons were
chosen for two reasons. They use different fast excitatory
transmitters, and their fields of innervation overlap extensively.
This allowed us to study the effects of peptide cotransmitters
released from neuronal terminals in the absence of large
postsynaptic effects elicited by the conventional transmitter.
Specifically, we could selectively inhibit excitatory junction
potentials (EJPs) and contractions produced by B9 using a
cholinergic antagonist. We used this preparation to investigate
the role of the peptide cotransmitters expressed in B3 and B9.
First, we studied the effects of the application of exogenous
FMRFamide and SCP on EJPs and contractions evoked by B3
and B9. This is the first example in buccal muscle of the
actions of peptide cotransmitters on two motor neurons that
innervate the same muscle and use different fast excitatory
transmitters. Next, we investigated the effects of stimulating
B9 on B3-evoked contractions in the presence of a cholinergic
antagonist. B9 was stimulated in a bursting pattern similar to
that observed during feeding-like activity (Church and Lloyd
1994). Application of SCP to buccal muscle caused large
increases in cAMP levels. Thus measuring the effects of B9
stimulation on cAMP levels in the muscle provides an indirect
0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society
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Keating, Christopher and Philip E. Lloyd. Differential modulation
of motor neurons that innervate the same muscle but use different
excitatory transmitters in Aplysia. J. Neurophysiol. 82: 1759 –1767,
1999. The medial portion of intrinsic buccal muscle 3 (I3m) is
innervated by two excitatory motor neurons, B3 and B9. B3 uses
glutamate as its fast transmitter and expresses the neuropeptide
FMRFamide, whereas B9 uses acetylcholine as its fast transmitter and
expresses the neuropeptide SCP. This preparation was used to study
peptidergic modulation of muscles innervated by neurons that use
different fast excitatory transmitters. First, we determined the effects
of the application of the neuropeptides expressed in these neurons on
excitatory junction potentials (EJPs) and contractions. FMRFamide
increased the amplitude of EJPs and contractions evoked by B3 while
decreasing those evoked by B9. This is the first observation in buccal
muscle of a substance that modulates two excitatory neurons innervating the same muscle in opposite directions. SCP increased EJPs
contraction amplitude, and the rate of muscle relaxation for both
motor neurons. We determined that SCP potently increased cAMP
levels in I3m as it does in other buccal muscles. Stimulation of B9 also
caused increased cAMP levels in I3m providing independent evidence
for SCP release. Finally, stimulation of B9 increased both the contraction amplitude and relaxation rate of B3-evoked I3m contractions
in a manner similar to that observed using exogenous SCP. By
inhibiting B9’s cholinergic transmission with an antagonist, we were
able to observe modulatory effects of B9 in the absence of fast
excitatory effects. We found that the magnitude of the modulation was
dependent on the firing frequency and did occur at frequencies and
patterns of firing recorded previously for B9 during ingestive-like
motor programs.
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C. KEATING AND P. E. LLOYD
but independent means of demonstrating the release of SCP
(Church et al. 1993; Cropper et al. 1990; Whim and Lloyd
1989, 1990). We found that stimulation of B9 both increased
cAMP levels in I3m and modulated contractions in a manner
very similar to the application of SCP. A major goal of our
research is to ascertain the behavioral consequences of peptide
release. Thus we determined that peptidergic modulation occurred at physiological firing rates using stimulation paradigms
similar to the patterns of activity recorded in B9 during fictive
feeding-like activity (Church and Lloyd 1994).
To date, despite extensive investigation, we have been unable to find an effective antagonist for the glutamatergic neuromuscular receptors (Fox and Lloyd 1999), so we have not
been able to carry out the symmetrical experiment of determining the effects of B3 stimulation of B9-evoked contractions.
Animals
Aplysia californica (90 –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.
Measurement of I3m EJPs
Neurons were impaled and stimulated as described above. EJPs
were recorded via a perfusion electrode (Church et al. 1993; Fox and
Lloyd 1997). Briefly, this electrode consisted of a small chamber (250
ml) with a small aperture (;1.5 mm diam) that was positioned to
firmly press down on a portion of I3m. The inside of this chamber was
superfused rapidly with ASW (;6 volumes/min). The remainder of
the muscle outside of the recording chamber was superfused with low
Ca21 (0.5 mM; 0.05 3 normal) high Mg21 (110 mM; 2 3 normal)
ASW (termed low Ca ASW) to block synaptic transmission and
muscle contractions. This procedure confined the contractions to the
small area of the muscle covered by the recording chamber and thus
markedly reduced movement artifacts in the recordings. Muscle contractions do not begin until after the 10th EJP so at least the 1st 10
EJPs of a burst (at 15 Hz) are recorded in the absence of any
movement. EJPs were recorded by extracellular electrodes placed
inside and just outside the wall of the perfusion apparatus. Stimulation
parameters were similar to those used to evoke contractions. Signals
were amplified using a Grass P15D A.C. amplifier. Peptides to be
tested for their actions on evoked EJPs were applied in ASW through
the perfusion electrode.
Identification of buccal motor neurons
Animals were immobilized with an injection of isotonic MgCl2 and
dissected in a high Mg21 (165 mM; 3 3 normal), high Ca21 (33 mM;
3 3 normal) ASW (termed high Ca, Mg ASW). The buccal mass/
buccal ganglia complex was removed and bisected along the midline,
and all nerves were severed except buccal nerves 2 and 3 (nerve
designations from Gardner 1971; muscle nomenclature from Howells
1942; see also Lloyd 1988). The hemiganglion was desheathed and
perfused with high Ca, Mg ASW to raise the firing thresholds of
neurons in the ganglion. The remainder of the bath containing the I3
muscle was separated by a barrier (except when a perfusion electrode
was used; see Measurement of I3m EJPs) through which nerves 2 and
3 ran, and was superfused with ASW. Neurons were impaled with two
microelectrodes (3– 4 MV; filled with 3 M K acetate, 30 mM KCl,
0.1% Fast green), one to inject current, and the other to monitor the
membrane potential. Motor neurons were identified by their muscle
innervation pattern, action potential shape, pharmacological properties, and axonal projections recorded with a suction electrode (Church
and Lloyd 1991).
Measurement of I3m contractions
Individual spikes were driven by brief (10 –20 ms) depolarizing
current pulses. The I3 muscle is contiguous but can be divided into
anterior (I3a), medial, and posterior (I3p) segments by the pattern of
contractions produced by identified motor neurons. For example, B3
causes contractions of the anterior and medial segments, B9 the
medial and posterior segments, and B38 only causes anterior contractions (Church and Lloyd 1991). The anterior extent of I3m is also
defined by the location of a white cartilage-like tissue that is confined
to the inner side of I3a. Once the region of I3m containing overlapping
B3 and B9 innervation was identified, it was mechanically isolated
from the other segments by a combination of small incisions and pins.
Reproducible submaximal I3m contractions were evoked by stimulating either motor neuron every 100 s at ;15 Hz in 0.5- to 1-s bursts.
The long interburst intervals were used to minimize the release of
endogenous peptides (Whim and Lloyd 1989). Contractions were
monitored with an isotonic transducer (Harvard Apparatus). Experiments were performed at room temperature (;22°C). Peptides to be
tested for their actions on evoked contractions were dissolved in ASW
Measurement of cAMP
Levels of cAMP in I3 muscle were measured as previously described for the I5 muscle (Whim and Lloyd 1989). Segments of I3m
were weighed, incubated at room temperature for 2 h in three changes
of ASW, and then for 20 min in ASW containing known concentrations of peptide. All incubations were carried out on a rotary shaker in
a volume of ASW at least 1,000-fold greater than that of the muscle
segment. Muscle segments were extracted in glass homogenization
tubes containing 300 ml 1% 1N HCl in ethanol, maintained at 230°C,
homogenized and stored at 220°C. The tubes were centrifuged at
10,000 g, and the supernatants were used for cAMP determinations
using a commercial cAMP-radioimmunoassay (Biotechnologies).
cAMP levels in I3 muscle segments were also measured following
stimulation of B9. These experiments were performed at 15°C because previous experiments carried out using other buccal neuromuscular preparations indicated that stimulated peptide release increased
at lower temperatures (Vilim et al. 1996; Whim and Lloyd 1990). B9
was either hyperpolarized, or stimulated (4-s 10-Hz bursts with 6-s
interburst intervals) for 10 min, and muscles were frozen with an
electronic component freezer spray (Miller-Stephenson Chemical,
Danbury, CT) and separated into anterior, medial, and posterior segments while frozen. Individual segments were then homogenized as
described above. cAMP was determined by radioimmunoassay. It was
not possible to weigh the frozen muscles so cAMP was normalized to
protein determinations using a BCA kit (Pierce Chemicals) carried out
on 10,000 g pellets extracted for 10 min in 0.1 N NaOH at 100°C.
RESULTS
Modulation of motor neuron evoked I3m EJPs and
contractions by neuropeptides
Superfusion of FMRFamide selectively over I3m differentially modulated EJPs in that B3-evoked EJPs were facilitated
and B9-evoked EJPs inhibited (Figs. 1 and 3). FMRFamide
also differentially modulated contractions. Figure 2 shows the
effects of 1028 M FMRFamide on I3m contractions. The
amplitude of I3m contractions evoked by B3 were potentiated,
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METHODS
and applied via the superfusion. Note that the ganglion was separately
superfused with high Ca, Mg ASW and was not exposed to these
substances (see Identification of buccal motor neurons).
DIFFERENTIAL MODULATION OF NEUROMUSCULAR SYNAPSES
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whereas those evoked by B9 were inhibited. These effects
increased with concentration for B9 but not for B3 (Fig. 3).
FMRFamide also moderately decreased resting tone of I3m.
The relaxation rates of I3m contractions evoked by both B3
FIG. 1. Effects of FMRFamide on I3m excitatory junction potentials (EJPs)
evoked by B3 or B9. In each pair of traces, the top traces are extracellular
recordings from I3m, the bottom traces are intracellular recordings from motor
neurons. A: EJPs were evoked by stimulation of B3 at 15 Hz for 0.9 s with
100 s interburst intervals. Superfusion of FMRFamide over I3m facilitated
EJPs. B: B3-evoked EJPs with an expanded time scale before (1) and after (2)
superfusion with FMRFamide. C: EJPs were evoked by stimulation of B9 at 15
Hz for 0.5 s with 100-s interburst intervals. Superfusion of FMRFamide
inhibited the amplitude of EJPs. D: B9-evoked EJPs before (1) and after (2)
superfusion with FMRFamide. In all experiments, the ganglia were separated
from the muscle by a barrier to prevent exposure to exogenously applied
peptides and selectively superfused with high Ca, Mg artificial seawater
(ASW) (see METHODS) to eliminate spontaneous activity. The effects of
FMRFamide reversed on wash out. Pooled data are presented in Fig. 3.
and B9 were unaffected by FMRFamide. For example, in
1026 M FMRFamide, the relaxation time constants were 99 6
1% of control for B3 and 101 6 3% of control for B9 (mean 6
SE, n 5 4 for each neuron). For B3, similar results were
obtained for the effects of FMRFamide on EJPs and contractions in I3a (Church et al. 1993). B9 does not innervate I3a, and
the effects of peptides on B9 have not previously been studied.
Among buccal muscles that have been studied, this is the first
example of a neuropeptide modulating the efficacy of motor
neurons innervating the same muscle in opposite directions. It
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FIG. 2. Effects of FMRFamide on I3m contractions evoked by B3 or B9. A:
top trace is a recording of contractions from I3m; bottom trace is an intracellular recording from B3. Contractions were evoked by stimulation of B3 at 15
Hz in 0.9-s bursts with 100-s interburst intervals. Superfusion of 1028 M
FMRFamide over the muscle increased the amplitude of contractions. A
decrease in the resting tone of the muscle was also observed. B: superimposed
B3-evoked I3m contractions with an expanded time scale before (1) and just
after (2) superfusion with 1028 M FMRFamide show that exogenous FMRFamide had no effect on the rate of relaxation of B3-evoked I3m contractions.
In this and subsequent figures, contractions were aligned by superimposing
motor neuron bursts (not shown). C: top trace is a recording of contractions
from I3m; bottom trace is an intracellular recording from B9. Contractions
were evoked by stimulation of B9 at 15 Hz for 0.9 s with 100-s interburst
intervals. Superfusion of 1028 M FMRFamide over the muscle decreased the
amplitude of B9-evoked contractions. D: B9-evoked I3m contractions with an
expanded time scale before (1) and just after (2) superfusion with 1028 M
FMRFamide show that there was no effect on the relaxation rate of I3m
contractions. The effects of FMRFamide reversed on wash out. Pooled data are
presented in Fig. 3.
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C. KEATING AND P. E. LLOYD
;1,000-fold over control. By contrast, 1026 M FMRFamide
had no effect on I3m cAMP levels (Fig. 7). We reasoned that
if B9 releases SCP from its terminals in I3m, this release
should lead to an increase in cAMP levels in the muscle.
Levels of cAMP normalized to protein in I3m were compared
with cAMP levels in I3a. I3a was used as a control because B9
does not innervate this region of the muscle (Church and Lloyd
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FIG. 3. Summary of the modulation of B3- and B9-evoked I3m EJPs and
contractions produced by application of FMRFamide at 2 concentrations.
Values are means 6 SE (n $ 4). Significance was calculated using the paired
Student’s t-test (NS, not significant; * P , 0.05; ** P , 0.01).
is also the first example of the effects of neuropeptides being
examined on two motor neurons that innervate the same muscle but use different fast excitatory transmitters.
Superfusion of SCP selectively over I3m modulated EJPs
and contractions evoked by both B3 and B9. At 1028 M, SCP
did not have a significant effect on EJPs evoked by B3 or B9,
whereas 1026 M SCP facilitated the amplitude of EJPs evoked
by both neurons (Figs. 4 and 6). Figure 5 shows the effects of
1028 M SCP on I3m contractions. SCP increased the amplitude
and relaxation rates of contractions evoked by both B3 and B9
(Figs. 5 and 6). The effect on contraction amplitude was
dose-dependent, whereas the effect on relaxation rate was
similar at 1028 M and 1026 M (Fig. 6). SCP also caused a
decline in the resting tone of the I3m. EJPs and contractions
evoked by B3 in I3a were also increased by SCP. Although the
effects on EJPs were similar, the potentiation of contractions
was larger in I3a (Church et al. 1993).
Effects of neuropeptides and motor neuron stimulation on
cAMP levels of I3m
As was the case with other buccal muscles, exogenous SCP
was very effective at increasing cAMP levels in I3m segments
(Fig. 7). Indeed at 1026 M, SCP increased cAMP levels
FIG. 4. Effects of SCP on I3m EJPs evoked by B3 or B9. In each pair of
traces, top traces are extracellular recordings from I3m, and bottom traces are
intracellular recordings from motor neurons. A: EJPs were evoked by stimulation of B3 at 15 Hz for 0.9 s with 100-s interburst intervals. SCP reversibly
increased the amplitude of B3-evoked EJPs. B: B3-evoked EJPs with an
expanded time scale before (1) and after (2) superfusion with SCP. C: EJPs
were evoked by stimulation of B9 at 15 Hz for 0.6 s with 100-s interburst
intervals. SCP increased the amplitude of B9-evoked EJPs. D: B9-evoked EJPs
with an expanded time scale before (1) and after (2) superfusion with SCP.
Pooled data are shown in Fig. 6.
DIFFERENTIAL MODULATION OF NEUROMUSCULAR SYNAPSES
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FIG. 5. Effect of SCP on I3m contractions evoked by B3 and B9. A: top
trace is a recording of contractions from I3m; bottom trace is an intracellular
recording from B3. Contractions were evoked by stimulation of B3 at 15 Hz
for 0.9 s with 100-s interburst intervals. Superfusion of 1028 M SCP over the
muscle reversibly increased B3-evoked contractions. B: B3-evoked I3m contractions with an expanded time scale before (1) and just after (2) superfusion
with SCP. Note that SCP increased the rate of relaxation. C: top trace is a
recording of contractions; bottom trace is an intracellular recording from B9.
Contractions were evoked by stimulation of B9 at 15 Hz in 0.6-s bursts with
100-s interburst intervals. Application of 1028 M SCP over the muscle reversibly increased B9-evoked contractions. D: B9-evoked I3m contractions with an
expanded time scale before (1) and just after (2) superfusion with SCP. Note
that SCP increased the rate of relaxation. Pooled data are shown in Fig. 6.
1991). When B9 was hyperpolarized for 10 min, the level of
cAMP in I3m was somewhat lower than in I3a (Fig. 8).
However, when B9 was stimulated in 10-Hz bursts for 10 min,
the level of cAMP in I3m was significantly higher than that in
I3a. The increase in cAMP levels produced by B9 stimulation
was quite variable (increases ranged from 3- to 10-fold). However, the effects of application of SCP were also quite variable.
For example, increases in cAMP caused by 1027 M SCP
ranged from 20 to over 100-fold. We did not look at the effects
FIG. 6. Summary of the modulation of B3- and B9-evoked I3m EJPs
and contractions produced by application of SCP at 2 concentrations. Effect
of superfusion of SCP on EJPs (top), contraction amplitude (middle), and
relaxation time constant of I3m contractions (bottom) evoked by B3 or B9.
Note that a decrease in time constant indicates an increase in the rate of
relaxation. Values are means 6 SE (n $ 4). Significance was calculated
using the paired Student’s t-test (NS, not significant; * P , 0.05; ** P ,
0.01).
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C. KEATING AND P. E. LLOYD
of higher stimulation frequencies (e.g., 15 Hz) because extracellular recordings from I3m indicated that B9-evoked EJPs
failed before the end of the 10-min stimulation period at
frequencies higher than 10 Hz. The failure of somatic spikes to
elicit EJPs has been observed previously for neurons innervating I3a and appears to be due to a failure of action potential
propagation into the axon (Church et al. 1993; Fox and Lloyd
1998). Failures at higher stimulation frequencies were not
observed during the much shorter trains of bursts used for
physiological experiments (see Effects of B9 stimulation on
contractions evoked by B3). Similar experiments using increases in cAMP levels to indirectly measure SCP release have
been carried out in other buccal neuromuscular preparations
and have been shown to correlate well with other methods of
measuring SCP release (Church et al. 1993; Cropper et al.
1990; Vilim et al. 1996; Whim and Lloyd 1989, 1990).
Finally, we wished to determine whether stimulation of B9
during B3 interburst intervals would have the same effect on
subsequent B3-evoked I3m contractions as application of exogenous SCP. We chose to concentrate on contractions rather
than EJPs because stimulation of B9 produced increased cAMP
levels in I3m that were well below those produced by application of 1027 M SCP. At lower concentrations, application of
SCP had little effect on EJPs but increased both the amplitude
and relaxation rate of contractions (Figs. 6 and 9). These
experiments were carried out in the presence of a cholinergic
antagonist (3 mM hexamethonium) to suppress B9-evoked I3m
contractions (Fig. 9). The degree of suppression varied with
stimulation parameters being less effective at higher stimulation frequencies (Whim and Lloyd 1990). Nevertheless, hexamethonium partly or completely blocked contractions evoked
by firing B9 at frequencies similar to those recorded in feedinglike motor patterns (Church and Lloyd 1994). In contrast,
hexamethonium had no significant effect on the amplitude of
B3-evoked I3m contractions. In 3 mM hexamethonium, B3evoked I3m contraction amplitudes were 95 6 4% of control
FIG. 7. Effects of FMRFamide and 2 concentrations of SCP on cAMP
levels in I3m segments. Incubations were for 20 min. cAMP in pmol/mg wet
weight were normalized to another I3m segment taken from the same animal
and incubated in ASW only. Note that cAMP levels are shown on a logarithmic
the scale. FMRFamide (at 1 mM) had no effect on cAMP levels, whereas SCP
(at 1 mM) increased cAMP ;1,000-fold. Values are means 6 SE (n 5 4).
(n 5 4). B3 was stimulated at 100-s intervals to evoke reproducible submaximal contractions. B9 was then stimulated in a
train of bursts during B3 interburst intervals. Stimulation of B9
caused an increase in the amplitude and relaxation rate of
subsequent B3-evoked contractions, and the magnitude of
these effects were dependent on the frequency of stimulation
(Fig. 9). These effects were reversible, with the B3-evoked I3m
contractions returning to control values within ;30 min. These
responses closely resembled the effects of exogenous SCP on
B3-evoked I3m contractions, and it appears likely that these
effects of B9 are mediated, at least in part, by the release of
SCP from its terminals in I3m.
DISCUSSION
Neuromuscular synapses between the buccal motor neurons
and their target muscle in Aplysia have been used extensively
to investigate the physiological role of cotransmitters in modulating synaptic plasticity. Modulation of neuromuscular transmission has now been examined at a variety of buccal muscles
in Aplysia (Evans et al. 1996; Scott et al. 1997; Weiss et al.
1978, 1992; Whim et al. 1993). A striking conclusion from
these studies is that, although there are some basic similarities,
there are also fundamental differences in the degree and type of
modulation observed, and even in the nature of the modulators
found in each of the neuromuscular preparations. Indeed, in the
present study, we found some significant differences with other
buccal neuromuscular systems. This is the first buccal muscle
found to be innervated by motor neurons that use different fast
excitatory transmitters. In other preparations, either the excitatory neurons use only acetylcholine (muscles I5 or ARC;
Cohen et al. 1978; and I7–10, Evans et al. 1996) or only
glutamate (I3a, Fox and Lloyd 1999). In I3m, B3 uses glutamate and B9 is cholinergic. In addition, FMRFamide is the first
modulator to have opposite effects on two excitatory motor
neurons innervating the same muscle. FMRFamide decreased
the amplitude of B9-evoked EJPs and contractions and increased the amplitude of B3-evoked EJPs and contractions. B3
and B9 fire simultaneous bursts during ingestive-like buccal
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Effects of B9 stimulation on contractions evoked by B3
FIG. 8. Effects of stimulating B9 on cAMP levels in I3m. cAMP normalized to protein of I3m (experimental) were compared with that of I3a (control)
from the same animal. B9 does not innervate I3a. B9 was either hyperpolarized
(Hyper) or stimulated at 10 Hz (Stim) in 4-s bursts with a 6-s interburst interval
for 10 min. Values are means 6 SE (n 5 4 for stimulation and n 5 3 for
hyperpolarization). Experiments were carried out at 15°C. Stimulation of B9
caused a significant increase in cAMP levels in I3m (P , 0.025).
DIFFERENTIAL MODULATION OF NEUROMUSCULAR SYNAPSES
motor programs (Church and Lloyd 1994). So FMRFamide
changes the relative efficacy of two motor neurons that fire
together during ingestion-like motor patterns and that have
overlapping but different fields of innervation. A modulator
that decreases the response to one neuron while increasing the
response to another may have little net effect where the fields
of innervation overlap but may change the shape of contractions in regions where the innervation does not overlap.
Currently little is known about the mechanisms underlying
FMRFamide’s effects. It is possible that the changes in EJP
amplitude caused by FMRFamide produce the changes in
contraction amplitude. FMRFamide increases both EJP and
contraction amplitude for B3 and decreases both for B9. We do
not know whether these effects are presynaptic or postsynaptic.
In another buccal muscle, the inhibitory effects of FMRFamide
has been shown to be due, at least in part, to an increase in a
K current in muscle fibers (Scott et al. 1997). B3 and B9
functionally innervate the same muscle fibers (Fox and Lloyd
1999), and EJPs and contractions evoked by the two neurons
are modulated in opposite directions. Thus it is likely that some
aspect of this modulation must occur at sites that are specific to
one of the neurons.
An interesting observation is that EJPs and contractions
evoked by cholinergic motor neurons (B9, B15, and B16) are
inhibited by FMRFamide, whereas those evoked by glutamatergic neurons (B3 and B38) are increased (Church et al. 1993;
Cohen et al. 1978; Weiss et al. 1986; L. E. Fox and P. E. Lloyd,
personal observation). Of course a much larger sample size is
needed to determine how strong this trend is. FMRFamide does
inhibit central glutamatergic excitatory postsynaptic potentials
between sensory and motor neurons (Mackey et al. 1987), and
these synapses are facilitated by SCP. FMRFamide has also
been shown to selectively inhibit the effects of a cholinergic
motor neuron that innervates the aorta in Aplysia while having
no effect on noncholinergic motor neurons innervating the
same tissue (Alevizos et al. 1989).
FMRFamide has been shown previously to inhibit EJPs and
contractions in the ARC (I5) buccal muscle (Weiss et al. 1986).
However, in this muscle, the neuronal source of FMRFamide
has not been identified, and the physiological ligands for this
inhibition may actually be three peptides, termed the RFamide
peptides, that have sequence similarity to FMRFamide and that
were found to be synthesized in one of the motor neurons
innervating the muscle (Cropper et al. 1994). We cannot exclude the possibility that these peptides also act in I3m, but it
is clear that at least three of the motor neurons that innervate
I3m synthesize authentic FMRFamide (Church and Lloyd
1991). However, in that study, peptides were identified by
labeling with radioactive methionine, and this residue is not
present in the RFamide peptides, so it remains possible that any
of the motor neurons innervating I3m could synthesize the
three peptides or indeed any other peptides that do not contain
methionine residues.
Application of SCP potentiated the amplitude of EJPs and
contractions evoked by both B3 and B9 in I3m. SCP may have
presynaptic actions but clearly has postsynaptic actions in
muscle fibers. It increases cAMP levels, and the magnitude of
this increase indicates that it must occur predominantly in
muscle fibers and not in neural elements that make up a very
small proportion of the muscle volume. In addition, SCP increases the relaxation rate of contractions. Relaxation occurs
after the EJPs have decayed and as such is a property of the
muscle fibers themselves. Indeed, in another buccal muscle, the
rate of relaxation has been correlated with the degree of cAMP-
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FIG. 9. Modulation of B3-evoked I3m contractions by stimulation of B9. A:
top trace is I3m contractions; middle trace is an intracellular recording from
B3; bottom trace is an intracellular recording from B9. I3m was superfused
with ASW containing 3 mM hexamethonium to suppress B9-evoked contractions. Stimulation of B3 at 15 Hz for 0.9 s every 100 s elicited reproducible
submaximal contractions. B9 was stimulated (3 series of 4-s bursts at 10 Hz
with 3-s interburst intervals for 60 s) during B3 interburst intervals. Subsequent
B3-evoked I3m contractions were increased in amplitude. B: B3-evoked I3m
contractions with an expanded time scale before (1) and after (2) B9 stimulation also show an increased relaxation rate. C: summary of the effects of
stimulating B9 on B3-evoked contraction amplitude and relaxation time constant. B9 was stimulated using the paradigm described above. Values are
means 6 SE (n 5 5). Significance was calculated using the paired Student’s
t-test (NS, not significant; * P , 0.05; ** P , 0.01).
1765
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C. KEATING AND P. E. LLOYD
1984). Apparently, the mechanisms of action of modulatory
neuropeptides in buccal muscles are varied.
We thank L. E. Fox for critically reading the manuscript and for carrying out
the cAMP assays.
This work was supported by IBN-9728453 to P. E. Lloyd.
Address for reprint requests: P. E. Lloyd, Committee on Neurobiology,
University of Chicago, 947 E. 58th St., Chicago, IL 60637.
Received 6 May 1999; accepted in final form 23 June 1999.
REFERENCES
ABRAMS, T. W., CASTELLUCCI, V. F., CAMARDO, J. S., KANDEL, E. R., AND
LLOYD, P. E. Two endogenous neuropeptides modulate the gill and siphon
withdrawal reflex in Aplysia by presynaptic facilitation involving cAMPdependent closure of a serotonin-sensitive potassium channel. Proc. Natl.
Acad. Sci. USA 81: 7956 –7960, 1984.
ALEVIZOS, A., BAILEY, C. H., CHEN, M., AND KOESTER, J. Innervation of
vascular and cardiac muscle of Aplysia by multimodal motoneuron L7.
J Neurophysiol. 61: 1053–1063, 1989.
BREZINA, V., EVANS, C. G., AND WEISS, K. R. Enhancement of Ca current in the
accessory radula closer muscle of Aplysia californica by neuromodulators
that potentiate contractions. J. Neurosci. 14: 4393– 4411, 1994.
BREZINA, V., OREKHOVA, I. V., AND WEISS, K. R. Functional uncoupling of
linked transmitter effects by combinatorial convergence. Science 273: 806 –
810, 1996.
CALABRESE, R. L. Modulation of muscle and neuromuscular junctions in
invertebrates. Semin. Neurosci. 1: 25–34, 1989.
CHURCH, P. J. AND LLOYD, P. E. Expression of diverse neuropeptide cotransmitters by identified motor neurons in Aplysia. J. Neurosci. 11: 618 – 625,
1991.
CHURCH, P. J. AND LLOYD, P. E. Activity of multiple identified motor neurons
recorded intracellularly during evoked feeding-like motor programs in Aplysia. J. Neurophysiol. 72: 1794 –1809, 1994.
CHURCH, P. J., WHIM, M. D., AND LLOYD, P. E. Modulation of neuromuscular
transmission by conventional and peptide transmitters released from excitatory and inhibitory motor neurons in Aplysia. J. Neurosci. 13: 2790 –2800,
1993.
COHEN, J. L., WEISS, K. R., AND KUPFERMANN, I. Motor control of buccal
muscles in Aplysia. J. Neurophysiol. 41: 157–180, 1978.
CROPPER, E. C., BREZINA, V., VILIM, F. S., HARISH, O., PRICE D. A., ROSEN, S.,
KUPFERMANN, I., AND WEISS, K. R. FRF peptides in the ARC neuromuscular
system of Aplysia: purification and physiological actions. J. Neurophysiol.
72: 2181–2195, 1994.
CROPPER, E. C., LLOYD, P. E., REED, W., TENENBAUM, R., KUPFERMANN, I., AND
WEISS, K. R. Multiple neuropeptides in cholinergic motor neurons of Aplysia: evidence for modulation intrinsic to the motor circuit. Proc. Natl. Acad.
Sci. USA 84: 3486 –3490, 1987.
CROPPER, E. C., PRICE, D., TENENBAUM, R., KUPFERMANN, I., AND WEISS, K. R.
Release of peptide cotransmitters from a cholinergic neuron under physiological conditions. Proc. Natl. Acad. Sci. USA 87: 933–937, 1990.
EVANS, G. C., ROSEN, S., KUPFERMANN, I., WEISS, K. R., AND CROPPER, E. C.
Characterization of a radula opener neuromuscular system in Aplysia.
J. Neurophysiol. 76: 1267–1281, 1996.
FOX, L. E. AND LLOYD, P. E. Serotonin and the small cardioactive peptides
differentially modulate two motor neurons that innervate the same muscle
fibers in Aplysia. J. Neurosci. 17: 6064 – 6074, 1997.
FOX, L. E. AND LLOYD, P. E. Serotonergic neurons differentially modulate the
efficacy of two motor neurons innervating the same muscle fibers in Aplysia.
J. Neurophysiol. 80: 647– 655, 1998.
FOX, L. E. AND LLOYD, P. E. Glutamate is the fast excitatory transmitter at
some buccal neuromuscular synapses in Aplysia. J. Neurophysiol. 82: 1477–
1488, 1999.
GARDNER, D. Bilateral symmetry and interneuronal organization in the buccal
ganglia of Aplysia. Science 173: 550 –553, 1971.
HOWELLS, H. H. The structure and function of the alimentary canal of Aplysia
punctata. Q. J. Microsc. Sci. 83: 357–397, 1942.
JORGE-RIVERA, J. C., SEN, K., BIRMINGHAM, J. T., ABBOTT, L. F., AND MARDER,
E. Temporal dynamics of convergent modulation at a crustacean neuromuscular junction. J. Neurophysiol. 80: 2559 –2570, 1998.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
dependent phosphorylation of a contractile protein (Probst et
al. 1994).
Stimulation of B9 mimics the actions of SCP on I3m. In
these experiments, B9 evoked EJPs, and contractions were
selectively inhibited using a cholinergic antagonist. Both application of SCP and stimulation of B9 caused increased cAMP
levels in the muscle. The magnitude of the increase in cAMP
produced by B9 was similar to those observed when other
SCP-containing motor neurons were stimulated at frequencies
observed during ingestion-like motor programs (Church et al.
1993; Cropper et al. 1990; Whim and Lloyd 1989). Both
application of SCP and stimulation of B9 produced very similar increases in the amplitude and rate of relaxation of muscle
contractions. Finally, the effects produced by stimulation of
SCP-containing motor neurons were independent of the the fast
excitatory transmitter used by the neurons. For example, the
effects on cAMP levels and muscle contractions were similar
for neurons that used ACh (B9) or glutamate (B38) (Church et
al. 1993). In the present study, we were able to to look for the
first time at the immediate effects of peptide cotransmitter
release from an excitatory motor neuron in the absence of large
contractions evoked by the neuron’s fast excitatory transmitter.
The potentiation of B3-evoked I3m muscle contractions was
observed when firing B9 at rates and in patterns similar to those
observed during ingestive-like buccal motor programs, suggesting that modulation of I3 contractions occurs during feeding behaviors. It is also likely that other motor neurons that
innervate I3m contribute to the modulation of EJPs and contractions. Indeed, of the the six motor neurons known to
innervate I3m, three synthesize FMRFamide (B3, 4, 5) and
three synthesize SCP (B6, 9, and 10) (Church and Lloyd 1991).
These neurons all fire bursts during ingestive-like buccal motor
programs (Church and Lloyd 1994). Thus it is likely that
peptides released from their terminals modulate their own
synaptic efficacy on muscle fibers as well as the efficacy of
other motor neurons that innervate I3m. It is difficult to estimate how the observed modulatory effects would affect contractions during feeding when all of the motor neurons innervating I3m fire bursts approximately at the same time (Church
and Lloyd 1994). It is possible that simultaneous activation of
the motor neurons would cause near maximal contraction amplitude and the modulatory effects on amplitude would not be
important. However, it is difficult to imagine that the increased
rate of relaxation would not have behavioral implications.
Indeed, modeling studies of another buccal muscle (ARC or I5)
indicate that increased relaxation rates are particularly important behaviorally (Weiss et al. 1992). However, this preparation differs from I3m in that each motor neuron releases a
complement of peptide cotransmitters that both increase and
decrease the amplitude of contractions, and increase the rate of
relaxation so that the net modulatory effect may be the increase
in relaxation rate.
B3 also innervates the I3a region, and the effects of SCP on
contraction amplitude are considerably larger in I3a than I3m
(Church et al. 1993). This raises the possibility that in the intact
I3, released SCP would not only increase the amplitude of a
B3-evoked contraction, but also change its shape by potentiating contractions more effectively in I3a than in I3m. SCP
also potentiated contractions evoked by motor neurons innervating another buccal muscle (I5 or ARC), but SCP did not
facilitate EJPs in this muscle (Brezina et al. 1994; Lloyd et al.
DIFFERENTIAL MODULATION OF NEUROMUSCULAR SYNAPSES
PROBST, W. C., CROPPER, E. C., HEIERHORST, J., HOOPER, S. L., JAFFE, H.,
VILIM, F., BEUSHAUSEN, S., KUPFERMANN, I., AND WEISS, K. R. cAMPdependent phosphorylation of Aplysia twitchin may mediate modulation of
muscle contractions by neuropeptide cotransmitters. Proc. Natl. Acad. Sci.
USA 91: 8487– 8491, 1994.
SCOTT, M. L., BREZINA, V., AND WEISS, K. R. Ion currents and mechanisms of
modulation in the radula opener muscle of Aplysia. J. Neurophysiol. 78:
2372–2387, 1997.
VILIM, F. S., CROPPER, E. C., PRICE, D. A., KUPFERMANN, I., AND WEISS, K. R.
Release of peptide cotransmitters in Aplysia: regulation and functional
implications. J. Neurosci. 16: 8105– 8114, 1996.
WEISS, K. R., BREZINA, V., CROPPER, E. C., HOOPER, S. L., MILLER, M. W.,
PROBST, W. C., VILIM, F. S., AND KUPFERMANN, I. Peptidergic cotransmission in Aplysia: functional implications for rhythmic behaviors. Experientia
48: 456 – 463, 1992.
WEISS, K. R., COHEN, J. L., AND KUPFERMANN, I. Modulatory control of buccal
musculature by a serotonergic neuron (metacerebral cell) in Aplysia. J Neurophysiol. 41: 181–203, 1978.
WEISS, K. R., LLOYD, P. E., CROPPER E. C., FRANKFURT M., AND KUPFERMANN,
I. FMRFamide is present in the ARC muscle of Aplysia and depresses its
contractions. Soc. Neurosci. Abstr. 12: 947, 1986.
WHIM, M. D., CHURCH, P. J., AND LLOYD, P. E. Functional roles of peptide
cotransmitters at neuromuscular synapses in Aplysia. Mol. Neurobiol. 7:
335–347, 1993.
WHIM, M. D. AND LLOYD, P. E. Frequency-dependent release of peptide
cotransmitters from identified cholinergic motor neurons in Aplysia. Proc.
Natl. Acad. Sci. USA 86: 9034 –9038, 1989.
WHIM, M. D. AND LLOYD, P. E. Neuropeptide cotransmitters released from an
identified cholinergic motor neuron modulate neuromuscular efficacy in
Aplysia. J. Neurosci. 10: 3313–3322, 1990.
WORDEN, M. K. Modulation of vertebrate and invertebrate neuromuscular
junctions. Curr. Opin. Neurobiol. 8: 740 –745, 1998.
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 18, 2017
KRAVITZ, E. A., GLUSMAN, S., HARRIS-WARRICK, R. M., LIVINGSTONE, M. S.,
SCHWARTZ, T., AND GOY, M. F. Amines and a peptide as neurohormones in
lobsters: actions on neuromuscular preparations and preliminary behavioral
studies. J. Exp. Biol. 89: 159 –176, 1980.
KUPFERMANN, I. Feeding behavior in Aplysia: a simple system for the study of
motivation. Behav. Biol. 10: 1–26, 1974.
LLOYD, P. E. Fast axonal transport of modulatory neuropeptides from central
ganglia to components of the feeding system in Aplysia. J. Neurosci. 8:
3507–3514, 1988.
LLOYD, P. E. AND CHURCH, P. J. Cholinergic neuromuscular synapses in
Aplysia have low endogenous acetylcholinesterase and a high affinity uptake
system for acetylcholine. J. Neurosci. 14: 6722– 6733, 1994.
LLOYD, P. E., FRANKFURT, M., STEVENS, P., KUPFERMANN, I., AND WEISS, K. R.
Biochemical and immunocytological localization of the neuropeptides
FMRFamide, SCPA and SCPB to neurons involved in the regulation of
feeding in Aplysia. J. Neurosci. 7: 1123–1132, 1987.
LLOYD, P. E., KUPFERMANN, I., AND WEISS, K. R. Evidence for parallel actions
of a molluscan peptide (SCPB) and 5HT in mediating arousal in Aplysia.
Proc. Natl. Acad. Sci. USA 81: 2934 –2937, 1984.
LOTSHAW, D. P. AND LLOYD, P. E. Peptidergic and serotonergic facilitation of
a neuromuscular synapse in Aplysia. Brain Res. 526: 81–94, 1990.
LU, B., FU, W. M., GREENGARD, P., AND POO, M.-M. Calcitonin gene-related
peptide potentates synaptic responses at developing neuromuscular junction.
Nature 363: 76 –79, 1993.
MACKEY, S. L., GLANZMAN, D. L., SMALL, S. A., DYKE, A. M., KANDEL, E. R.,
AND HAWKINS, R. D. Tail shock produces inhibition as well as sensitization
of the siphon-withdrawal reflex of Aplysia: possible behavioral role for
presynaptic inhibition mediated by the peptide Phe-Met-Arg-Phe-NH2.
Proc. Natl. Acad. Sci. USA 84: 8730 – 8734, 1987.
O’SHEA, M. AND SCHAFFER, M. Neuropeptide function: the invertebrate contribution. Annu. Rev. Neurosci. 8: 171–198, 1985.
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