1Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029; 2Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52 900, Israel; and 3Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York, New York 10032
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ABSTRACT |
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Hurwitz, I., E. C. Cropper, F. S. Vilim, V. Alexeeva, A. J. Susswein, I. Kupfermann, and K. R. Weiss. Serotonergic and Peptidergic Modulation of the Buccal Mass Protractor Muscle (I2) in Aplysia. J. Neurophysiol. 84: 2810-2820, 2000. Plasticity of Aplysia feeding has largely been measured by noting changes in radula protraction. On the basis of previous work, it has been suggested that peripheral modulation may contribute to behavioral plasticity. However, peripheral plasticity has not been demonstrated in the neuromuscular systems that participate in radula protraction. Therefore in this study we investigated whether contractions of a major radula protraction muscle (I2) are subject to modulation. We demonstrate, first, that an increase in the firing frequency of the cholinergic I2 motoneurons will increase the amplitude of the resulting muscle contraction but will not modulate its relaxation rate. We show, second, that neuronal processes on the I2 muscle are immunoreactive to myomodulin (MM), RFamide, and serotonin (5-HT), but not to small cardioactive peptide (SCP) or buccalin. The I2 motoneurons B31, B32, B61, and B62 are not immunoreactive to RFamide, 5-HT, SCP, or buccalin. However, all four cells are MM immunoreactive and are capable of synthesizing MMa. Third, we show that the bioactivity of the different modulators is somewhat different; while the MMs (i.e., MMa and MMb) and 5-HT increase I2 muscle relaxation rate, and potentiate muscle contraction amplitude, MMa, at high concentrations, depresses muscle contractions. Fourth, our data suggest that cAMP at least partially mediates effects of modulators on contraction amplitude and relaxation rate.
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INTRODUCTION |
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Rhythmic movements driven by neural networks play a major role in the life of both vertebrates and invertebrates. These behaviors may appear to be simple and fixed, e.g., to be driven by a "loop program" that elicits the same phasic movements again and again. Nevertheless, the relationship between the magnitude and the timing of each act can be quite flexible. In part this flexibility results from complex properties of central pattern generating circuits, which have been studied in a number of preparations. Additionally, however, plasticity can be mediated peripherally, i.e., in the neuromuscular systems that execute behavior. Central and peripheral plasticity have been characterized in model systems, e.g., within the context of feeding in Aplysia.
Ingestive feeding behavior in Aplysia consists of
stereotypic repetitive biting or swallowing movements
(Kupfermann 1974). The rate and specific features of
swallowing movements can vary to compensate for changes in the load and
width of the ingested food (Hurwitz and Susswein 1992
).
Both biting and swallowing are also altered as a function of the
motivational state of the animal (Weiss et al. 1981
).
For instance, as animals become food aroused there are progressive
increases in the size and speed of biting responses (Kupfermann
1974
; Susswein et al. 1978
). Food-induced arousal, in part, results from the release of serotonin from the metacerebral cells (MCCs) (Eisenstadt et al. 1973
;
Gerschenfeld and Paupardin-Tritsch 1974
;
Gerschenfeld et al. 1978
; Rosen et al.
1983
; Weinreich et al. 1973
; Weiss et al.
1975
, 1979
, 1986
). These neurons
make extensive central and peripheral connections and exert modulatory
actions. As arousal is developed, there are changes in the movements
that constitute a bite. For example, when animals bite they initially
weakly protract the radula, a chitinous structure that grasps food. As
animals become aroused, the extent of radula protraction increases.
These effects are partially mediated by MCC activity (Rosen et
al. 1983
).
Previous experiments have demonstrated that I2 (intrinsic muscle 2) is
the major muscle that mediates radula protraction (Hurwitz et
al. 1996). On the basis of the widespread serotonergic
innervation of the buccal mass, we hypothesized that serotonin would be
present in the I2 neuromuscular system, and that it would modulate
parameters of motor neuron elicited muscle contractions that are likely
to be altered when food-induced arousal is developed.
Studies of other buccal muscles have indicated that in addition
to serotonin released by the MCCs, Aplysia neuromuscular
systems can also be modulated by peptides present in the feeding motor neurons themselves (Brezina et al. 1995; Church
et al. 1993
; Cropper et al. 1987a
,b
,
1988
, 1990b
, 1994
;
Fox and Lloyd 1997
; Lloyd et al. 1984
;
Whim and Lloyd 1990
). Thus another major goal of
this study was to determine whether there are intrinsic (Cropper
et al. 1987a
; Katz 1995
) modulators present in
I2 motor neurons.
The I2 muscle (Howells 1942) was of particular interest
because it mediates the protraction phase of feeding (Hurwitz et
al. 1996
) while previously characterized muscles control other
movements, i.e., the accessory radula closer (ARC) controls radula
closing, the I7-10 controls radula opening, and the I3a control jaw
closure (Church et al. 1993
; Cohen et al.
1978
; Cropper et al. 1987a
,b
, 1988
, 1990a
,b
, 1994
;
Evans et al. 1996
, 1999
; Fox and
Lloyd 1997
; Lloyd et al. 1984
). The fact that
the I2 muscle is a radula protractor is important because measurements
of bite magnitude during food-induced arousal and satiation have been
based on the extent of radula protraction only, since during retraction
the radula recedes into the buccal cavity and cannot be visualized.
Therefore characterization of the neuromuscular system that controls
protraction is central to the attempt to establish a connection between
overt behavior and physiological studies performed in vitro. Although
there are extensive data regarding modulation of the ARC muscle, recent work indicates that individual buccal muscles exhibit significantly different contractile properties (Evans et al. 1996
).
Thus one cannot assume that different muscles are modulated in a
similar fashion. To determine whether the muscles that generate
protraction are also subject to modulation that enhances this phase of
behavior, we undertook an investigation of the I2 muscle.
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METHODS |
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The experimental subjects for this study were Aplysia californica weighing 150-400 g. They were obtained from Marinus (Long Beach, CA) and from the National Resource for Aplysia at the University of Miami. They were maintained at 14-16°C in holding tanks containing aerated, filtered seawater. Animals were initially immobilized by injection of isotonic MgCl2 (50% of body weight) and dissected. In neuromuscular preparations the buccal ganglion and I2 muscle were removed with the I2 nerve intact and attached to the I2 muscle. The buccal ganglion was pinned with the caudal surface up. The sheath overlying the uppermost surface of the ganglion was surgically removed.
Recording apparatus and bathing solutions
Intracellular recordings were obtained from isolated ganglia or
from neuromuscular preparations maintained at room temperature (18-22°C). Neuromuscular preparations, which consisted of the buccal
ganglion, I2 nerve, and I2 muscle, were transferred to silicone
elastomer (Sylgard)-lined plastic culture dishes (Fig. 1A). The I2 muscle was pinned
along the edge that was originally attached to the I1/I3 muscle (the
edge of the I2 muscle that was originally attached to the esophagus was
left free). A plexiglass subchamber was gently placed around
the I2 muscle, on top of the I2 nerve, and was sealed with petroleum
jelly (Vaseline). A force transducer (Isotonic Transducer
"60-3000," Harvard Apparatus) was used to monitor muscle
contractions and was attached to the free end of the I2 muscle. In most
experiments, continuous stretching of the muscle was prevented since
the free end of the transducer arm was supported (Evans et al.
1996). In general, both the outside compartment and the muscle
subchamber contained artificial seawater (ASW; in mM: 460 NaCl, 10 KCl,
11 CaCl2, 55 MgCl2, and 5 NaHCO3). In some experiments, however, a solution
containing high divalent cations (HiDi) was used for blocking
polysynaptic activity in the buccal ganglion (in mM: 311 NaCl, 9 KCl,
33 CaCl2, 132 MgCl2, and 5 NaHCO3). This solution blocks polysynaptic
activity by raising the threshold for triggering action potentials.
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I2 muscle contractions can be elicited when any of its four motor
neurons (B61, B62, B31, and B32) are stimulated (Hurwitz et al.
1994). Neurons B31 and B32 cannot be distinguished
morphologically. Moreover, because they are strongly electrically
coupled, they are difficult to manipulate individually. Consequently,
it is difficult to use these motor neurons to elicit a series of
controlled muscle contractions with similar parametric features. When
B61 is stimulated alone, the "bands" of the I2 muscle contract
first, i.e., a response to neuronal stimulation is initially observed along the edge of the I2 that is adjacent to the esophagus (Fig. 1B1). In contrast, when B62 is stimulated, the part of the
I2 muscle that is adjacent to the I1/I3 muscles contracts first (Fig. 1B2). Because of the anatomical locus of contractions
produced by B62 stimulation, these contractions were more difficult to measure than contractions elicited by B61. When the duration of firing
of either B61 or B62 is extended, both parts of the I2 muscle contract
(Fig. 1B3; n = 37) perhaps because B61 and
B62 are both activated through their electrical coupling. Therefore in
most quantitative experiments we triggered I2 contractions by
stimulating B61 (at ~18 Hz) in relatively short-duration bursts (~1
s). Results obtained with B61 stimulation were qualitatively confirmed
with B62 and B31/B32.
Electrophysiology
For intracellular recording and stimulation, I2 motor neurons
were impaled with double-barreled microelectrodes that were made of
thin-walled glass and contained 1.9 M potassium acetate and 0.1 M
potassium chloride. Electrodes were beveled so that their impedance
ranged from 6 to 10 M. In double labeling experiments the potassium
acetate in the stimulating electrode was replaced by a 3% solution of
biocytin (Sigma) in 1 M potassium acetate. Biocytin electrodes were
beveled so that the impedance of the barrel containing the dye was
~10 M
and the impedance of the potassium acetate barrel was ~6
M
.
Morphology
Immunohistochemistry was performed on whole-mount preparations
as previously described (Miller et al. 1991;
Vilim et al. 1996a
,b
; Xin et al. 1999
).
Preparations were fixed in 4% paraformaldehyde (4°C for 24 h).
They were then washed for 2 days in Triton X-100 (diluted 1 to 100).
Goat-serum was added at a dilution of 1:200, and 1 h later the
first antibody was added at a dilution of 1:250. Primary antisera were
as follows: myomodulin (MM) (Miller et al. 1991
),
buccalin (Miller et al. 1992
), RFamide (Cropper
et al. 1994
), small cardioactive peptide (SCP; kind gift from
Dr. R. Scheller, Stanford University), and serotonin (5-HT; kind gift from Dr. Hadassah Tamir, Columbia University). Preparations were incubated for 24 h at 4°C and then washed for 24 h.
Secondary antibodies (fluorescently labeled with CY-3) were diluted 1 to 500 and were added for 24 h. Ganglia were then washed for
48 h. In double-labeling experiments two secondary antibodies were
used; for MM immunocytochemistry we used an antiserum fluorescently labeled with CY-3, and for RFamide or 5-HT immunocytochemistry we used
an antiserum fluorescently labeled with fluorescein. Absorption controls for MM and RFamide were performed by preincubating antibodies with MMa and RFamide A at
10
5 M. Preincubation
controls were not performed for 5-HT.
When specific neurons needed to be identified for immunocytochemistry, the cells were first physiologically identified and were then iontophoretically filled with biocytin (500-ms, 0.5-nA current pulses with alternating polarity for ~10 min), and preparations were kept for 24 h at 14°C. Following the fixation and permeabilization described above, cells were fluorescently labeled by incubating tissue with a streptavidin Bodipy FL conjugate (50 µg/ml; Molecular Probes) in RIA buffer. Ganglia were then processed for immunocytochemistry as described above and were cleared in 50% glycerol in RIA buffer. Preparations were viewed with a Nikon fluorescence microscope and photographed with Tri-X (ASA 400) film.
Biochemical identification of peptides contained in the B61/B62 and B31/B32 neurons
Identified B61/B62 and B31/B32 neurons (Hurwitz et al.
1994; Susswein and Byrne 1988
) were marked by
intracellular iontophoresis (constant 4-nA hyperpolarizing current) of
Fast green. Buccal ganglia containing labeled neurons were incubated at
14°C for 24 h in 1 ml of a mixture of 50% ASW and 50%
Aplysia hemolymph that had been filtered through a 0.22-µm
filter. The incubation solution contained 0.5 mCi of
[S35] methionine, 100 µl antibiotics
(penicillin and streptomycin each at 50 U/ml), and 2.5 µl of 1 M
colchicine dissolved in dimethyl sulfoxide (DMSO). Colchicine was added
to inhibit axonal transport, thereby eliminating labeled peptides that
might be transported to fibers and terminals near the somata of neurons
of interest. At the end of incubation, ganglia were rinsed with 10 ml
of the incubation solution without [35S]
methionine and were left in this solution for another 30 min. Individual B61/B62 and B31/B32 neurons were then dissected using a
freeze substitution method (Ono and McCaman 1980
). Cells
were transferred to glass microtubes, and peptides were extracted by heating for 10 min in 100 µl of 0.01 M trifluoroacetic acid (TFA) containing nanomolar quantities of synthetic peptides (Cropper et al. 1987b
; Lloyd et al. 1987
).
Extracted material was subjected to two sequential stages of reverse phase high performance liquid chromatography (RP-HPLC), which were performed using an RP-300 column. Synthetic peptides were identified by absorbance measurements at 215 nm. In the first stage of chromatography, the column was developed at 1 ml/min with 5-5% solvent B in 5 min, followed by 5-50% solvent B in 45 min. Solvent A was 0.01 M TFA in H2O and solvent B was 0.01 M TFA in acetonitrile. Fractions were collected every 30 s. Radioactive peptides were identified by scintillation counting of fraction aliquots (20% of 9 B31/B32s and 10% of 12 B61/B62s). In the second stage of chromatography, the column was developed at 1 ml/min with 5-5% solvent B in 5 min, followed by 10-50% solvent B in 40 min. Solvent A was 0.01 M HFBA in H2O and solvent B was 0.01 M heptafluorobutyric acid (HFBA) in acetonitrile. Fractions were collected every 30 s. Radioactive peptides were detected by counting whole fractions.
cAMP measurements
I2 muscles were dissected, weighted, and incubated for 2 h
in ASW before treatment with neuromodulators for 10 min. No
phosphodiesterase inhibitors were used. After muscles were exposed to
modulators, they were homogenized in 65% ethanol and 35%
H2O and heated to 90°C for 5 min. Homogenates
were spun in a clinical centrifuge for 2 min, and the supernatant was
stored at 80°C. cAMP levels were quantified using a commercially
available RIA (Amersham).
Data analysis
When multiple group comparisons were performed, we used a one-way ANOVA. In post hoc comparisons we used a t-test. The level of statistical significance was set at P < 0.05.
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RESULTS |
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Acetylcholine as the putative transmitter of the I2 motoneurons
Many motor neurons in the buccal ganglion utilize acetylcholine as
their primary transmitter, and many muscles contract in response to
acetylcholine application (Cohen et al. 1978;
Evans et al. 1996
; Fox and Lloyd 1997
;
Lloyd and Church 1994
). We therefore sought to determine
whether hexamethonium, the acetylcholine antagonist that blocks
synaptic potentials and contractions of other buccal muscles, could
block I2 neuromuscular activity. In the presence of a HiDi solution,
firing of B61/B62 induced 3- to 10-mV excitatory junction potentials
(EJPs) in I2 muscle fibers, and firing of B31/B32 induced 1- to 5-mV
EJPs. These EJPs had a constant latency and a fixed amplitude, i.e., we
did not observe facilitation or depression. EJPs were reversibly
abolished when hexamethonium was added to I2 subchambers (Fig.
2; n = 4 for each motor
neuron group). These experiments suggest that the connections of
B61/B62 and B31/32 are monosynaptic and cholinergic.
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To monitor muscle contractions the cut end of the I2 muscle was
attached to a force transducer, and I2 motor neurons were stimulated in
fixed bursts. When hexamethonium was added to the I2 subchamber at
concentrations of 106,
10
5, and
10
4 M, muscle
contractions induced by stimulation of B61/B62 were reduced in size
(Fig. 3). When the hexamethonium
concentration was increased to
10
3 M, contractions were
abolished (n = 5). The blocking effect of hexamethonium
was reversible. Similar results were obtained when muscle contractions
were elicited by stimulation of B31/B32 (not shown).
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We also tested the effect of applying acetylcholine directly to the I2
muscle. At 105 M
acetylcholine induced a powerful long-lasting muscle contraction (Fig.
4A; n = 7).
Furthermore, we observed that when the ACh-elicited contractions
decremented, presumably because of desensitization of ACh receptors,
stimulation of the motor neuron produced a much smaller response than
it did before ACh application. The decrement of the motor neuron
elicited contractions was unlikely to be a result of muscle fatigue.
The size of muscle contraction elicited by motor neuron stimulation
also decreased in the presence of low concentrations (i.e.,
10
9 to
10
7 M) of ACh, although
at these concentrations ACh itself did not elicit muscle contractions
(Fig. 4B) (see also Cohen et al. 1978
). Together, these pharmacological experiments support the idea that ACh
is a primary neurotransmitter in the I2 neuromuscular system.
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Neuromodulators are present in the I2 muscle
Many of the muscles of the buccal mass in Aplysia
receive modulatory input (Cropper et al. 1987b,
1988
, 1994
; Lloyd et al. 1984
). To determine whether the same is true for the I2 muscle, we examined this muscle for the following types of immunoreactivity: MM-like, buccalin-like, SCP-like, RFamide-like, and 5-HT-like. MM
immunocytochemistry revealed a dense network of processes (Fig. 5, A1 and B1;
n = 5). This immunoreactivity was abolished when the MM
antibody was preincubated with synthetic MMa (not shown). This is
consistent with MM being the epitope that is recognized by our
antibody. In addition, processes in the I2 muscles stained positively
with an antiserum that nonspecifically recognizes peptides with the
C-terminal RFamide sequence (Fig. 5A2; n = 3). The I2 also stained positively with a 5-HT antiserum (Fig.
5B2; n = 4). To determine whether
immunoreactivity was present in the same or different sets of
processes, we performed double-labeling experiments. Fibers that were
MM immunoreactive were not immunoreactive to either RFamide or 5-HT
(Fig. 5, A1 vs. A2, and Fig. 5, B1 vs. B2; n = 2).
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I2 muscles did not show buccalin-like or SCP-like immunoreactivity even
when buccalin and SCP immunoreactivity could be seen in ARC muscles
(Cropper et al. 1987a; Miller et al.
1992
) that were processed in parallel with I2 muscles
(n = 2; data not shown). We did, however, observe
SCP-like immunoreactivity in processes innervating the junction of the
I2, I4, and I1/I3 muscles. These processes did not, however, extend
into the rest of the I2 muscle. This may account for previous
observations of SCP-like immunoreactivity in the I2 muscle
(Church et al. 1991
).
B31/B32 and B61/B62 synthesize modulatory neuropeptides
To determine whether any of the I2 muscle motoneurons contain 5-HT
or neuropeptides (i.e., MM or RFamide), these neurons were filled with
biocytin, preparations were fixed, and tissue was incubated with avidin
labeled with fluorescein. Buccal ganglia were first exposed to those
antibodies that stained neuronal processes in the periphery, and then
visualized using a secondary antibody labeled with CY-3. Interestingly,
all four I2 motoneurons were immunoreactive to MM (Fig.
6) but not to RFamide (not shown;
n = 3), or 5-HT (not shown; n = 3).
Notice that the signal in B31/B32 and B61/B62 is not due to a
breakthrough of fluorescent labeled avidin as neuron B4, which does not
contain MM (Church and Lloyd 1991), shows no rhodamine
fluorescence. Because various MMs share their C-terminus, the
antibodies we used do not distinguish between them.
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To determine whether both sets of I2 motor neurons (B31/32 and B61/62)
actually synthesize authentic MM, we performed biochemical experiments.
Although the gene for MM encodes seven structurally related
neuropeptides, only one peptide, MMa, is present in multiple copies
(i.e., 10) and is the most abundantly expressed (Miller et al.
1993). I2 motoneurons were incubated in
[S35] methionine and then subjected to two
sequential stages of RP-HPLC. The counterions used in these experiments
were previously employed to purify the MMs (Brezina et al.
1995
; Cropper et al. 1987b
). Radiolabeled
peptides synthesized by B61/B62 and by B31/B32 coeluted with synthetic
MMa in both stages of chromatography (Fig.
7). Thus B31/B32 and B61/B62 neurons
synthesize MMa. These data are also supported by immunostaining.
Previous work has shown that neurons in which MM is not detected
chromatographically do not stain for MM (e.g., Cropper et al.
1987b
). Furthermore, we show in Fig. 6 the absence of
immunostaining of neuron B4, in which MM is not detected
chromatographically (Church and Lloyd 1991
).
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Effects of I2 modulators on contraction size and relaxation rate
Because both 5-HT and MM were detected in the innervation of the
I2 muscle, we characterized the effects of these substances on muscle
contractions elicited by the I2 motoneurons. (Although RFamide-like
immunoreactivity was detected in the I2 system, we could not
characterize the bioactivity of this peptide since the structure of the
specific RFamide that is present in the I2 innervation is not known.)
MM experiments were performed with MMa and MMb since both are encoded
by the MMa precursor and previous studies have shown that these two MMs
exert the most differing actions (Brezina et al. 1995).
These differences are particularly apparent when
concentration-dependent effects are compared. For example, in the ARC
muscle both MMa and MMb increase the amplitude of muscle contractions
at low concentrations, but at higher concentrations MMa begins to
depress muscle contractions while MMb continues to enhance contraction
amplitude. In this study, therefore we tested the effects of modulators
at concentrations ranging from ~10
9 to
10
5 M.
MMa increased contraction amplitude at concentrations lower than
107 M and decreased the
amplitude of contractions at higher concentrations (Fig.
8A1; also see Fig. 11 for
group data). In addition to increasing the amplitude of muscle
contractions, MMa also increased the rate of relaxation of muscle
contractions (Fig. 8A2). To determine whether the MM-evoked
increase of the relaxation rate was due to the increase of the
amplitude of muscle contraction, we increased the amplitude of muscle
contractions by increasing the frequency of B61 firing (Fig.
8B1). Figure 8B2 demonstrates that when the amplitude of muscle contractions was increased, relaxation rate was not
changed.
|
At concentrations of up to
107, MMb exerted actions
that qualitatively resembled the actions of MMa, i.e., MMb increased
the amplitude and the relaxation rate of muscle contractions (Fig. 9, A and B). In
this series of experiments, we could again dissociate the modulation of
relaxation rate from modulation of the amplitude of muscle contractions
since increases of contraction amplitude did not account for increases
in relaxation rate (Fig. 9, B and C). Analysis of
group data revealed significant quantitative differences between the
effects of MMa and MMb on the amplitude of muscle contractions. Unlike
MMa, which increases the amplitude of muscle contractions at
concentrations up to 10
8
M and at higher concentrations depresses the contraction amplitude, MMb
potentiates muscle contractions throughout the range tested (up to
10
5 M). Indeed the
potentiating effects of MMb grew progressively larger until the
concentration of 10
6 M
was reached (group data in Fig. 11).
|
The actions of 5-HT were similar to those of the two MMs, i.e., 5-HT potentiated the amplitude (Fig. 10A) and the relaxation rate of muscle contractions (Fig. 10, B and C). The actions of 5-HT on contraction amplitude were more similar to those of MMb than MMa as 5-HT potentiated I2 muscle contractions at all concentrations tested (group data in Fig. 11). In fact, the two modulators appeared to be approximately equipotent on the amplitude of muscle contractions (Fig. 11).
|
|
Serotonin and myomodulins stimulate cAMP synthesis in the I2 muscle
cAMP has been implicated as a second messenger in several studies
of peptidergic and serotonergic modulation of neuromuscular function in
Aplysia (Cropper et al. 1990b; Evans
et al. 1999
; Lloyd et al. 1984
; Weiss et
al. 1979
). To investigate the possible role that cAMP may play
in the modulation of the I2 muscle we took two approaches. First, we
tested the effect of a cAMP analogue [8-(4-chlorophenylthio)-adenosine
3':5'-cyclic mono phosphate] (8-CPT) on motor neuron elicited muscle
contractions. We found that perfusion with this cAMP analogue increased
contraction amplitude (Fig.
12A) and enhanced relaxation
rate (Fig. 12B; n = 4). Second, we measured
cAMP levels after I2 muscles were incubated for 10 min in ASW,
10
5 M 5-HT and
10
5 M MMa (Fig.
13A). In general, levels of
stimulation found in this study are within the range of cAMP
stimulation that has been reported for other buccal muscles in
Aplysia (e.g., Cropper et al. 1990b
; Evans et al. 1999
; Lloyd et al. 1984
;
Weiss et al. 1979
). More specifically, one-way ANOVA
revealed an overall statistically significant difference (f = 106.4; P < 0.001; df = 2.12) between the three
treatments (controls, MMa treated, and 5-HT treated). Individual
comparisons using a two-tailed t-test also revealed statistically significant differences. Specifically, when the MMa-treated muscles were compared with controls, the level of cAMP was
found to be significantly increased (P < 0.05).
Similarly, the level of cAMP in 5-HT-treated muscles was also
significantly increased (P < 0.001). The observations
that 5-HT is a stronger stimulator of cAMP than MMa parallels the
observation that 5-HT is also a stronger potentiator of muscle
contractions than MMa. Since MMb is also a stronger potentiator of
contraction amplitude than MMa, we sought to determine whether MMb is
also a stronger stimulator of cAMP (Fig. 13B). Five
different MM concentrations were tested,
10
9,
10
8,
10
7,
10
6, and
10
5 M. Both MMs produced
statistically significant increases in cAMP (MMa df = 4.27; f = 4.8; P < 0.005, and for MMb df = 4.27; f = 6.5; P < 0.001). Maximal stimulation occurred at
10
7 M for MMb and
10
8 M for MMa. There was
statistically significant overall difference between the effects of the
two MMs (t-test df = 1.52; f = 2.06; P < 0.05).
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DISCUSSION |
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Behavioral observations of biting have been limited to radula
protraction (when the radula retracts, it recedes from view into the
buccal cavity). In contrast, physiological studies that were used to
support the hypothesis that modulatory processes contribute to the
enhancement of muscle contractions were performed on muscles that
control other movements, i.e., the ARC and I7-I10, which mediate radula
opening and closing, and the I3a, which mediates jaw closure
(Church et al. 1993; Cohen et al. 1978
;
Cropper et al. 1987a
,b
, 1988
,
1990a
,b
, 1994
; Fox and Lloyd
1997
; Lloyd et al. 1984
). In this study we
investigated modulatory control of the I2 muscle, a major buccal muscle
that protracts the radula (Hurwitz et al. 1996
).
Importantly, recent work on different buccal muscles has indicated that
muscles display distinct contractile and biophysical properties
(Evans et al. 1996
). Thus these observations make it
imperative that the organization of modulation of protraction muscles
be determined as a first step toward establishing a connection between
the overt behavior of the animal and the modulatory processes that may
underlie the behavior.
In view of the widespread serotonergic innervation of the buccal
musculature, we expected that neural processes on the I2 muscle would
contain 5-HT. We also expected that 5-HT would increase the size of
motor neuron elicited I2 contractions. This is indeed what we found.
Additionally, we found that as in other buccal muscles (Fox and
Lloyd 1997; Lloyd et al. 1984
; Rosen et
al. 1983
; Weiss et al. 1975
,
1979
) 5-HT increased relaxation rate and therefore shortened contraction duration. Modeling and experimental studies in
the ARC neuromuscular system have suggested that increases in
relaxation rate are important to accommodate the increase in the speed
of biting that occurs in parallel with the increase in bite magnitude
(Deodhar et al. 1994
; Weiss et al. 1992
,
1993
). In particular, experimental studies have shown
that when contraction amplitude is increased in unmodulated ARC
muscles, relaxation rate does not change (Cropper et al.
1990b
). Consequently, contraction duration increases. We found
that this is also true for the I2 muscle. Modeling studies have
suggested that these types of increases in contraction duration may
become a problem when behavior is executed rapidly (Brezina and
Weiss 2000
; Brezina et al. 2000a
,b
; Deodhar et al. 1994
; Weiss et al. 1992
).
Thus behavior becomes inefficient because individual muscles fail to
completely relax between contractions and antagonistic muscles are
coactive, and therefore the two sets of muscles work against each
other. Experimental studies (Yu et al. 1999
) of the
visco-elastic properties of the I2 muscle indicate that this muscle may
exert braking, i.e., opposing actions on retraction movements,
especially when the retraction movements are rapid. The braking actions
of I2 on the retraction phase will depend on the speed with which the
I2 muscle relaxes. In this study we demonstrated that the relaxation
rate of I2 can be modulated by 5-HT and by the MMs. Since the two forms
of modulation also enhance the size of contractions, it is likely that
the two modulatory actions will act jointly to assure that in face of increased amplitude of I2 contractions this muscle will not provide additional braking of the retraction phase that follows I2 contractions.
Considerable evidence indicates that all serotonergic input to the
buccal muscles has as its source the extrinsic modulatory neuron (the
MCC). Thus the 5-HT in I2 is also presumably from this
extrinsic source. We sought to determine whether there are also
modulatory transmitters intrinsic to the I2 neuromuscular system. To
localize modulatory peptides we initially used immunocytological techniques. We found that antibodies raised against RFamide and MM
stained neural processes and varicosities on I2 muscles. Immunopositive neurons were not detected in the muscle, indicating that somata of
immunopositive neurons were located elsewhere. The I2 motor neurons
were not, however, RFamide immunopositive. A similar situation exists
in the ARC neuromuscular system where RFamide peptides are present in
terminals on the ARC muscle but are not present in the ARC motor
neurons. The RFamide like peptides in the ARC neuromuscular system may
originate from the S cluster of buccal mechano-sensory neurons, which
send their process to buccal muscle (Fiore and Meunier
1979; Lloyd et al. 1987
). Since the S cells project extensively to buccal muscles, they may also innervate the I2 muscle.
We found that both sets of I2 motor neurons, i.e., B31/32 and B61/62,
are MM immunoreactive. Although the antiserum used in this study does
not distinguish between the nine members of the MM family, our
biochemical data are consistent with the idea that MMa is present in
B31/32 and B61/B62. At least six of the other MMs are likely to be
present in the I2 motor neurons since multiple copies of MMa are
present on the MM precursor that additionally encodes single copies of
MMb, MMd, MMf, MMg, MMh, and MMi (Miller et al. 1993).
We did not find evidence that the other two MMs (C and E) are present
in the I2 motor neurons. A peak of radioactivity with a relatively long
retention time that could correspond to MMc was not observed in B31/B32
or B61/B62. We would not have expected to see MMe in biochemical
experiments since MMe does not contain methionine and therefore would
not be radiolabeled. Thus immunocytochemical and biochemical
experiments indicate that I2 motor neurons do in fact contain intrinsic
modulators, i.e., members of the MM peptide family.
Interestingly low doses of the most abundant MM, MMa exert modulatory
effects that are very similar to those of 5-HT; they increase the size
and relaxation rate of motor neuron elicited I2 contractions. This
might seem redundant. Data, however, suggest that the release of
extrinsic and intrinsic modulators may predominately occur at different
times during a feeding sequence. For example, recordings from intact
animals have indicated that the MCCs are active during the appetitive
phase of feeding (Horn et al. 1999; Kupfermann
and Weiss 1982
), but that their firing slows down precipitously when food is ingested (Kupfermann and Weiss, unpublished observation). Radula protraction does not occur during appetitive behaviors; consequently, for the most part the I2 motor neurons are not likely to
be active until consummatory feeding begins. Thus the extrinsic modulator 5-HT is likely to be present in the highest concentrations during appetitive feeding, whereas the intrinsic I2 modulators, the
MMs, are likely to be present during consummatory feeding.
An understanding of the relative role of the extrinsic (5-HT) and
intrinsic (peptide) modulation of buccal muscles must take into
consideration that the effects of these modulators are apparent only
when the muscle begins to contract. Consequently, although 5-HT is
released in the I2 neuromuscular system during appetitive behaviors,
its effects will not be manifested until consummatory feeding is
triggered. Since the released 5-HT will dissipate as consummatory
feeding progresses (Horn et al. 1999; Kupfermann and Weiss 1982
), 5-HT might primarily modulate I2 muscle
contractions that occur at the beginning of a feeding sequence. In
contrast, studies of peptide release in other Aplysia
neuromuscular systems have suggested that peptide release progressively
increases when motor neurons are repeatedly activated (Vilim et
al. 1996a
,b
; Whim and Lloyd 1992
,
1994
). In fact, peptide release may not occur at all
during a single burst of activity (Vilim et al. 1996a
,b
, 2000
). Thus although effects of 5-HT and MM on the I2
muscle will both be manifested during consummatory feeding,
serotonergic effects may predominate early in an ingestive sequence,
whereas effects of MM may predominate as feeding progresses.
This work has established that the I2 muscle contains two types of
modulators: those that are intrinsic (i.e., originate in the
motoneurons) and those that are extrinsic (i.e., originate in cells
that are not motoneurons) (Cropper et al. 1987a;
Katz 1995
; Katz and Frost 1996
).
Previously, intrinsic and extrinsic modulators were shown to elevate
the levels of cAMP in other muscles: the ARC, the I7-10 complex, and
the I3 muscles (Church et al. 1993
; Cropper et
al. 1990b
; Evans et al. 1999
; Fox and
Lloyd 2000
; Lloyd et al. 1984
; Probst et
al. 1994
; Weiss et al. 1978
,
1979
; Whim and Lloyd 1989
,
1990
). In these muscles, cAMP was shown to potentiate
muscle contractions and to increase muscle relaxation rate. In this
paper, we established that incubation of the I2 muscle with 8-CPT-cAMP
also increases the amplitude and the relaxation rate of muscle
contractions. In addition, the extrinsic modulator (5-HT) and the
intrinsic modulators (MMa or MMb) elevate cAMP levels in the I2 muscle.
Thus cAMP is a candidate second messenger for the actions of both the
intrinsic and extrinsic modulators.
However, our data concerning the mechanisms by which I2 muscle
contractions/relaxations are modulated suggest that cAMP may not be the
sole player. Several lines of evidence suggest that other unknown
second messengers might be involved. First, different modulators
produced similar effects in the I2 muscle, but do not produce similar
levels of cAMP synthesis. For example, 5-HT and MMb both potentiate I2
muscle activity, but yet, 5-HT increase cAMP synthesis more than MMb
did. Second, increases in the potentiation of I2 muscle do not always
go along with increases in cAMP synthesis. Namely, increasing MMb
concentration increases the muscle potentiation but decreases cAMP
synthesis levels. Third, in the ARC muscle, MMa was shown to activate
both cAMP synthesis that up-modulates an L-type calcium current
(Brezina et al. 1994a) and, in addition, an unknown
second messenger responsible for potassium current activation
(Brezina et al. 1994b
). High concentrations of MMa decrease the contraction size of I2 muscle, which can be related to
activation of K current that tends to decrease the depolarization of
the muscle and therefore to diminish the activation of the voltage-dependent L-type calcium current (Brezina et al.
1994a
). The fact that MMa produce similar bioactive effects
both in the I2 and ARC muscles suggests that similar currents driven by
similar second messengers may be activated. Other studies in the I7-I10 and the I3 muscle describe some degrees of dissociation between modulation of muscle contractions and cAMP elevations (Evans et al. 1999
; Fox and Lloyd 2000
). Independent of
the detailed explanations of the dissociations of cAMP synthesis and
potentiation of muscle contractions of the I2 muscle, it is important
to note that when one combines the finding that intrinsic and extrinsic
modulators stimulate cAMP with the finding that cAMP application mimics
the actions of modulators, the most parsimonious interpretation of our
data suggests that, at least in part, cAMP mediates the actions of
modulators on muscle contractions.
In summary, in this study we sought to determine the identity and
define the actions of modulators that may be present in the
neuromuscular system that plays a major role in generating the
protraction phase of feeding. We showed that the MM peptides are
present in the motoneurons that innervate this muscle and that the
muscle also receives serotonergic modulation. The peptides as well as
5-HT increase the amplitude and relaxation rate of the I2 contractions.
Because the I2 muscle is a major protractor and observations of the
buccal mass during feeding are largely restricted to this phase of
behavior, these findings begin to bridge the gap between the behavior
and the in vitro physiology of the animal. Thus these findings will be
incorporated into computer simulations (e.g., Brezina and Weiss
2000; Brezina et al. 2000a
,b
) that are being
developed to understand how activity of different buccal muscles
becomes integrated to generate appropriate output of the buccal mass
during feeding behavior.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Mental Health Grants MH-36730, MH-51393, K02 MH-01267, and K05 MH-01247 and Human Frontier Science Program Grant LT-0464/1997. Aplysia were partially provided by the National Resource for Aplysia at the University of Miami under National Center for Research Resources Grant RR-10294.
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FOOTNOTES |
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Address for reprint requests: I. Hurwitz, Box 1218, Dept. of Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029 (E-mail: itay.hurwitz{at}mssm.edu).
Received 24 May 2000; accepted in final form 22 August 2000.
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REFERENCES |
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