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Introduction
The neuromuscular junction’s (NMJ) primary
responsive element, the nicotinic acetylcholine
receptor (nAChR), is the best characterized of all
ligand-gated channels. Its expression on the surface
of the cell membrane, at the mature NMJ, is mainly
restricted to areas directly opposite the nerve
endings. This regionalization is induced and main-
tained by the motor nerve via the activity that it
confers upon the muscle, as well as trophic substances
released into the synaptic cleft during periods of
activity.1These control mechanisms alter the level
of nAChR synthesis throughout the muscle, prolong
the nAChR half-life at the motor endplate, and
cause the receptor’s cell surface mobility to become
largely restricted to the postsynaptic area of the
muscle.2
There is increasing evidence that the function
and morphology of the mammalian NMJ may
demonstrate adaptations to physiological levels of
increased activation. For example, endurance training
increases presynaptic terminal size3and quantal
content.4These adaptations are probably supported
by the increased synthesis and fast axonal transport
of proteins,5some of which may have trophic effects
on neuromuscular components. In addition, we have
previously shown that SNAP-25, which is destined
for the nerve terminal and is instrumental in quantal
release of acetylcholine,6is increased in abundance
among fast-transported proteins in sciatic nerves
of endurance-trained rats.7Such adaptations could
constitute a strategy to reduce the possibility of
neuromuscular transmission failure which is sug-
gested to occur during continuous neuromuscular
activity.8
Knowledge of the response of postsynaptic
elements to increased activity is limited. Prakash
et al.9have demonstrated that the size of the pre-
and postsynaptic elements at the NMJ are tightly
coupled, and that the extent to which the overlap
occurs can be adjusted in situations of altered
levels of activity. Also, Deschenes et al.3have shown
that exercise training increases the area occupied
by both the pre- and postsynaptic elements of the
NMJ. However, whether increased activity results in
a concomitant increase in endplate nAChR number
or in a redistribution of existing nAChR is not
known. The purpose of the present study was to
test the hypothesis that the abundance of endplate-
associated nAChR would be altered by chronic
exercise.
Motor Systems
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0959-4965 © 1998 Lippincott Williams &Wilkins Vol 9No 1616 November 1998 3549
T
HE
aim of the study was to test the hypothesis that
a 16 week endurance training program would alter
the abundance of endplate-associated nicotinic acetyl-
choline receptors (nAChR) in various rat skeletal
muscles. We found a 20% increase in endplate-specific
[125I]␣-bungarotoxin binding in several muscles of
trained rats, accompanied by equal susceptibility of toxin
binding to the inhibitory effect of
D
-tubocurarine in
sedentary and trained muscles. We conclude that the
neuromuscular junction adaptations that occur with
increased chronic activation include an increase in
nAChR number. Results of experiments designed to
determine nAChR turnover also suggest that this effect
is mediated by an alteration in the receptor’s metabolic
state. The potential implications and mechanisms of this
adaptation are discussed. NeuroReport9: 3549–3552 ©
1998 Lippincott Williams &Wilkins.
Key words: Acetylcholine receptor; Alpha-bungarotoxin;
Endurance training; Motor endplate; Neuromuscular junc-
tion; Rat skeletal muscle
Endurance training
increases acetylcholine
receptor quantity at
neuromuscular junctions
of adult rat skeletal
muscle
Patrice Desaulniers,
Pierre-André Lavoie1and
Phillip F. GardinerCA
Départements de Kinésiologie et
1Pharmacologie, Université de Montréal,
C.P. 6128, Succ. Centre-Ville, Montréal, Québec,
Canada H3C 3J7
CACorresponding Author
Website publication 19 November 1998 NeuroReport 9, 3549–3552 (1998)
Materials and Methods
Treatment of animals: During these experiments,
animals were treated according to the guidelines of
the Canadian Council of Animal Care, and all
procedures were approved by the University of
Montreal animal ethics committee. Female Sprague–
Dawley rats were endurance-trained on a treadmill
for 12–16 weeks (30 m/min, 5% grade, 2 h/day,
5 days/week). This training program evokes adapta-
tions in muscle metabolism and in fast transport of
proteins in motor axons.5,7 Control group animals
were kept cage confined. At least 24 h after the last
training session, rats were anaesthetized with sodium
pentobarbital (40 mg/kg, i.p.), and several muscles of
various fiber-type composition (diaphragm, tibialis
anterior, soleus, adductor longus, vastus lateralis
(white)) were quickly excised. A sedentary animal
was always treated in parallel with a trained animal.
nAChR quantity: nAChR number was determined
by incubating the muscles for 60 min in an
oxygenated mammalian-Krebs solution containing
1g/ml of [125I]Tyr54-␣-bungarotoxin ([125I]-␣-Btx;
Dupont Canada). The excess unbound toxin was
subsequently eliminated by wash-out periods con-
sisting of a single 1 h wash, two 2 h washes, and
an overnight wash, all of which were carried out
in 100 ml mammalian-Krebs solution at 4°C. The
following morning, the endplates were identified
by staining for acetylcholinesterase10 and, under
×10 magnification, the muscles were dissected into
endplate-containing and non-endplate segments
which were individually assayed for radioactivity
with a LKB Universal gamma counter. Endplate-
specific binding of [125I]-␣-Btx was determined by
subtracting c.p.m. of the non-endplate segments from
c.p.m. of the endplate-containing segments on a
weight-adjusted basis. The mean endplate-specific
binding of each pair of muscles was then used for
analysis purposes. Evidence that the toxin was truly
binding to nAChR in the trained and sedentary
muscles was provided by a series of competition
experiments in which incubation with [125I]-␣-Btx in
one member of each pair of muscles was both
preceded by a 20 min incubation in a Krebs solution
containing 1 mM
D
-tubocurarine chloride and carried
out in the presence of 1 mM
D
-tubocurarine chlor-
ide, all other procedures being as described above.
nAChR turnover: The rate of nAChR synthesis
was determined in a separate group of animals by
injecting the tibialis anterior (TA) muscle of seden-
tary and trained rats with a saturating dose of
unlabelled ␣-Btx.11 Twenty or 40 h after the injec-
tion the muscles were excised and assayed for nAChR
quantity as described above. nAChR degradation
was evaluated by injecting the TA muscles with a
saturating dose of [125I]-␣-Btx. Four days after the
injection the muscles were excised and processed for
endplate-specific binding determination.
Statistics: Data are expressed as mean ± s.e.m. A
two-way analysis of variance (ANOVA) was used
for comparison between the groups except when indi-
cated. The critical level of probability was set at 0.05.
Results
We found a significant increase in endplate-specific
[125I]-␣-Btx binding in the trained muscles (20%;
p< 0.002); this increase occurred in the absence
P. Desaulniers, P.-A. Lavoie and P. F. Gardiner
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3550 Vol 9 No 16 16 November 1998
FIG. 1. (A) End-plate specific (125I)-␣-Btx binding in skeletal muscles of sedentary (
n
= 6) and endurance trained (
n
= 6) rats. The mean from
the left and right hindlimbs (and both hemi-diaphragms) were used for each animal. Data for individual muscles (± s.e.m.), along with a
2-way ANOVA on the pooled results (ALL) are provided. (B) D-tubocurarine chloride-induced endplate-specific (125I)-␣-Btx binding inhibition
in sedentary (
n
= 6) and trained (
n
= 6) rats. Percent inhibition was obtained by comparing endplate-specific binding from muscles which
were exposed to 1 mM D-tubocurarine chloride with that of their contralateral counterparts which were not exposed to the D-tubocurarine
chloride.
of any change in toxin binding to non-endplate
muscle regions (p> 0.50) and presumably reflects an
increased number of nAChR at the endplates. Figure
1a depicts the magnitude of this endplate-specific
up-regulation in the various muscles. The degree
of endplate-specific binding inhibition caused by
exposure to
D
-tubocurarine was similar in trained
and control muscles (p> 0.75, Fig. 1b), confirming
the nAChR nature of the increased endplate-specific
radiolabelled toxin binding to trained muscles.
Table 1 outlines the effect of the training program
on whole muscle weight and shows that in four of
the five muscles, endplate-specific toxin binding/mg
muscle is increased by chronic exercise.
Figure 2 shows that the quantity of receptors
synthesized 20 and 40 h after blockade of existing
receptors was 38% and 45% greater, respectively, in
the trained TA. We found an overall training effect
(p< 0.03) on nAChR synthesis, but no statistical
difference in the rate of synthesis between 20 and
40 h (there is no interaction between the training
and injection time effects; p> 0.55). The amount of
labelled nAChR remaining 4 days after a saturating
[125I]-␣-Btx injection was identical for both groups
(sedentary = 65623 ± 7107 c.p.m., trained = 65190 ±
4010 cpm; t-test p> 0.96). Thus, endurance training
appeared to decrease nAChR half-life.
Discussion
The increase in nAChR number by 20% is consis-
tent with the 22% (soleus) and 32% (extensor digi-
torum longus) increase that has been reported
for nerve terminal size with endurance training.12 A
corresponding increase in endplate area, the pre-
and postsynaptic elements of the neuromuscular
junction having been shown to be tightly coupled,9
would therefore imply no change in receptor density.
The increased nAChR number may therefore indi-
cate an increased endplate area to accommodate
the increased terminal size. Evidence corroborating
this interpretation has been reported by Deschenes
et al.,3who found that pre- and postsynaptic area is
increased by endurance exercise. If muscle fiber diam-
eter had increased following the training program,
the increased endplate size would probably occupy
a similar proportion of the total muscle surface.
However, the type of training employed in this study
has been shown to have little effect on muscle fiber
diameter3and we found no difference in whole
muscle weights between the two groups (Table 1. All
muscles; p> 0.85).
The increased nAChR quantity in the trained
muscles is, therefore, probably accompanied by an
increased endplate size, itself occupying a larger
proportion of the muscle surface than its sedentary
counterpart. Since nAChR are in considerable excess
at the NMJ13 the functional consequences, if any, of
the endurance training-induced increase in nAChR
may be related to the phenomenon of receptor desen-
sitization which occurs under conditions of high
ACh release.14 Thus, the increased nAChR number,
along with the increase in the G4 form of acetyl-
cholinesterase which we have previously documented
in this model,15 may limit the potential of the higher
quantal content to produce receptor desensitization
during sustained ACh release.
Interestingly, the increase in nAChR was a
phenomenon which appeared to occur independently
of the recruitment level of the muscles during
training. We have previously shown that endurance
training increases fast axonal transport to a similar
extent in nerves innervating muscles with differing
recruitment levels,5and we have recently found that
the content of calcitonin gene-related peptide in
motoneurons innervating fast and slow muscles also
increases similarly in trained rats.16 The fact that the
muscles we examined in this study all behaved analo-
gously (Fig. 1a), regardless of fiber-type composition
Endurance training increases endplate nAChR
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Vol 9 No 16 16 November 1998 3551
Table 1. The effect of chronic exercise on whole muscle
weights and endplate-specific [125I]-␣-Btx binding/mg muscle
in five rat skeletal muscles.
Muscle (
n
) Sedentary Trained
Weight c.p.m. Weight c.p.m.
(mg) (mg)
Diaphragm (6) 234 ± 15 287 ± 13 233 ± 11 348 ± 21*
TA (6) 542 ± 24 126 ± 18 548 ± 28 153 ± 14*
Adl (6) 40 ± 7 415 ± 23 43 ± 10 421 ± 37
Sol (6) 108 ± 13 256 ± 11 102 ± 9 324 ± 18*
VLW (6) 268 ± 37 79 ± 11 271 ± 33 123 ± 16*
*
p
< 0.05
FIG. 2. [125I]-␣-Btx endplate-specific binding in sedentary (
n
= 5) and
trained (
n
= 4) TA muscle 20 and 40 h following an unlabelled ␣-Btx
injection i.m. designed to saturate existing nAChR. The increased
radiolabelling in the trained muscles reflects an enhanced rate of
nAChR synthesis in these muscles, although the effect seems to
primarily manifest itself within the intial 20 h period.
or recruitment level, may therefore indicate that this
adaptation is related to these previously reported
adaptations, and is mediated by trophic neural
substances.
We also observed that exercise training may
increase the rate at which nAChR are synthesized
and inserted into the endplate area, and decrease
receptor half-life. Figure 2 shows that the amount of
nAChR was consistently greater at the trained NMJ,
although the effect seems to manifest itself primarily
in the first 20 h of recuperation from the unlabelled
␣-Btx injection. Because the amount of toxin injected
into the trained and sedentary groups was identical,
it is arguable that we failed to bind all nAChR in the
trained muscles given the increase in total receptor
number we have found in this study. However,
visual inspection of the animals’ locomotive ability
following the injections confirmed that the procedure
induced paralysis of ankle flexion in both groups of
animals. Four days after having saturated the muscles
with [125I]-␣-Btx, the amount of persisting radio-
labelled receptors was found to be identical in both
experimental groups. Inferring a larger amount of
receptors in the trained TA at the time of injection,
we interpret this result as an indication that nAChR
degradation is accelerated in trained muscles. The
balance of the apparent enhanced synthesis and
decreased half-life results in a steady-state 20%
increase in nAChR at the NMJ of trained rats.
Conclusion
Endurance training increased nAChR quantity in
endplate-rich regions of various rat skeletal muscles.
This effect seems to be accompanied by an increased
rate of nAChR synthesis by the muscle fiber. Such
an adaptation may occur via increased motoneuronal
synthesis, transport and secretion of neuropeptides
such as calcitonin gene-related peptide, which
elevates the level of nAChR synthesis during the
developmental stages of the neuromuscular junction2.
This altered state of nAChR turnover on the surface
of the muscle cell membrane of endurance-trained
rats is indicative of a neuromuscular system having
effective remodeling capabilities.
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ACKNOWLEDGEMENTS: This research was supported by a grant from the
Natural Sciences and Engineering Research Council of Canada to P.F.G.
Received 24 June 1998;
accepted 7 September 1998
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