1Department of Physiology and Biophysics and
2The Fishberg Center for Research in
Neurobiology,
Evans, Colin G.,
Ferdinand S. Vilim,
Orna Harish,
Irving Kupfermann,
Klaudiusz R. Weiss, and
Elizabeth C. Cropper.
Modulation of Radula Opener Muscles in Aplysia.
J. Neurophysiol. 82: 1339-1351, 1999.
We observed fibers immunoreactive (IR) to serotonin (5-HT), the
myomodulins (MMs), and FMRFamide on the I7-I10 complex in the marine
mollusk Aplysia californica. The I7-I10 muscle complex, which produces radula opening, is innervated primarily by one motor
neuron, B48. B48 is MM-IR and synthesizes authentic MMA. When B48 is stimulated in a physiological manner, cAMP levels are
increased in opener muscles. cAMP increases also are seen when the MMs
are applied to opener muscles but are not seen with application of the
B48 primary neurotransmitter acetylcholine (ACh). Possible
physiological sources of 5-HT and FMRFamide are discussed. When
modulators are applied to resting opener muscles, changes in membrane
potential are observed. Specifically, 5-HT, MMB, and low
concentrations of MMA all depolarize muscle fibers. This
depolarization is generally not sufficient to elicit myogenic activity
in the absence of neural activity under "rest" conditions. However,
if opener muscles are stretched beyond rest length, stretch- and
modulator-induced depolarizations can summate and elicit contractions. This only occurs, however, if "depolarizing" modulators are applied alone. Thus other modulators (i.e., FMRFamide and high concentrations of MMA) hyperpolarize opener muscle fibers and can prevent
depolarizing modulators from eliciting myogenic activity. All
modulators tested affected parameters of motor neuron-elicited
contractions of opener muscles. MMB and 5-HT increased
contraction size over the range of concentrations tested, whereas
MMA potentiated contractions when it was applied at lower
concentrations but decreased contraction size at higher concentrations.
FMRFamide decreased contraction size at all concentrations and did not
affect relaxation rate. Additionally, the MMs and 5-HT increased muscle
relaxation rate, decreased contraction latency, and decreased the rate
at which tension was developed during motor neuron-elicited muscle
contractions. Thus these modulators dramatically affect the ability of
opener muscles to follow activity in the opener motor neuron B48. The possible physiological significance of these findings is discussed.
Many investigators that have sought to
characterize the neural mechanisms important for plasticity in rhythmic
behaviors have studied the neural circuits that generate these
behaviors. Changes in the firing patterns of the circuits that mediate
behavior are presumed to be indicative of changes in the behavior
itself. Although this assumption is likely to be valid under some
circumstances, it may not be valid under others. For example, it is not
likely to be valid when muscle response dynamics are slow, i.e., when muscle tension cannot accurately follow changes in neural activity (Hooper et al. 1999 It has been shown that the relationship between motor neuron activity
and the magnitude of the resulting muscle contraction can be quite
complex even under steady-state conditions. For example, the peak or
mean contraction amplitude may not be solely determined by the mean
firing frequency of the motor neuron in that the particular firing
pattern of the motor neuron also may be important (e.g., Brezina
et al. 1997 To further complicate matters, it has become apparent that the NMT does
not have to be a fixed filter that always operates in the same manner
(Brezina et al. 1999 In the research described in this paper, we examined effects of
modulatory neurotransmitters on the muscles that are antagonistic to
the ARC muscles (the I7-I10 (radula opener) muscles in
Aplysia (Evans et al. 1996 An abstract of this work has appeared (Evans et al.
1993 Animals
Aplysia californica (200-400 g) were maintained at
14-16°C in 150-gallon holding tanks containing aerated, artificial
sea water (ASW). In all experiments animals were anaesthetized with isotonic magnesium chloride (50% wt/vol).
Methods used in physiological experiments
Physiological experiments were conducted in reduced preparations
that have been described in detail (Evans et al. 1996 When contractions of the I7 muscle were recorded, an isotonic
transducer was used to detect muscle movements (Harvard Apparatus, MA)
(Evans et al. 1996 To obtain intracellular recordings from I7 muscle fibers, about
one-third of the length of the I7 muscle was immobilized, with pins, on
a raised piece of Sylgard within the Lucite chamber that separated the
buccal ganglion from the I7 muscle. Electrodes used to record from
muscle fibers were single barreled glass pipettes with resistances of
10-25 M Methods used in immunocytochemical experiments
In most experiments, immunocytochemical experiments were
performed using standard whole-mount methods (Longley and
Longley 1986 In all cases, tissues were viewed with a Nikon microscope equipped with
epifluorescence and photographed with Tri-X (ASA 400) film. In
experiments where immunocytochemistry was performed on B48 neurons,
cells were identified by their position in the buccal ganglion and
their ability to produce contractions of the I7 muscle (using
physiological methods described in the preceding section). They then
were injected with Lucifer yellow dye.
Methods used to radiolabel B48 neurons
In situ radiolabeling was done as in previous studies (see e.g.,
Cropper et al. 1987a Coelution of radiolabeled and synthetic material was tested through two
sequential reverse phase high-performance liquid chromatography (RP-HPLC) passes. In the first pass, an Aquapore RP-300 column was
developed at 1 ml/min with a linear gradient of 5-50% solvent B in 45 min. Solvent A was 100% H20, 0.01 M
trifluoroacetic acid (TFA) and solvent B was 100%
CH3CN, 0.01 M TFA. In the second RP-HPLC pass,
the same column was developed with a linear gradient of 15-45%
solvent B in 30 min. Solvent A was 100% H2O,
0.01 M heptafluoroacetic acid (HFBA) and solvent B was 100%
CH3CN, 0.01 M HFBA. In both passes, synthetic
peptides were detected by absorbence measurements using a V-4 flow
spectrophotometer (ISCO) at 215 nm. In the first pass, radiolabeled
peptides were detected by scintillation counting of 10% of each
fraction. After the second pass, whole fractions were counted.
Methods used for measuring cAMP levels in I7 muscles
In experiments with exogenous modulators, I7 muscles were
removed and placed in ASW for 2 h to stabilize preparations.
Muscles then were exposed to modulators at different concentrations for different periods of time (see RESULTS for descriptions of
specific experiments). In experiments in which cAMP elevations were
induced by the release of endogenous modulators, buccal ganglia were
desheathed, and preparations were rested for 2 h. B48 neurons then
were stimulated in a pattern that mimicked physiological activity,
i.e., B48 was fired at 3 Hz for 2 s followed by a pause of 4 s (Evans et al. 1996 In all experiments, we extracted cAMP from I7 muscles by homogenizing
them in 65% ethanol, 35% H2O, heating them to
90°C for 5 min, and then spinning them in a clinical centrifuge for 2 min. We removed the resulting supernatant and stored samples at
Reagents
The ASW used in these experiments had the following composition
(in mM): 460 NaCl, 10 KCl, 11 CaCl2, 55 MgCl2, and 5 NaHCO3. The pH
was adjusted to 7.6. All salts, Fast Green dye, Lucifer yellow CH, and
the N-methyl-D-glucamine were obtained from
Sigma. Forskolin was obtained from Calbiochem and was dissolved in
DMSO. The final concentration of DMSO during experiments with forskolin was 0.01%. Control experiments established that this concentration was
not bioactive at the neuromuscular junction.
Modulators are present in the opener neuromuscular system
To identify potential modulators in the I7-I10 neuromuscular
system, we used immunocytochemical techniques. Specifically, we sought
to determine whether the I7-I10 muscles contain fibers that are
immunoreactive to 5-HT and peptides that modulate neuromuscular activity in Aplysia, i.e., MM (Cropper et al.
1987b
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
; Morris and Hooper
1998
). Systems with slow response dynamics that have been
extensively investigated include the accessory radula closer (ARC)
neuromuscular system in the marine mollusk Aplysia
californica (e.g., Brezina et al. 1997
) and
stomatogastric muscles of the lobster Panulirus
interruptus (e.g., Morris and Hooper 1997
, 1998
).
; Morris and Hooper 1997
). Pattern
dependence can be predicted from quantitative modeling (Brezina
et al. 1997
; Morris and Hooper 1997
) but it is
not always intuitively obvious. The term neuromuscular transform (NMT)
has been introduced to refer to the complex nonlinear filter through
which motor commands must pass before they are translated into muscle
contractions (Brezina et al. 1999
). Physiologically, the
NMT comprises multiple steps including presynaptic
Ca+2 elevation, neurotransmitter release,
postsynaptic Ca+2 elevation, and activation of
the contractile machinery. Thus the muscle contractions that will
result from a particular pattern of neuronal activity often cannot be
predicted unless the relevant NMT is understood.
). Instead it can be dynamic and can
be modified. This plasticity is likely to be important because models
have suggested that when the NMT is fixed, a system may not be able to
generate behaviors with certain parameters (Brezina et al.
1999
). When the NMT is "tuned," however, these behaviors
become possible. To fully appreciate how neuronal activity is
translated into a functional movement, therefore it has become apparent
that it may be important to describe how the NMT can be altered.
Modulatory neurotransmitters clearly can be important in this context.
These modulators can be intrinsic, i.e., present as
cotransmitters in the behavior-generating motor neurons themselves, and/or extrinsic, i.e., released as hormones or present in
specialized modulatory neurons (Cropper et al. 1987a
).
Effects of modulatory neurotransmitters on neuromuscular function have
been investigated in a number of preparations (Calabrese
1989
) including the ARC neuromuscular system. Experiments in
the ARC system have concentrated on effects of modulatory
neurotransmitters that are the most striking in this preparation, i.e.,
effects of modulators on contraction amplitude and muscle relaxation
rate. Work in the ARC neuromuscular system therefore has provided
insights into how effects of modulators on certain aspects of the NMT
are likely to be important for behavioral plasticity.
). Modulators that
are present in the I7-I10 complex were identified and their effects
studied in whole muscle preparations. In a previous paper (Scott
et al. 1997a
), experiments were conducted on dissociated opener
muscle fibers and the specific ion currents that were modulated were
identified. Because I7-I10 muscles display different contraction
characteristics than the ARC muscle, the studies described in this
paper are designed to characterize additional aspects of modulation
that will be incorporated into models of the experimentally
advantageous radula closer-opener complex.
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
).
Briefly, one side of the buccal mass was cut away and the buccal
ganglia were left attached to the remaining half of the buccal mass
through buccal nerve 3. Preparations were placed in silicone elastomer (Sylgard)-lined dishes, and a small (5 ml) Lucite chamber was placed
over the opener muscles to pharmacologically isolate them from the
buccal ganglion. Preparations were grounded routinely using a chlorided
silver wire.
). Briefly, to connect I7 muscles to
the transducer, a wooden beam was attached, approximately at its
midpoint, to the rotating arm of the transducer. One end of the beam
had a metal hook to which the odontophoral end of the I7 muscle was tied using a silk suture. The other half of the beam was marked with a
centimeter scale, along which a known weight could be moved to vary the
load on the muscle. When contractions of the I7 muscle were elicited by
intracellular stimulation of motor neuron B48, we used intracellular
double-barreled electrodes that were filled with a solution of 3 M
potassium acetate containing 10 mM KCl. Electrodes had resistances of
~10 M
. When muscle contractions were elicited by stretching the I7
muscle, different loads were used for different muscles because stretch
was elicited more readily in some cases than others. In general loads
on muscle ranged from 36 to 410 mg. A "stop" was placed under the
wooden beam to control muscle length (Evans et al.
1996
). Experiments began with the stop at its highest point.
The stop then was lowered to stretch muscles. Changes in relaxation
rate were quantified by measuring the time it took for contractions to
relax to two-thirds of their original size.
. In ion substitution experiments, preparations were
grounded with a seawater agar bridge connected to a reservoir
containing 3 M KCl and an Ag/AgCl pellet.
; Miller et al. 1991
; Vilim
et al. 1996b
). Primary antisera were as follows: serotonin
(5-HT)-rabbit host (kind gift from Dr. Hadassah Tamir, Columbia
University), FMRFamide-rabbit host (Dia Sorin, Stillwater, MN),
buccalin-rabbit host (Miller et al. 1992
), SCP-rabbit
host (kind gift from Dr. H. R. Morris Empire College), and
myomodulin (MM)-rat host (raised against a peptide conjugated to bovine
thyroglobulin with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Vilim et al. 1996b
). Primary antisera were applied for
48 h at room temperature at a dilution of 1:250. The secondary
antibodies [lissamine rhodamine donkey anti-rat, and fluorescein
donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA)] were
applied for 24 h (1:500 dilution; room temperature).
;Lloyd et al. 1987
).
Briefly, 7 B48 neurons were physiologically identified (using
physiological methods described in the preceding text). Neurons were
marked by iontophoretic injection of Fast Green dye. Buccal ganglia
were incubated for 24 h in 1 ml of 50% ASW, 50%
Aplysia hemolymph containing 0.5 mCi of
[35S]methionine, 2.5 µl of 1 M colchicine
(dissolved in DMSO), and 100 µl antibiotics (penicillin and
streptomycin each at 50 units/ml). B48 neurons were dissected
individually from the labeled ganglia (Ono and McCaman
1980
), and radioactive MM was extracted in the presence of
synthetic MM.
). B48 then was fired at 14 Hz for
1.2 s followed by a pause of 1.5 s. The 3-Hz stimulation then
was repeated. Neurons were stimulated for a total of 5 min at 15°C.
During the last burst of stimulation I7 muscles were frozen, i.e., the
ASW bathing muscles was replaced with liquid nitrogen.
20°C until we made cAMP measurements. cAMP levels were quantified
using a commercially available RIA (Amersham). Proteins were measured using a BCA protein assay reagent (Pierce, Rockford, IL).
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
), buccalin (Cropper et al. 1988
),
FMRFamide (Weiss et al. 1986
), and SCP (Lloyd et
al. 1984
). Fibers were 5-HT immunoreactive (IR) and MM-IR.
Specifically, 5-HT and MM antisera stained dense networks of finely
dividing processes that had numerous varicosities that extended over
the surface of muscles (Fig. 1,
A and B). Muscles were also immunoreactive to
FMRFamide, but staining was confined to a much more sparsely
distributed network of fibers (Fig. 1C). There was no
detectable SCP-like or buccalin-like immunoreactivity (not shown).
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Fig. 1.
Whole-mount immunocytochemistry of I7 muscles. Serotonin (5-HT)-like
(A), myomodulin (MM)-like (B), and
FMRFamide-like (C) immunoreactive fibers on the surface
of the I7 muscle. Note that 5-HT and MM stain dense networks of finely
dividing fibers, whereas FMRFamide stains a much more sparsely
distributed network of fibers. A and B
are from the same preparation and were visualized with different
secondary antibodies (i.e., fluorescein in A and
lissamine rhodamine in B). C is from a
different preparation. Scale bar 200 µm.
In a previous study, we demonstrated that the I7-I10 muscle complex is
innervated primarily by one motor neuron (Evans et al.
1996). This neuron was similar in size and location to B48, a
neuron described by Church and Lloyd (1994)
.
Additionally, Church and Lloyd found that stimulation of neuron B48
elicited radula opening/protraction as our neuron did and that B48
innervated the I8 muscle. If our motor neuron and B48 are in fact the
same cell, we would expect our motor neuron to contain MM as B48 does (Church and Lloyd 1994
). To determine whether this was
the case, we initially performed immunocytochemical experiments on
buccal ganglia. We found that our motor neuron was MM-IR but was not 5-HT or FMRFamide IR (not shown).
To determine whether our motor neuron synthesizes authentic MM, buccal ganglia were incubated in a radiolabeled form of an amino acid precursor of MM, namely [35S]methionine. Motor neurons were dissected individually from ganglia and supplemented with quantities of synthetic MM, which are detected easily by optical measurements. Mixtures of native and synthetic material were subjected to sequential RP-HPLC. Native radioactivity did in fact precisely coelute with synthetic material through both stages of chromatography (Fig. 2).
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Modulators are bioactive in whole muscle preparations
Physiological experiments were conducted on the I7 muscles, which
are the longest and most experimentally advantageous of the opener
complex. Modulators tested were those that are physiologically relevant. Thus 5-HT and FMRFamide were tested because fibers on opener
muscles are 5-HT-IR and FMRFamide-IR. Two MMs were tested (MMA and MMB) because we
specifically localized MMA to B48 in biochemical
experiments, and we have found that MMA is
cleaved from a precursor protein that also encodes
MMB (Miller et al. 1993).
Actually, the MM precursor encodes five other MMs in addition to
MMA and MMB (Miller
et al. 1993
). We chose MMA and
MMB because experiments in the ARC neuromuscular
system have suggested that differences in the bioactivity of the MMs
are most striking if these two peptides are compared (Brezina et
al. 1995
).
Previous experiments have shown that vigorous contractions of opener
muscles are elicited if the motor neuron B48 is stimulated (Evans et al. 1996). In this study therefore, we sought
to characterize effects of modulators on parameters of motor
neuron-elicited muscle contractions. Additionally, previous experiments
have shown that opener contractions can be elicited in the absence of
neural activity if muscles are counterweighted so that they are
stretched beyond their resting length (Evans et al.
1996
). In unmodulated muscles, this does not, however, appear
to occur unless muscles are stretched in an unphysiological manner
(Evans et al. 1996
). A second goal of these experiments,
however, was to determine whether contractions of opener muscles are
induced more readily by stretch in the presence of modulators. Finally,
in some neuromuscular systems modulators themselves induce myogenic
activity (e.g., Meyrand and Marder 1991
). We, therefore
also conducted experiments to determine whether similar effects would
occur in the opener complex.
EFFECTS OF MODULATORS ON THE MEMBRANE POTENTIAL OF I7. When modulators were applied to I7 muscles, changes in membrane potential were observed. These effects were concentration dependent and reversible (Fig. 3). 5-HT and MMB both induced a concentration-dependent depolarization of muscle fibers (Fig. 3, A and B; Table 1), whereas FMRFamide induced a concentration-dependent hyperpolarization (Fig. 3D; Table 1).
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EFFECTS OF MODULATORS ON STRETCHED OPENER MUSCLES.
In a previous study we demonstrated that opener muscles were
depolarized in the absence of neural activity if they were stretched beyond resting length (Evans et al. 1996). When muscles
were stretched in a physiological range, slight tension increases were
observed but vigorous contractions were not elicited (Evans et
al. 1996
). Thus modulators and stretch can both depolarize
opener muscle fibers but neither manipulation alone elicits myogenic
activity. What if the two manipulations are interacted? To answer this
question, I7 muscles were attached to a movement transducer and
stretched in a physiological range (Evans et al. 1996
)
so that a contraction was not elicited. 5-HT
(10
6 M) then was applied to stretched muscles
and muscle contractions were elicited (n = 8 of 10 preparations; Fig. 5B). These contractions were rhythmic,
and each contraction was preceded by what appeared to be a "spike."
These spikes were not large in amplitude, therefore they were
presumably generated in regions of the muscle that were stretched and
were only electrotonically conducted to fibers from which recordings
were made.
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EFFECTS OF MODULATORS ON B48-INDUCED CONTRACTIONS OF THE I7 MUSCLE.
We next characterized the effects of various modulators on contractions
induced by firing the opener motor neuron B48. We found that
MMB and 5-HT potentiated contractions over the
range of concentrations tested, whereas MMA
potentiated contractions when it was applied at
109 to
10
7 M but decreased contraction
size at 10
6 M (Fig.
9, B and C).
FMRFamide decreased contraction size at all concentrations (Fig. 9,
B and C).
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5-HT and MM increase muscle cAMP levels
We found that MMA, MMB, and 5-HT all increased cAMP levels in opener muscles in a concentration-dependent (Fig. 11A), and time-dependent (Fig. 11B) manner. FMRFamide (Fig. 11A) and the B48 primary neurotransmitter ACh (Fig. 11B) did not increase cAMP levels. Additionally, we found that forskolin and 8-CPT-cAMP mimicked effects of 5-HT and the MMs on motor neuron evoked muscle contractions (n = 3; Fig. 12). Thus they increased contraction size (Fig. 12A) and relaxation rate (Fig. 12B).
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Indirect evidence for release of modulators under physiological conditions
To indirectly determine if MM might be released during motor
neuron firing, we took advantage of the fact that MM increases cAMP
levels in opener muscles. Previous experiments have shown that B48
fires at least twice during the opening/protraction phase of ingestive
motor programs (Evans et al. 1996). One relatively high-frequency burst of activity occurs during visible
opening/protractions, the second burst of low-frequency activity occurs
at peak retraction. We mimicked this firing pattern, i.e., we
stimulated B48 neurons at 14 Hz for 1.2 s followed by a pause of
1.5 s. We then stimulated B48 at 3 Hz for 2 s followed by a
pause of 4 s. The burst of 14 Hz activity then was repeated. After
periods of stimulation, we measured the resulting levels of cAMP in the
I7 muscle. Other preparations were processed in a similar manner
except that we did not stimulate B48 neurons. (B48 bilaterally
innervates the I7-I10 complex (Evans et al. 1996
) so we
could not make within animal comparisons, i.e., use one I7 as a control
(unstimulated) muscle and one as an experimental (stimulated) muscle.)
We found that physiological stimulation of B48 produced a significant
increase in cAMP levels in the I7 muscle (Fig.
13).
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Source of modulatory input to the I7-I10 complex
We show that there are MM-IR neural processes and varicosities on
opener muscles. The opener motor neuron B48 synthesizes authentic
MMA (Fig. 2) (Church and Lloyd
1994) and is therefore one physiological source of MM input to
the I7-I10 complex. We also demonstrate that there are neuronal
processes on opener muscles that are FMRFamide-IR. Our data and data of
Church and Lloyd (1994)
indicate that the FMRFamide-like
peptide is not a cotransmitter in B48. We showed that FMRFamide-IR
processes had an appearance that was clearly different from the
appearance of MM-IR processes. Namely, the MM antibody stained a dense
network of finely dividing processes that had numerous varicosities
(Fig. 1B). In contrast, the FMRFamide antibody stained a
much more sparsely distributed network of fibers (Fig. 1C).
Church and Lloyd (1994)
have shown that B48 does not
synthesize FMRFamide, and we found that B48 is not FMRFamide-IR. One
source of the FMRFamide-like input to the opener complex is likely to
be the multifunction neurons B4/B5. These neurons innervate the opener
complex (Evans et al. 1996
) and synthesize FMRFamide
(Church and Lloyd 1991
). Additionally, FMRFamide-like
peptides may originate from buccal S (sensory) cells, which are
strongly FMRFamide-IR (Lloyd et al. 1987
) and innervate
many muscles of the buccal mass (Jahan-Parwar et al. 1983
).
In addition to peptide immunoreactivity, there is 5-HT immunoreactivity
in neuronal processes on opener muscles. One source of this
immunoreactivity is likely to be the serotonergic (Eisenstadt et
al. 1973; Weinreich et al. 1973
) metacerebral
cells (MCCs). The MCCs have been studied extensively, and it has become
apparent that these neurons act both centrally on neurons that generate feeding behavior and peripherally on the muscles of the buccal mass
that execute feeding behavior (e.g., Fox and Lloyd 1998
; Lotshaw and Lloyd 1990
; Weiss et al.
1978
). Neurons B48 and B4/B5 are not 5-HT-IR, and 5-HT is not
present in buccal neurons.
Modulation of stretch-induced contractions of opener muscles
In this study we show that some of the modulatory
neurotransmitters in the opener neuromuscular system (e.g., 5-HT and
MMB) can induce rhythmic muscle contractions if
muscles are stretched. The induction of myogenic activity in the
absence of neural activity has been described in other systems, e.g.,
in the pyloric dilator muscle of the shrimp Palaemon
(Meyrand and Marder 1991; Meyrand and Moulins
1986
), and in cardiac muscle of the leech Hirudo
(Li and Calabrese 1987
). In the shrimp there are times
when the pyloric dilator motor neuron is silent and rhythmic
contractions of the pyloric dilator muscle could occur naturally
(Meyrand and Moulins 1988
). In contrast, we expect that
myogenic activity in the I7-I10 complex is not likely to occur in the
absence of neural activity, at least during ingestive motor programs.
During this type of activity, large presumably motor neuron-induced
excitatory junctional currents (EJCs) are always recorded from I7
muscles during the radula opening/protraction phase of behavior
(Evans et al. 1996
). We also demonstrate that myogenic
activity is only elicited in the I7-I10 complex when modulators that
depolarize muscle fibers are applied to stretched muscles alone, i.e.,
when they are not applied with modulators that hyperpolarize muscle fibers.
Our results taken together with those of Scott et al.
(1997a) suggest that the induction of myogenic activity
results, at least in part, from the activation of an
IMod(cat), which is primarily a Na
current (Scott et al. 1997a
). This inward current can be activated at resting membrane potentials and can summate with the
inward current that results from stretch, which also appears to be
primarily a Na current (Evans et al. 1996
). Together
these inward currents depolarize muscle fibers; this is likely to
indirectly increase intracellular Ca levels. A direct effect of
modulators on the characterized ICa is
unlikely to occur in this context because
ICa is activated at relatively
depolarized membrane potentials (Scott et al. 1997a
). It
should be noted, however, that recent data suggest that there may be a
second source of calcium in opener muscle fibers, i.e., muscle
contractions appear to be activated at more negative voltages than the
characterized ICa (Scott et al.
1997b
). Because this source of Ca has not been specifically identified, it has not been possible to determine whether it is activated or enhanced by modulators.
Modulation of motor-neuron elicited contractions of the I7-I10 muscles: mechanisms of action of modulatory neurotransmitters
In this study, we show that the parameters of motor neuron elicited contractions of the opener muscles are altered by modulatory transmitters. Namely, we show that contraction size can be increased or decreased, muscle relaxation rate can be increased, contraction latency can be decreased, and the rate at which tension is developed can be increased. In the following text, we discuss likely mechanisms for these effects.
INCREASES AND DECREASES IN CONTRACTION SIZE.
As described in the preceding text, Scott et al. (1997a)
have shown that some modulators activate the inward Na current
IMod(cat) at resting membrane
potentials. Although activation of this current is likely to play a
role in producing increases in motor-neuron-elicited muscle
contractions, a second inward current is also likely to be important in
this context. Specifically, modulators that activate IMod(cat) also enhance a
dihydropyridine-sensitive "L"-type Ca current that is observed at
relatively depolarized membrane potentials (Scott et al.
1997a
). Scott et al. (1997a)
suggested therefore that modulators that increase contraction size do so in part by their
effect on ICa. They postulated that
this effect is augmented by the activation of
IMod(cat).
INCREASES IN MUSCLE RELAXATION RATE.
Biophysical correlates of modulator-induced increases in relaxation
rate have not been observed in either opener muscles or ARC muscles. In
the ARC neuromuscular system, data suggest that these types of effects
result from a direct effect of modulators on the contractile machinery
(Probst et al. 1994). More specifically, modulators
appear to phosphorylate a large (i.e., >750 kDa) protein that is
structurally related to the muscle protein twitchin (Heirerhorst et al. 1994
; Probst et al. 1994
). The
phosphorylation state of this protein is well correlated with the
relaxation rate of the muscle (Probst et al. 1994
).
Effects of modulators on twitchin are at least partially mediated
through cAMP (Probst et al. 1994
). A similar mechanism
may be important in the opener neuromuscular system. We show that cAMP
analogues do in fact produce increases in muscle relaxation rate.
EFFECTS ON CONTRACTION LATENCY AND THE RATE AT WHICH TENSION IS
DEVELOPED.
As is typical for molluscan muscle, a single motor neuron spike
(Evans et al. 1996) generally does not elicit
contractions of opener muscles. The opener motor neuron B48, however,
fires in bursts during ingestive feeding behavior (Evans et al.
1996
). Consequently, motor-neuron-elicited EJPs summate so that
although a contraction is not elicited by the first EJP, it is elicited by subsequent EJPs. Contraction latency therefore can be decreased if
EJPs that were previously subthreshold for eliciting contractions of
muscle fibers become suprathreshold. This is one effect we observed
(Fig. 10). In addition to decreases in contraction latency, modulators
also increase the rate at which tension is developed in opener muscles.
This appears to result from the fact that modulated EJPs continue to be
more effective at producing tension increases in muscles than
unmodulated EJPs. Because tension increases produced by modulated EJPs
are larger, they summate more efficiently. This effect of 5-HT on
contraction latency is different from the effect that is observed on
the I3 muscle of Aplysia (Fox and Lloyd 1997
, 1998
). In the I3 muscle 5-HT increases EJP size, which results in greater EJP summation. In contrast, in the opener system, EJP size
is not significantly increased and the EJP time constant actually is
decreased. Consequently the summation of modulated EJPs in the opener
muscle is actually reduced. Enhancement of tension occurs in spite of
this because the effects of 5-HT on tension development are so powerful.
Functional consequences of modulation of motor-neuron-elicited contractions of the I7-I10 muscles
Functional consequences of neuromuscular modulation have been
modeled in the ARC system of Aplysia. The ARC muscles are
radula closers and function as antagonists of the I7-I10 muscles. One important finding that has guided current conceptualizations of modulator function in the ARC neuromuscular system is that modulator release is enhanced as the rate at which feeding behavior is executed is increased, i.e., as animals are aroused (Cropper et al.
1990; Vilim et al. 1996a
; Whim and Lloyd
1989
). When animals are aroused, feeding behavior not only
occurs more rapidly but bite strength also is increased (Weiss
and Kupfermann 1977
; Weiss et al. 1980
). Consequently, muscle contractions must be increased in amplitude but
must have limited durations because interbite intervals are decreased.
If contraction duration is not limited, one muscle will not have
relaxed before its antagonistic begins to contract (Weiss et al.
1992
) and individual contractions will occur before the
previous contraction has returned to baseline (Deodhar
1999
). Modulators appear to be important in this context
because they alter the NMT so that both contraction amplitude and
muscle relaxation rate are increased (Cropper et al. 1988
,
1990
). Previous work in the ARC neuromuscular system therefore
has suggested that modulators can be important because they can affect
muscle relaxation rate and determine when a contraction ends.
In this study, we show that modulators in the radula opener complex can additionally affect contraction duration by strongly affecting other parameters of motor neuron elicited muscle contractions. More specifically we show that modulators can decrease contraction latency and the rate at which tension is developed as a result of motor neuron activity. These effects actually are observed in conjunction with the modulatory effects that are striking in the ARC neuromuscular system, i.e., modulators affect contraction amplitude and muscle relaxation rate in the I7-I10 system as they do in the ARC neuromuscular system. Thus in the most general sense, modulators in the I7-I10 neuromuscular system presumably also act to alter the NMT so that contraction duration is decreased when behavior is executed more rapidly. In the opener complex, however, effects on both tension development and muscle relaxation presumably decrease contraction duration.
Increases in the rate at which tension is developed in the opener neuromuscular system are not, however, likely to exclusively result from the release of modulatory neurotransmitters. Posttetanic potentiation (PTP) is observed in this system. Consequently, even when intraburst stimulation parameters are kept constant and EJPs are larger and summate more efficiently, contractions are potentiated if the interburst interval is decreased. We therefore can speculate that modulators are released in the opener system when behavior is executed more rapidly, and it is important that contraction dynamics are adjusted so that contractions can be increased in size without corresponding increases in contraction duration. In considering effects on muscle relaxation rate, it is intuitively obvious why modulator release may be important. Current data indicate that increases in muscle relaxation rate cannot be produced by changes in primary transmitter release. In contrast, in considering effects of modulators on tension development, it is more difficult to appreciate why modulator release is necessary. When behavior is executed more rapidly interburst intervals obviously will decrease and tension development will be increased by the resulting PTP. Thus there appears to be convergence in the opener system in that as behavior is executed more rapidly the rate at which tension is developed will presumably increase both as a result of PTP and as a result of modulator release.
In summary, like modulators in the ARC neuromuscular system, modulators in the opener neuromuscular system are likely to be released when animals are aroused and behavior is executed relatively rapidly. Under these conditions, modulators are likely to alter the NMT so that muscles contract and relax more rapidly. These changes in muscle dynamics are likely to be important since they enable muscles to more faithfully "follow" neural activity.
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ACKNOWLEDGMENTS |
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We thank V. Brezina and M. Scott for comments on an earlier version of this manuscript.
This work was supported by an Irma T. Hirschl Career Scientist Award, Research Scientist Development Awards (MH01267 and MH01427), and National Institute of Mental Health Grants (MH-51393 and MH-36730). Some of the Aplysia used in this study were provided by the National Resource for Aplysia of the University of Miami under Grant RR-10294 from the National Center for Research Resources.
Present address of C. G. Evans: Phase V Communications, 114 Fifth Ave., New York, NY 10011.
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FOOTNOTES |
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Address for reprint requests: E. C. Cropper, Dept. of Physiology and Biophysics, Box 1218, Mt. Sinai Medical School, One Gustave L. Levy Place, New York, NY 10029.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 March 1999; accepted in final form 17 May 1999.
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REFERENCES |
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