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The Journal of Neuroscience, March 1, 2000, 20(5):2036-2042
Peptide Cotransmitter Release from Motorneuron B16 in
Aplysia californica: Costorage, Corelease, and
Functional Implications
Ferdinand S.
Vilim1,
Elizabeth C.
Cropper1,
David A.
Price2,
Irving
Kupfermann3, and
Klaudiusz R.
Weiss1
1 Department of Physiology and Biophysics, Mount Sinai
School of Medicine, New York, New York 10029, 2 C. V. Whitney Laboratory, University of Florida, St. Augustine, Florida
32086, and 3 Center for Neurobiology and Behavior, College
of Physicians and Surgeons of Columbia University, New York, New York
10032
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ABSTRACT |
Many neurons contain multiple peptide cotransmitters in addition to
their classical transmitters. We are using the accessory radula closer
neuromuscular system of Aplysia, which participates in
feeding in these animals, to define the possible consequences of
multiple modulators converging on single targets. How these modulators
are released onto their targets is of critical importance in
understanding the outcomes of their modulatory actions and their
physiological role. Here we provide direct evidence that the partially
antagonistic families of modulatory peptides, the myomodulins and
buccalins, synthesized by motorneuron B16 are costored and coreleased
in fixed ratios. We show that this release is calcium-dependent and
independent of muscle contraction. Furthermore, we show that peptide
release is initiated at the low end of the physiological range of
motorneuron firing frequency and that it increases with increasing
motorneuron firing frequency. The coordination of peptide release with
the normal operating range of a neuron may be a general phenomenon and
suggests that the release of peptide cotransmitters may exhibit similar
types of regulation and plasticity as have been observed for classical
transmitters. Stimulation paradigms that increase muscle contraction
amplitude or frequency also increase peptide release from motor neuron
B16. The net effect of the modulatory peptide cotransmitters released
from motorneuron B16 would be to increase relaxation rate and therefore
allow more frequent and/or larger contractions to occur without
increased resistance to antagonist muscles. The end result of this
modulation could be to maximize the efficiency of feeding.
Key words:
Aplysia; neuropeptide; cotransmitter; buccalin; myomodulin; immunolocalization; RIA; release; motorneuron
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INTRODUCTION |
Neuropeptides, the most diverse
class of signaling molecules in the nervous system, are often
hypothesized to function as cotransmitters. However, despite their
pervasiveness and undoubtedly important function in the nervous system,
most neuropeptides retain the status of putative rather than
established cotransmitters. Furthermore, many neurons contain multiple,
functionally distinct peptides (Kupfermann, 1991 ; Iversen, 1995).
Without detailed knowledge of the actions and release parameters of
peptide cotransmitters, it is virtually impossible to determine their
physiological function. It has been suggested that the task of
understanding the functional role of neuropeptides may be facilitated
by studying relatively simple preparations where the biological context
is well understood (Iversen, 1995).
One preparation in which both the biological context and the nature of
the primary transmitters and cotransmitters have been extensively
studied is the accessory radula closer (ARC) muscle of Aplysia
(Weiss et al., 1992). This muscle is innervated by two cholinergic
motorneurons, B15 and B16. Each of these motor neurons also contains
multiple neuropeptides, which are members of two distinct neuropeptide
families that have partially antagonistic effects on muscle. The
cellular mechanisms of action of acetylcholine, the primary
transmitter, and of the neuropeptides have been well characterized.
Furthermore, because the firing of motorneurons in freely behaving
animals has been characterized (Cropper et al., 1990b), previous work
provides an excellent background for more advanced studies of peptide
release and peptide action in this system.
Motorneuron B15 contains the SCPs and buccalins (BUCs), whereas
motorneuron B16 contains the myomodulins (MMs) and BUCs (Weiss et al.,
1992). The SCPs and MMs act postsynaptically to increase contraction
amplitude and relaxation rate, whereas the BUCs depress contraction
amplitude by reducing ACh release. How these motorneurons release these
peptides critically affects the interpretation of the biological
function of the neuromuscular system (Brezina et al., 1996). In a
recent series of studies, we conclusively demonstrated that neuron B15
coreleases SCPs and BUCs in a fixed ratio and that release occurs
within the normal range of firing frequencies of B15 (Vilim et
al., 1996a,b). However, motorneuron B16 contains a different set of
partially antagonistic peptide families and exhibits a different range
of firing frequencies (Cropper et al., 1990b). Because motorneuron B16
is coactive with motorneuron B15, it is also important to determine the
release parameters for the modulators contained in B16 if an
understanding of their role in the functioning of the system is to be achieved.
In the present study we sought to achieve three goals. First, to
measure the release of MMs from motorneuron B16. This is of particular
importance because despite the presence of MMs and related peptides in
a number of systems of a variety of species (Brezina et al., 1995;
Kellett et al., 1996; Greenberg et al., 1997; Wang et al., 1998), no
direct evidence exists for their release. Second, we sought to
determine if the MMs and BUCs are released in a fixed or a variable
ratio from motorneuron B16. Third, we sought to determine whether or
not release of the peptides is coordinated with the normal range of
firing frequencies for B16.
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MATERIALS AND METHODS |
Animals. Specimens of Aplysia californica
were maintained at 14-16oC on a 12 hr
light/dark cycle and fed every 3 d. Before dissection animals were
injected with isotonic MgCl2 (25-50% body wt).
Antibodies. The rabbit antibody to buccalin used for
immunocytochemistry and RIA was raised against BUCa coupled to bovine serum albumin (BSA), as has been described previously (Miller et al.,
1991, 1992; Vilim et al., 1996a,b). The rat antibody to MMa used for
immunocytochemistry was raised against MMa coupled to bovine
thyroglobulin (BTG). The rabbit antibody to MMc described previously
(Miller et al., 1991) was not sufficiently sensitive for RIA.
Consequently, a new antibody was made. Because we had previously
obtained the best sensitivity with BSA-coupled antigens, we decided to
make two new antibodies, one against MMa coupled to BSA and the other
against MMa coupled to BTG. RIA tests of these antibodies indicated
that the titer (and sensitivity) of the BSA-coupled antigen kept
increasing with additional boosts, whereas the titer of the BTG-coupled
antigen leveled off after about the third boost. In the end, the tenth
and final bleed of the BSA-coupled MMa antigen had the best sensitivity
and was used in all the release experiments. The peptides were coupled
to the carrier protein with
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). All the antibodies
were prepared by BABCO (Richmond, CA), and the peptides were
synthesized by Applied Biosystems (Foster City, CA).
Immunocytochemistry. Immunocytochemistry was performed as
previously described (Vilim et al., 1996a). Tissues were fixed in freshly prepared fixative (4% paraformaldehyde, 0.2% picric acid, 25% sucrose, and 0.1 M
NaH2PO4, pH 7.6) for either
3 hr at room temperature (RT) or overnight at 4°C. After
washes with PBS to remove the fixative, ganglia from larger animals
were desheathed to expose the neurons whereas ganglia from smaller
animals (10-15 gm) were processed without desheathing. All subsequent
incubations were done at RT with rocking. Tissue was permeabilized and
blocked by overnight incubation in blocking buffer (BB: 10% normal
donkey serum, 2% Triton X-100, 1% BSA, 154 mM NaCl, 10 mM Na2HPO4, 50 mM EDTA, and 0.01% thimerosal, pH 7.4). The primary
antibody was diluted 1:250 in BB and incubated with the tissue for 4-7
d. The tissue was then washed twice a day for 2-3 d with washing
buffer (WB: 2% Triton X-100, 1% BSA, 154 mM NaCl, 10 mM Na2HPO4, 50 mM EDTA, and 0.01% thimerosal, pH 7.4). After the washes,
the tissue was incubated with 1:500 dilution of secondary antibody
(lissamine-rhodamine donkey anti-rat; Jackson ImmunoResearch, West
Grove, PA) for 2-3 d. The tissue was then washed twice with WB for
1 d and four times with storage buffer (SB: 1% BSA, 154 mM NaCl, 10 mM
Na2HPO4, 50 mM
EDTA, and 0.01% thimerosal, pH 7.4) for 1 d. The tissues were then stored at 4°C or viewed and photographed on a Nikon microscope equipped with epifluorescence.
EM immunocytochemistry. Procedures used for these
experiments were adapted from those of others (Reed et al., 1988;
Merighe et al., 1989), as has been described (Vilim et al., 1996a).
Briefly, tissue was fixed (4% glutaraldehyde, 10% sucrose, 11 mM magnesium chloride, and 0.2 M Na HEPES, pH
7.6) for 3 hr (RT), post-fixed with 1% osmium tetroxide in buffer at
4°C for 1 hr, and embedded in EMbed 812 (EM Sciences). Ultrathin
sections were etched with saturated sodium metaperiodate, washed with
TBS, then blocked with 8% normal goat serum in TBS for 1 hr.
The sections were stained overnight with a 1:100 dilution of primary
antibody, washed, then stained for 1 hr in a 1:4 dilution of
gold-labeled secondary antibody (Amersham, Arlington Heights, IL). The
sections were lightly counterstained with uranyl acetate and lead
citrate, then examined and photographed with a JEOL 100 CX at 80 kV.
Electron microscopy supplies and reagents were obtained from EMS (Fort
Washington, PA).
Radioimmunoassays. Desaminotyrosinated MMa and BUCa were
iodinated (125I) using the chloramine T
method. Iodinated stocks were repurified using reverse-phase HPLC and
diluted in RIA buffer (154 mM NaCl, 10 mM
Na2HPO4, 50 mM EDTA, 0.25 mM thimerosal, and 1% BSA, pH 7.5) to a final activity of
10,000-15,000 cpm/100 µl. Antibodies were diluted in RIA buffer so
that 100 µl bound ~50% of the counts in 100 µl of iodinated
peptide trace (with a sample volume of 50 µl). The reaction was
performed for 2-3 d at 4°C and terminated by addition of 2 ml of RIA
charcoal (10 mM Na2HPO4, 0.25 mM thimerosal, 0.25% activated charcoal, 0.025% 70,000 kDa dextran, pH 7.5). The samples were spun and the supernatant,
containing the bound peptide, was decanted and counted in a gamma
counter. Standard curves were generated using serial dilutions of
peptide in 50 µl of artificial sea water containing 1% BSA to
prevent sticking. A spreadsheet program (Kaleidagraph 2.1) was used to
plot the standard curves, to convert counts bound to femtomoles of
peptide in the unknowns, and display the data as graphs. Because
the RIAs are probably measuring the release of more than one
peptide cotransmitter within a family (Price, 1990), the release
of the family is referenced (i.e., MM = MMa, b, etc. and BUC = BUCa, b, etc.; Miller et al., 1993a,b). Because different
preparations released differing amounts of peptide, the data were
normalized to the average release for all the stimulation periods in a
single experiment. The percentage of average release was calculated by
adding all the released peptide detected in the experiment and dividing
by the total number of stimulation periods. The release for each
stimulation period was then divided by the average release for that
experiment and multiplied by 100. The normalized data were then
combined for the same experimental conditions across different
preparations, and statistical analysis was performed. A statistical
analysis program (StatView 4.5) was used to perform a within-subject
repeated-measures ANOVA on relevant data to assess the overall level of
statistical significance. Individual comparisons were performed using
paired t tests.
Release preparation. The ARC buccal ganglion preparation was
isolated as described elsewhere (Vilim et al., 1996a). Briefly, the
buccal ganglion was pinned in a dish containing 25% isotonic MgCl2 to prevent spontaneous activity, and the
nerve was passed through a slit in the side of the dish. The ARC was
suspended outside the dish and encased with a combination of silicone
grease (Dow Corning, Corning, NY) and parafilm. The ARC was perfused through an artery, and the perfusate was collected (drops, every 2.5 min) directly into the RIA tubes. The motorneuron was impaled with two
independent glass microelectrodes, one for recording voltage, and one
for injecting current. The temperature of the ARC was maintained at
15 ± 0.5°C by cooling the room with an air conditioner. For the
experiments examining the effects of calcium and hexamethonium on
peptide release, the agents were introduced into the ARC via the
perfusion line. The perfusing solutions were exchanged simply by
turning a peristaltic pump off and transferring the perfusion inlet
into another solution, and turning the perfusion line on again. This
method maintained a constant rate of perfusion for the different
perfusing solutions, and the small dead volume (<100 µl) enabled
fairly rapid (<5 min) exchange of solutions. All reagents were
obtained from Sigma (St. Louis, MO) except where otherwise noted.
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RESULTS |
The accessory radula closer muscle is innervated by two
cholinergic motorneurons, B15 and B16 (Cohen et al., 1978). Motorneuron B15 contains SCPs (Cropper et al., 1987a), motorneuron B16 contains MMs
(Cropper et al., 1987b, 1991), and both contain BUCs (Cropper et al.,
1988; Cropper et al., 1990a; Vilim et al., 1994). This is illustrated
by the LM immunocytochemistry in Figure
1. Here you can see processes
immunostaining for buccalin and myomodulin that are indicative of B16
processes. (In addition, there are processes immunostaining for
buccalin but not myomodulin that are indicative of B15 processes.) To
subcellularly localize the B16 peptides, we performed EM
immunocytochemistry. We found that MM is present in dense core vesicles
(DCVs), which had not been shown. In fact we found that vesicles that
are myomodulin-immunoreactive are also buccalin-immunoreactive. Data
from three preparations with 13 terminals containing a total of 122 DCVs showed that 97 (79.5%) were costained with myomodulin and
buccalin. In contrast, 10 B15 terminals from the same sections
contained 220 DCVs staining for buccalin, and only four of those
vesicles had gold particles corresponding to myomodulin staining. These
results demonstrate that the overall background levels were low and
that the immunostaining is specific. Thus, LM and EM evidence indicates
that the BUCs and MMs are not differentially packaged and targeted.
However, this evidence cannot determine whether the relative amounts of MMs and BUCs are fixed in all DCVs.
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Figure 1.
A, Myomodulin immunostaining in the
ARC muscle using lissamine-rhodamine-conjugated donkey anti-rat
secondary antibody. B, Buccalin immunostaining of the
same field in the ARC muscle using fluorescein-labeled donkey
anti-rabbit secondary antibody. Short arrows point to
processes that stain for buccalin alone and correspond to B15
processes. Longer arrows point to processes that stain
for both myomodulin and buccalin, corresponding to B16 processes. Scale
bar, 100 µm (applies to A and B).
C, Post-embedding immunogold dual labeling of BUC and MM
in a B16 neuromuscular junction of the ARC. BUC is labeled with a
rabbit antibody and a 5 nm gold-labeled secondary antibody. MM is
labeled with a rat antibody and a 10 nm gold-labeled secondary
antibody. Most of the DCVs are labeled with both small (5 nm) and large
(10 nm) gold particles, indicating the presence of both BUCs and MMs in
the same DCVs. Scale bar, 100 nm.
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To approach this issue we first sought to determine whether peptides
were released from B16 constitutively or in an activity-dependent manner. Both peptides were in fact released in a calcium-dependent manner when B16 was stimulated (Fig.
2A) (n = 4) (intraburst firing frequency, 20 Hz; burst duration, 3.5 sec;
interburst interval, 3.5 sec). Because low calcium solutions not only
block release but also prevent muscle contractions, the above results
could be obtained if the peptides were constitutively released and
squeezed into the eluate by muscle contractions. We therefore used
hexamethonium, a cholinergic antagonist that blocks muscle contractions
but does not inhibit the release process (Vilim et al., 1996a). Under
these conditions we again observed that the release of both peptides depended on motorneuron firing (Fig. 2B)
(n = 4) (firing rate, 20 Hz; burst duration, 3.5 sec;
interburst interval, 3.5 sec). Because the motorneuron in these
experiments was stimulated within its normal firing rate and pattern,
this experiment indicates that peptide release is physiologically
relevant. Furthermore, it is the first direct demonstration that MMs
are released in any system.
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Figure 2.
Effect of extracellular calcium and hexamethonium
on peptide release from B16 in the ARC. A1, Results from
a single experiment in which MM release is measured with and without
normal calcium in the perfusate. Motorneuron B16 was stimulated during
four 5 min periods (indicated by the black bars) with a
physiologically relevant frequency and pattern (20 Hz for 3.5 sec every
7 sec). A2, The average of four such experiments for
each peptide. For each peptide, the mean ± SE of four separate
experiments is plotted at each calcium concentration. The results show
that release of both peptides is dependent on extracellular calcium
(p < 0.0001). B1, Results
from a single experiment in which BUC release is measured without and
with 0.1 mM hexamethonium (which completely abolished the
contraction of the muscle) in the perfusate. Motorneuron B16 was
stimulated during four 5 min periods (indicated by the black
bars) with a physiologically relevant frequency and pattern (20 Hz for 3.5 sec every 7 sec). B2, The mean ± SE of
the normalized release from four such experiments for each peptide. The
results show that release of both peptides is not dependent on
contraction of the muscle (p > 0.5).
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We next wanted to define whether or not the ratio was fixed during
release. To accomplish this, we stimulated for a longer period of time
and analyzed alternate samples for MM or BUC content. By comparing the
kinetics of BUC and MM release, we can determine if the ratio is fixed
or not. When the motorneuron is stimulated at a constant frequency over
time, even though the amount of peptides released changes, the ratio of
the two peptides remains constant (Fig.
3) (n = 4) (firing rate,
20 Hz; burst duration, 3.5 sec; interburst interval, 3.5 sec; total
period of stimulation, 1 hr). This observation was confirmed by further
experiments in which we manipulated the parameters of motorneuron
stimulation and found that although the total amount of peptides
released from B16 changed as a function of the parameters of
stimulation, the ratio of the two peptides remained constant (see
below).
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Figure 3.
Corelease of MMs and BUCs within the ARC in
response to stimulation of motorneuron B16. During the 1 hr period
indicated by the bar, the neuron was fired at 20 Hz for 3.5 sec every 7 sec, which is within the physiological range of B16 firing. Samples of
ARC perfusate were collected every 2.5 min and analyzed by
radioimmunoassay for their peptide content. Alternate samples were
analyzed for their MM or BUC content. A1, MM and BUC
release in a single experiment. A2, Same as
A1 except that BUC was scaled so those total amounts
released were equal for both MM and BUC, enabling a more direct
comparison of their profiles. B1, MM and BUC release
from four experiments was expressed as a percentage of the total amount
of peptide (MM + BUC) released in the experiment, and the percentages
were averaged for each 5 min (2 sample) period. B2, Same
as B1, except that BUC was scaled so that total
percentages were equal for both MM and BUC, enabling a more direct
comparison of their profiles. The ratio of MM and BUC remains constant
even though the absolute peptide content of the samples varies
considerably over the course of B16 stimulation.
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Because the two peptides are released together and they are released in
a constant ratio, the functional role of corelease in this system
requires that the action of the two peptides be conceptualized jointly.
As indicated before, the joint action of these two peptides is to
preferentially accelerate the relaxation rate and thus to shorten the
duration of the relaxation of the contraction. Accelerated relaxation
rate promotes unopposed action of antagonistic muscles, especially when
muscle contractions occur frequently and/or are large (Fig.
4A1,B1). We therefore
investigated the relationship between peptide release and those
parameters of motorneuron stimulation that produce frequent or large
contractions. Different frequencies of muscle contractions were
produced by changing the interburst interval of motorneuron firing
while maintaining a constant intraburst frequency and burst duration.
All of these parameters used were within the physiological range of
motorneuron firing, i.e., the intraburst firing frequency was 20 Hz,
the burst duration was 3.5 sec, and interburst intervals were 3.5, 5.0, and 7.5 sec (Cropper et al., 1990b). Data from an individual experiment in which we measured the release of buccalin are shown in Figure 4A2. We observed that indeed peptide release was
greater when a motorneuron was stimulated to produce more frequent
contractions. Because there are more action potentials per unit of time
when the interburst interval is shorter, we expressed peptide release as femtomoles per action potential. The plot of these data is shown in
Figure 4A3, and it indicates that indeed release per action potential increased as the frequency of contractions increased (n = 4). The second set of conditions under which
faster relaxation rate is advantageous occurs when muscle contractions
are large (Fig. 4B1). We varied the size of muscle
contractions by changing the intraburst frequencies while maintaining a
constant burst duration and interburst interval. Again all of these
parameters were maintained within the physiological range (Cropper et
al., 1990b), i.e., intraburst firing frequencies were 10, 15, and 20 Hz, the burst duration was 3.5 sec, and the interburst interval was 3.5 sec. We found that indeed peptide release was largest when motorneurons
were stimulated at frequencies that produced large contractions
(n = 4) (Fig. 4B2,B3). Importantly,
in experiments in which we varied either the interburst interval or the
intraburst frequency, peptide release occurred not only at the high end
but also at the low end of our stimulation parameters. These parameters were selected to mimic the range of motorneuron activity recorded in
freely behaving animals. Larger amounts of peptides were released under
conditions that produced large or frequent contractions conditions in
which appropriate shortening of the relaxation has advantageous behavioral consequences.
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Figure 4.
Effect of increasing either contraction
frequency or amplitude on peptide release from motorneuron B16.
A1, Hypothetical consequences of increased rate of
feeding with no concomitant alteration of muscle properties. The closer
is not sufficiently relaxed and opposes the contractions of opener
muscles. Consequently, the radula does not open, and functional feeding
is compromised. When the relaxation rate of the closer muscle is
increased, functional feeding can be restored. A2,
Results from a single experiment in which BUC release is measured at
different interburst intervals, simulating changes in the rate of
feeding, whereas the intraburst frequency (20 Hz) and the duration of
bursts (3.5 sec) are kept constant. The duration of stimulation in this
type of experiment was 5 min. The physiological range of interburst
intervals for B16 varies from 3.5 to 10 sec (or more) (Cropper et al.,
1990b). BUC release decreases as the interval between bursts increases.
A3, Peptide release is normalized to the total release
during an experiment and for the total number of action potentials
delivered in each condition. The results from four separate experiments
for each peptide were averaged and plotted (± SE) for each of the
three interburst intervals. There is a statistically significant (BUC:
F = 23.13; df = 2,6; p < 0.002 MM: F = 25.1; df = 2,6;
p < 0.002) inverse relationship between peptide
released per action potential and the duration between bursts. The fact
that this relationship is nearly identical for the two peptides
indicates that they are released at a fixed ratio. B1,
Hypothetical consequences of increased contraction amplitude of the
radula closer with no concomitant alteration of muscle properties. The
closer is not sufficiently relaxed and opposes the contractions
of opener muscles. Consequently, the radula does not open, and
functional feeding is compromised. As in A1, when the
relaxation rate of the closer muscle is increased, functional feeding
can be restored. B2, Results from a single experiment in
which MM release is measured at three different intraburst frequencies,
which produce contractions of differing amplitudes, while the duration
of bursts (3.5 sec) and the interburst interval (3.5 sec) are kept
constant. The physiological range of firing frequencies for B16 varies
between 10-20 Hz (Cropper et al., 1990b). The duration of stimulation
in this type of experiment was 5 min. MM release is lower at the lower
intraburst frequencies. B3, Normalized release per
action potential at each of the three intraburst frequencies corrected
to give the release per action potential. The results from four
separate experiments for each peptide were averaged and plotted (± SE)
for each of the three intraburst frequencies of stimulation. There is a
statistically significant (BUC: F = 47.36; df = 2,6; p < 0.0005; MM: F = 126.78; df = 2,6; p < 0.0001) increase of
peptide released per action potential and as the frequency of action
potentials increases. The fact that this relationship is nearly
identical for the two peptides also indicates that they are released at
a fixed ratio.
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DISCUSSION |
Our results provide direct evidence that the BUCs and MMs are
costored and coreleased from motorneuron B16 terminals in the ARC
muscle. This release is calcium-dependent and contraction-independent, demonstrating that it is not artifactual. In addition, several observations suggest that the amounts of peptides that are recovered and measured are a good reflection of the amounts released.
Specifically, we are measuring the release of two different peptide
families, and peptides from each of these families differ in their
length and chemical structure. Despite these differences, the
myomodulins and buccalins are recovered in constant ratios under high-
and low-release conditions. Furthermore, despite the fact that
individual preparations differed in the absolute amounts of peptides
detected, the error bars of the combined normalized data from several
preparations are all very small.
We demonstrate that peptide cotransmitter release from B16 increases in
response to changes in firing patterns that result in contraction
amplitude increases or decreases in the interval between contractions.
Furthermore, we show that the peptide release is initiated at the low
end of the physiological range of B16 firing frequency and then
increases with increasing B16 firing frequency in the range recorded
in vivo during feeding. Previously, we reported (Vilim et
al., 1996b) that release of SCPs and BUCs from motorneuron B15 is
coordinated with the range of firing frequencies observed in
vivo when the animal feeds. However, Cropper et al. (1990b)
reported that the range of firing frequencies for B15 and B16 are
somewhat different (7-12 Hz for B15 and 10-20 Hz for B16). In Figure
5 we compare the release per spike from
B15 and B16 as a function of the overall average frequency of
motorneuron firing. It is clear that the release as a function of
frequency is different for the two motorneurons, but increases for both as the frequency increases (above a certain threshold frequency, which
appears to be different for the two neurons). Release of SCPs from
neurons B1 and B2 in culture suggests that release from these neurons
also appear to be coordinated with their in vivo firing
(Whim and Lloyd, 1994). However, these neurons fire single action
potentials in vivo (Lloyd et al., 1988) and also release peptide in response to single action potentials. These results support
the general conclusion that neuropeptide release properties appear to
be neuron-specific (as opposed to peptide-specific). Moreover, as
hypothesized (e.g., Vilim et al., 1996a,b) peptide cotransmitter
release is coordinated with the normal range of firing frequencies for
neurons in Aplysia, and probably in other species as well.
This is a more attractive model of peptide release than one that
postulates that peptide release only occurs at high frequencies
(Hökfelt, 1991) because it allows for greater flexibility in
output.
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Figure 5.
Comparison of the peptide release per action
potential from motorneurons B15 and B16 as a function of intraburst
frequency and interburst interval. A, Normalized release
per action potential at each of the three intraburst frequencies
corrected to give the release per action potential. Burst duration (3.5 sec) and interburst interval (3.5 sec) were identical in all
experiments. The results from eight separate experiments for each
motorneuron were averaged and plotted (± SE) at three different
intraburst frequencies of stimulation. The physiological range of
firing frequencies for B15 varies between 7.5 and 12 Hz, and B16 varies
between 10 and 20 Hz. Peptide release appears to be coordinated with
the normal operating range of frequencies of each motorneuron.
B, Normalized release per action potential at each of
the three interburst intervals corrected to give the release per action
potential. Burst duration (3.5 sec) and intraburst frequencies (12 Hz
for B15, 20 Hz for B16) were identical in all experiments. The results
from eight separate experiments for each motorneuron was averaged and
plotted (± SE) at three interburst intervals of stimulation (3.5, 5, and 7 sec). The data for B15 have been described previously (Vilim et
al., 1996b). The data for B16 were recalculated from Figure 4.
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The peptide cotransmitters present in motorneuron B15 and B16 produce
dose-dependent responses when applied exogenously to the preparation
(Weiss et al., 1992; Brezina et al., 1995, 1996). They do not operate
in an all or none manner. The peptide cotransmitters from these
motorneurons are also released in a frequency-dependent rather than in
an all or none manner within the normal range of firing frequencies for
these two motorneurons. Furthermore, the release also changes gradually
in response to changes in the interval between bursts or contractions
(Fig. 5). Thus, the peptide cotransmitters in this system can alter the
characteristics of muscle contractions, amplitude and relaxation rate,
in a motorneuron firing-dependent manner. This can allow the
coordination of the amplitude and relaxation rate of the muscle
contractions to maximize the efficiency of the output of the system
(Weiss et al., 1992). By altering the relaxation rate in response to
the changing contraction amplitude and interval, inappropriate actions
of antagonist muscles can be minimized, and the proper force can be
maintained throughout a given phase of the cycle. This could be a
built-in mechanism to adjust the response characteristics of the muscle
to a wide range of amplitude and interval combinations.
The ARC is a nonspiking muscle in which the contraction amplitude is a
function of the amount of ACh released (Cohen et al., 1978). Thus,
release of classical transmitter correlates with contraction amplitude
that in turn correlates with peptide cotransmitter release. In
addition, release of ACh appears to be frequency-dependent (Lloyd and
Church, 1994) as is peptide cotransmitter release. Hence, peptide
cotransmitter and classical transmitter release appear to be
functionally coupled in this system. Therefore, it is likely that there
are key shared mechanisms for the release of classical and peptide
cotransmitters. For example, both release processes are
calcium-dependent. The calcium requirements for peptide and classical
transmitter release, however, are different (Verhage et al., 1991;
Llinas et al., 1992; Peng and Zucker, 1993), and the two can be
differentially released (Matteoli et al., 1988), so some important
differences in their mechanisms of release must exist.
In summary, our findings have several general implications for the
understanding of peptidergic cotransmission. Peptide cotransmitters have been suggested to function as amplifiers of the actions of conventional transmitters, being released only at very high levels of
neural firing (Hökfelt, 1991; Kupfermann, 1991). In many ways our
findings are not consistent with this view and instead support the
suggestions we have reached in our studies of peptide release from
motorneuron B15 (Vilim et al., 1996a,b). Thus, our findings demonstrate
that activity-dependent release of two modulatory peptide
cotransmitters occurs throughout the normal firing range of a neuron,
and although release becomes larger as the frequency of motorneuron
firing increases, the release does not require extreme conditions in
this system. Second, in this system the actions of the two peptides on
the size of muscle contractions, the parameter regulated by the primary
transmitter, tend to cancel each other and therefore the actions of
these peptides are not easily conceptualized as simply amplifying the
actions of the primary transmitter. Instead, the effect that emerges
from the joint effect of these peptides is to modify the relaxation
rate of contraction, an action that is not regulated by the primary transmitter. In view of the differences in the information transmitted by peptide cotransmitters and primary transmitters, these two classes
of molecules are best conceptualized as constituting parallel but
functionally distinct pathways of information transmission. Third, we
found that peptides were preferentially released when motorneurons were
stimulated to produce strong and frequent contractions, conditions that
prevent full relaxation of the muscle before its antagonist muscle
begins to contract. An incompletely relaxed muscle offers resistance to
its antagonist, which may result in a disruption of the functional
integrity of behavior. Furthermore, at higher rates of contraction a
slow relaxation rate can result in inefficient stepwise summation and
resultant tonic contractions. Because the amount of peptides that are
released is an automatic consequence of the firing pattern of the
motorneuron that produces the contraction, this arrangement obviates
the need for complex sensory functions that would monitor on-line the
state of muscle contraction to alter its rate of relaxation by engaging
other neuronal input to the muscle. In more general terms, the use of peptide cotransmitters appears to be a simple and elegant way to
automatically modify the characteristics of the response to the primary
transmitter without the added computational complexity that would
result from the incorporation of additional neuronal elements.
| |
FOOTNOTES |
Received Oct. 11, 1999; revised Dec. 10, 1999; accepted Dec. 20, 1999.
This work was supported by the National Institute of Mental Health
through Grants MH36730 and K05MH01427 to K.R.W. and MH01267 and MH51393
to E.C.C. Aplysia californica were partially provided by
the National Resource for Aplysia at the University of
Miami under National Institutes of Health National Center for Research Resources Grant RR10294. We thank Dr. Paul Church for critical reading
of this manuscript.
Correspondence should be addressed to F. S. Vilim, Box 1218, Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029. E-mail: vilim{at}inka.mssm.edu.
| |
REFERENCES |
-
Brezina V,
Bank B,
Cropper EC,
Rosen S,
Vilim FS,
Kupfermann I,
Weiss KR
(1995)
Nine members of the myomodulin family of peptide cotransmitters at the B16-ARC neuromuscular junction of Aplysia.
J Neurophysiol
74:54-72[Abstract/Free Full Text].
-
Brezina V,
Orekhova IV,
Weiss KR
(1996)
Functional uncoupling of linked neurotransmitter effects by combinatorial convergence.
Science
273:806-810[Abstract].
-
Cohen JL,
Weiss KR,
Kupfermann I
(1978)
Motor control of buccal muscles in Aplysia.
J Neurophysiol
41:157-180[Free Full Text].
-
Cropper EC,
Lloyd PE,
Reed W,
Tenenbaum R,
Kupfermann I,
Weiss KR
(1987a)
Multiple neuropeptides in cholinergic motor neurons of Aplysia: evidence for modulation intrinsic to the motor circuit.
Proc Natl Acad Sci USA
84:3486-3490[Abstract/Free Full Text].
-
Cropper EC,
Tenenbaum R,
Kolks MAG,
Kupfermann I,
Weiss KR
(1987b)
Myomodulin: a bioactive neuropeptide present in an identified cholinergic buccal motor neuron of Aplysia.
Proc Natl Acad Sci USA
84:5483-5486[Abstract/Free Full Text].
-
Cropper EC,
Miller MW,
Tenenbaum R,
Kolks MAG,
Kupfermann I,
Weiss KR
(1988)
Structure and action of buccalin: a modulatory neuropeptide localized to an identified small cardioactive peptide-containing cholinergic motor neuron of Aplysia californica.
Proc Natl Acad Sci USA
85:6177-6181[Abstract/Free Full Text].
-
Cropper EC,
Miller MW,
Vilim FS,
Tenenbaum R,
Kupfermann I,
Weiss KR
(1990a)
Buccalin is present in the cholinergic motor neuron B16 of Aplysia and it depresses accessory radula closer muscle contractions evoked by stimulation of B16.
Brain Res
512:175-179[Web of Science][Medline].
-
Cropper EC,
Kupfermann I,
Weiss KR
(1990b)
Differential firing patterns of the peptide-containing cholinergic motor neurons B15 and B16 during feeding behavior in Aplysia.
Brain Res
522:176-179[Web of Science][Medline].
-
Cropper EC,
Vilim FS,
Alevizos A,
Tenenbaum R,
Kolks MAG,
Rosen SC,
Kupfermann I,
Weiss KR
(1991)
Structure, bioactivity, and cellular localization of myomodulin B: a novel Aplysia peptide.
Peptides
12:683-690[Web of Science][Medline].
-
Greenberg MJ,
Doble KE,
Lesser W,
Lee TD,
Pennell NA,
Morgan CG,
Price DA
(1997)
Characterization of myomodulin-related peptides from the pulmonate snail Helix aspersa.
Peptides
18:1099-106[Web of Science][Medline].
-
Hökfelt T
(1991)
Neuropeptides in perspective: the last ten years.
Neuron
7:867-879[Web of Science][Medline].
-
Iversen LL
(1995)
Neuropeptides: promise unfulfilled?
Trends Neurosci
18:49-50[Web of Science][Medline].
-
Kellett E,
Perry SJ,
Santama N,
Worster BM,
Benjamin PR,
Burke JF
(1996)
Myomodulin gene of Lymnaea: structure, expression, and analysis of neuropeptides.
J Neurosci
16:4949-4957[Abstract/Free Full Text].
-
Kupfermann I
(1991)
Functional studies of cotransmission.
Physiol Rev
71:683-732[Free Full Text].
-
Llinas R,
Sugimori M,
Silver RB
(1992)
Microdomains of high calcium concentration in a presynaptic terminal.
Science
256:677-679[Abstract/Free Full Text].
-
Lloyd PE,
Church PJ
(1994)
Cholinergic neuromuscular synapses in Aplysia have low endogenous acetylcholinesterase activity and a high-affinity uptake system for acetylcholine.
J Neurosci
14:6722-6733[Abstract].
-
Lloyd PE,
Kupfermann I,
Weiss KR
(1988)
Central peptidergic neurons regulate gut motility in Aplysia.
J Neurophysiol
59:1613-1625[Abstract/Free Full Text].
-
Matteoli M,
Haimann C,
Torri-Tarelli F,
Polak JM,
Ceccarelli B,
DeCamilli P
(1988)
Differential effect of a-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction.
Proc Natl Acad Sci USA
85:7366-7370[Abstract/Free Full Text].
-
Merighe A,
Polak JM,
Fumagalli G,
Theodosis DT
(1989)
Ultrastructural localization of neuropeptides and GABA in the rat dorsal horn: a comparison of different immunogold labeling techniques.
J Histochem Cytochem
37:529-540[Abstract].
-
Miller MW,
Alevizos A,
Cropper EC,
Vilim FS,
Karagogeos D,
Kupfermann I,
Weiss KR
(1991)
Localization of myomodulin-like immunoreactivity in the central nervous system and peripheral tissues of Aplysia californica.
J Comp Neurol
314:627-644[Web of Science][Medline].
-
Miller MW,
Alevizos A,
Cropper EC,
Kupfermann I,
Weiss KR
(1992)
Distribution of buccalin-like immunoreactivity in the central nervous system and peripheral tissues of Aplysia.
J Neurosci
320:182-185.
-
Miller MW,
Beushausen S,
Cropper EC,
Eisinger K,
Stamm S,
Vilim FS,
Vitek A,
Zajc A,
Kupfermann I,
Brosius J,
Weiss KR
(1993a)
The buccalin-related neuropeptides: isolation and characterization of an Aplysia: cDNA clone encoding a family of peptide cotransmitters.
J Neurosci
13:3346-3357[Abstract].
-
Miller MW,
Beushausen S,
Vitek A,
Stamm S,
Kupfermann I,
Brosius J,
Weiss KR
(1993b)
The myomodulin-related neuropeptides: Characterization of a gene encoding a family of peptide cotransmitters in Aplysia.
J Neurosci
13:3358-3367[Abstract].
-
Peng Y,
Zucker RS
(1993)
Release of LHRH is linearly related to the time integral of presynaptic Ca+ elevation above a threshold level in bullfrog sympathetic ganglia.
Neuron
10:465-473[Web of Science][Medline].
-
Price DA,
Lesser W,
Lee TD,
Doble KE,
Greenberg MJ
(1990)
Seven FMRFamide-related and two SCP-related cardioactive peptides from Helix.
J Exp Biol
154:421-437[Abstract/Free Full Text].
-
Reed W,
Weiss KR,
Lloyd PE,
Kupfermann I,
Chen M,
Bailey CH
(1988)
Association of neuroactive peptides with the protein secretory pathway in identified neurons of Aplysia californica: immunolocalization of SCPa and SCPb to the contents of dense core vesicles and the trans face of the golgi apparatus.
J Comp Neurol
272:358-369[Web of Science][Medline].
-
Verhage M,
McMahon HT,
Ghijsen WEJM,
Boomsma F,
Scholten G,
Weigant VM,
Nicholls DG
(1991)
Differential release of amino acids, neuropeptides, and catecholamines from isolated nerve terminals.
Neuron
6:517-524[Web of Science][Medline].
-
Vilim FS,
Cropper EC,
Rosen SC,
Tenenbaum R,
Kupfermann I,
Weiss KR
(1994)
Structure, localization and action of buccalin b - a bioactive peptide from Aplysia.
Peptides
15:959-969[Web of Science][Medline].
-
Vilim FS,
Price DA,
Lesser W,
Kupfermann I,
Weiss KR
(1996a)
Costorage and corelease of modulatory peptide cotransmitters with partially antagonistic actions on the accessory radula closer muscle of Aplysia californica.
J Neurosci
16:8092-8104[Abstract/Free Full Text].
-
Vilim FS,
Cropper EC,
Price DA,
Kupfermann I,
Weiss KR
(1996b)
Release of peptide cotransmitters in Aplysia: regulation and functional implications.
J Neurosci
16:8105-8114[Abstract/Free Full Text].
-
Wang Y,
Price DA,
Sahley CL
(1998)
Identification and characterization of a myomodulin-like peptide in the leech.
Peptides
19:487-493[Web of Science][Medline].
-
Weiss KR,
Brezina V,
Cropper EC,
Hooper S,
Miller MW,
Probst WC,
Vilim FS,
Kupfermann I
(1992)
Peptidergic co-transmission in Aplysia: functional implications for rhythmic behaviors.
Experientia
48:456-463[Web of Science][Medline].
-
Whim MD,
Lloyd PE
(1994)
Differential regulation of the release of the same peptide transmitters from individual identified motor neurons in culture.
J Neurosci
14:4244-4251[Abstract].
Copyright © 2000 Society for Neuroscience 0270-6474/00/2052036-07$05.00/0
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ABSTRACT
INTRODUCTION
