J Appl Physiol
89: 1928–1936, 2000.
Downhill running preferentially increases CGRP in fast
glycolytic muscle fibers
DARLENE A. HOMONKO AND ELIZABETH THERIAULT
The Toronto Hospital Research Institute, Toronto Western Division, Toronto, Ontario, Canada M5T 2S8
Received 12 February 1999; accepted in final form 6 June 2000
Homonko, Darlene A., and Elizabeth Theriault.
Downhill running preferentially increases CGRP in fast
glycolytic muscle fibers. J Appl Physiol 89: 1928–1936,
2000.—Calcitonin gene-related peptide (CGRP) is present in
some spinal cord motoneurons and at neuromuscular junctions in skeletal muscle. We previously reported increased
numbers of CGRP-positive (CGRP⫹) motoneurons supplying
hindlimb extensors after downhill exercise (Homonko DA
and Theriault E, Inter J Sport Med 18: 1–7, 1997). The
present study identifies the responding population with respect to muscle and motoneuron pool and correlates changes
in CGRP with muscle fiber type-identified end plates. Twentyseven rats were divided into the following groups: control and
72 h and 2 wk postexercise. FluoroGold was injected into the
soleus, lateral gastrocnemius, and the proximal (mixed fiber
type) or distal (fast-twitch glycolytic) regions of the medial
gastrocnemius (MG). Untrained animals ran downhill on a
treadmill for 30 min. The number of FluoroGold/CGRP⫹
motoneurons within proximal and distal MG increased by
72 h postexercise (P ⬍ 0.05). No significant changes were
observed in soleus or lateral gastrocnemius motoneurons
postexercise. The number of ␣-bungarotoxin/CGRP⫹ motor
end plates in the MG increased exclusively at fast-twitch
glycolytic muscle fibers 72 h and 2 wk postexercise (P ⬍ 0.05).
One interpretation of these results is that unaccustomed
exercise preferentially activates fast-twitch glycolytic muscle
fibers in the MG.
neuropeptides; neuromuscular plasticity; activity; rat
(23, 35) and in situ hybridization
(6) studies in control animals have shown that motoneurons supplying fast-twitch muscles (e.g., extensor
digitorum longus) show higher levels of calcitonin
gene-related peptide (CGRP) staining than do motoneurons innervating muscles of slow-twitch fiber
type [e.g., soleus (Sol)]. A similar pattern of CGRP
expression is observed in the muscle, with CGRP found
predominantly at the motor end plates of fast-twitch
muscle fibers (23, 24). However, none of these studies
has correlated CGRP expression patterns in identified
(i.e., retrogradely labeled) motoneurons with motor
end plates identified according to muscle fiber type.
Whereas the role of CGRP in the normal adult motor
system is not entirely clear, the present framework of
evidence suggests that it is associated with presynaptic
IMMUNOCYTOCHEMICAL
Address for reprint requests and other correspondence: E. Theriault, Dean, School of Science and Technology, Sheridan College, 7899
McLaughlin Road, Brampton, Ontario, Canada L6V 1G6 (E-mail:
elizabeth.theriault@sheridanc.on.ca).
1928
sprouting and postsynaptic structural changes at the
neuromuscular junction (21, 22, 33, 39, 45). Any form
of experimental intervention that disrupts the connection between the motor nerve and the neuromuscular
junction, either surgically (3, 36) or pharmacologically
(39, 45), results in an upregulation of CGRP peptide
and/or its mRNA. CGRP expression also increases after spinal cord transection (2, 36) or androgen deprivation (37, 38). To further investigate the role of CGRP in
the normal, intact adult neuromuscular system, our
approach was to develop a “noninterventional” experimental paradigm, which provided a physiological challenge to the motoneuron and its target. We previously
demonstrated that CGRP expression in rat hindlimb
motoneurons increased after an acute bout of downhill
running exercise in sedentary animals (27). The results
showed that CGRP expression remained elevated over
a 2-wk period, returning to baseline by 4 wk, in motoneurons of the knee extensors (triceps surae; e.g.,
muscles performing mostly lengthening contractions
while loading) but not in the knee flexors (anterior
crural; e.g., muscles performing mostly shortening contractions).
We now identify the responding motoneurons, their
fiber type association, and the time course of change in
CGRP expression after unaccustomed downhill exercise. Intramuscular injections of FluoroGold were used
to retrogradely identify motoneurons supplying the
Sol, lateral gastrocnemius (LG), and the proximal
(pMG) and distal regions (dMG) of the medial gastrocnemius (MG). Changes between control and experimental groups were quantified by using double-labeling immunofluorescence techniques, identifying the
MG as the muscle within the triceps surae with a
significant increase in the numbers of CGRP-positive
(CGRP⫹) motoneurons after exercise. Interestingly, in
the MG muscle, there was a significant elevation in
CGRP levels at fast-twitch glycolytic (FG) motor end
plates exclusively.
MATERIALS AND METHODS
Retrograde labeling of motoneurons. To identify the responding population(s) of motoneurons, we used a total of 34
female Wistar rats (250–275 g) for this study. All animals,
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EXERCISE INCREASES CGRP AT FAST-TWITCH MOTOR END PLATES
with the exception of the animals used in the glycogen depletion study, were given intramuscular injections of 4%
FluoroGold (Fluorochrome, Inglewood, CA). Identification of
the three-dimensional topographic locations of the Sol, LG,
and MG motor nuclei in the spinal cord was completed in a
series of retrograde labeling experiments, which have been
described previously (27). Briefly, FluoroGold (10 ␮l) was
injected into the belly of the left Sol muscle of 11 animals and
into the right LG muscle (15 ␮l; belly portion) of 9 animals.
The pMG contains a mixture of fiber types [10% slow-twitch
oxidative (SO), 10% fast-twitch oxidative glycolytic (FOG),
35% FG; cf. Ref. 10], whereas the dMG is composed of FG
muscle fibers (80% FG; Ref. 10; Fig. 1). Therefore, in 14
animals, the proximal-medial region of the MG (pMG) of the
left leg and the distal-medial region of the MG (dMG) of the
right were injected with 15 ␮l of FluoroGold (Fig. 1). A single
injection of tracer was delivered into each muscle with the
use of an adapted 100-␮l Hamilton syringe (Fisher, Mississauga, ON). PE-20 Silastic tubing was placed onto the syringe needle with the other end of the tubing supporting a
30G1/2-gauge needle (Baxter Canlab, Mississauga, ON) attached to a micromanipulator. Care was taken during these
injections to prevent leakage of tracer into other muscles by
using a localized microsurgical approach and by using petroleum jelly and tiny gauze pads to isolate each muscle (27).
The injection site was then sealed with a drop of cyanoacrylate glue (Baxter Canlab). Three days after the muscle injections, animals were exercised. All experimental procedures
were completed according to the Canadian Council on Animal Care Guidelines on the Use of Animals in Research, with
ethics approval granted by the Toronto Hospital Animal Care
Committee.
Exercise protocol and tissue processing. Animals were randomly placed into three groups: control (nonexercise) and
Fig. 1. Schematic representation of the rat hindlimbs [left and right
medial gastrocnemius (MG)] as viewed from the dorsal surface.
Speckled areas indicate FluoroGold injection sites into the proximal
region of the left MG (pMG) (speckled region overlaying light gray
area illustrating the mixed-fiber-type composition of the region) and
the distal region of the right MG (dMG) [speckled region overlaying
white area illustrating exclusive fast-twitch glycolytic (FG) composition]. Muscle fiber type is indicated by the white regions that
contain FG fibers, and the light gray indicates mixed-fiber-type
region containing fast-twitch glycolytic, fast-twitch oxidative glycolytic, and slow-oxidative (FG, FOG, SO).
1929
72 h and 2 wk postexercise. These time points were selected
based on the results of our previous study (27). Untrained
(i.e., sedentary) animals ran continuously on a motor-driven
treadmill at a speed of 12 m/min for one 30-min period on a
⫺20° slope. After exercise, runners from each group were
returned to their cages where they were given food and water
ad libitum.
At the time of death, animals were administered an overdose of pentobarbital sodium (1.0 ml; 60 mg/ml). Before
fixation perfusion and under anesthesia, the MG was quickly
excised, and the pMG and dMG were separated, sliced into
1-mm-thick cross sections, mounted in optimum cutting temperature embedding media on cardboard, and then snap
frozen in a 2-methylbutane (Baxter Canlab) bath immersed
in liquid nitrogen. The spinal cord was harvested after
transcardial perfusion with 500 ml of 4% paraformaldehyde
(pH 7.35–7.45; BDH, Oakville, ON). The lumbar region of the
spinal cord (L3 –L5) was removed intact, postfixed in 4%
paraformaldehyde for 18 h, and cryoprotected overnight in
20% sucrose in 0.1 M phosphate buffer, pH 7.2 (BDH). Tissues were frozen in 2-methylbutane at ⫺80°C.
Immunocytochemistry of the spinal cord. The immunocytochemical methodologies have been previously reported (27)
and are briefly described here. Frozen spinal cord lumbar
regions (L3 –L4) were serially sectioned at 10 ␮m. In an
attempt to reduce interexperimental variability, spinal cord
cross sections from each experimental group were placed on
the same slide (i.e., control, 72 h, 2 wk). Sections were then
washed in 0.1 M PBS, pH 7.1, blocked with 10% normal goat
serum (Gibco, Baxter Canlab), and then incubated overnight
at 4°C with a polyclonal antibody to CGRP [rabbit anti-CGRP
(Rat, 1–37); Genosys, The Woodlands, TX] at a 1:2,000 dilution. Tissues were then processed with the use of the avidinbiotin complex kit with a goat anti-rabbit secondary antibody
(Vectastain, Vector, Mississauga, ON) followed by incubation
with avidin-conjugated Texas red fluorophore (Vector). Slides
were coverslipped with Mowiol.
Acetylcholinesterase histochemistry and immunocytochemistry of muscle tissue. Motor end plates were identified by acetylcholinesterase (AChE) histochemistry to determine the innervation pattern and location of neuromuscular junctions in the
two regions of MG. Frozen, unfixed muscle tissue was serially
sectioned at 12 ␮m. Three series of samples were collected every
200 ␮m and placed directly onto slides. The first series was
processed for AChE histochemistry, the second series for immunocytochemistry, and the third series for myofibrillar ATPase
(myosin ATPase) determination (see Myofibrillar ATPase histochemistry below). Briefly, for AChE histochemistry (34), cross
sections on slides were incubated in 20% sodium sulfate (BDH)
for 3 min followed by a wash in deionized water, incubated in
reaction solution [pH 7.2; 5-bromoindoxyl acetate, ethanol,
K3Fe(CN)6, K4Fe(CN)6 䡠3H2O, Tris䡠HCl, Tris base, and CaCl2;
Sigma Chemical] for 15 min, washed in deionized water,
quickly dipped in eosin (Sigma Chemical), and then defatted
and coverslipped with Entellan (BDH).
For immunocytochemistry, frozen serial sections from unfixed muscle tissue were collected on slides as described
above. Slides were washed in 0.1 M PBS, pH 7.1, and immersed in 4% paraformaldehyde fixative (pH 7.4; BDH) for
30 min. Slides were then washed in 0.1 M PBS, pH 7.1,
blocked with 10% normal goat serum (Gibco, Baxter Canlab),
and incubated overnight at 4°C with the CGRP antisera at a
1:1,000 dilution. Tissue sections were processed by using the
avidin-biotin complex kit with a goat anti-rabbit secondary
antibody (Vectastain, Vector) followed by incubation with
avidin-conjugated Texas red (Vector). After a washing in 0.1
M PBS, the tissues were incubated overnight in fluorescein-
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EXERCISE INCREASES CGRP AT FAST-TWITCH MOTOR END PLATES
Table 1. Number of muscle fibers analyzed per fiber
type in the proximal and distal regions of MG
for glycogen depletion studies
Region of MG
Proximal
Distal
Fiber
Type
No. of
Animals
No. of Fibers
Analyzed per Animal
Average No. of
Fibers per Cross
Section
ST
FOG
FG
FG
8
8
8
8
100
100
100
500
2,900
2,900
2,900
4,000
MG, medial gastrocnemius; ST, slow-twitch; FOG, fast-twitch oxidative glycolytic; FG, fast-twitch glycolytic.
conjugated (FITC) ␣-bungarotoxin (␣-BuTx) (FITC-␣-BuTx;
1:1,500; Sigma Chemical). Slides were then washed and
coverslipped with Mowiol as described above.
Glycogen study: tissue sampling and analysis. To evaluate
whether the exercise protocol may have preferentially activated the pMG or the dMG, we examined the pattern of
glycogen depletion in both regions of the muscle after downhill running. Two groups of animals were divided into control
(n ⫽ 4) and downhill runners (n ⫽ 4). The running group
completed the exercise protocol and was killed 20 min later.
The MG (2 per animal) were quickly dissected out and snap
frozen as described above. Frozen control and exercise pMG
and dMG muscle were serially sectioned, placed on the same
slide, histochemically analyzed for glycogen content using
the periodic acid Schiff (PAS) stain (14), and placed on separate slides for myosin ATPase.
Myofibrillar ATPase histochemistry. Muscle fibers were
identified and classified as SO, FOG, and FG from the
ATPase stain (8). Briefly, frozen muscle sections mounted on
slides were placed in coplin jars containing acid medium
(NaC2H3O2 and KCl, pH 4.6; BDH) for 4 min, washed in basic
medium (C2H5NO2, CaCl2, NaCl, and NaOH, pH 9.4; BDH)
for 30 s, and placed in incubation medium (basic medium ⫹
ATP, pH 9.4, 30 min, 37°C; Sigma Chemical), followed by a
series of washes in CaCl2 (BDH), rinses in CoCl2 (Fisher),
and rinses in distilled H2O, ending with a 1-min incubation
in 20% (NH4)2 (Fisher). Slides were then cleared and coverslipped with Entellan (BDH).
Fig. 2. Photomicrographs of double-labeled pMG motoneurons 72 h after
downhill exercise. A: FluoroGold-filled
motoneuron in rat lumbar (L4) spinal
cord retrogradely labeled from the
pMG that is calcitonin gene-related
peptide negative (CGRP⫺) (B). Arrow
indicates the location of the CGRP⫺
motoneuron. C: FluoroGold-filled motoneuron retrogradely labeled from the
pMG that is CGRP positive (CGRP⫹)
(D). Calibration bars, 30 ␮m.
Fiber populations were quantified by counting and categorizing 100–120 fibers in pMG and 500–600 fibers in dMG
from each sample in randomly chosen fascicles distributed
throughout the entire cross-sectional area of the sample (see
Table 1). These values represent 10% of the muscle fibers in
the proximal compartment (300/2,900) and 12.5% of the total
number of muscle fibers in the distal compartment (500/
4,000) (42). With the use of bright-field settings with a Leica
DM/RB microscope, staining intensities for glycogen in the
fibers were automatically analyzed by relative optical density
measurement using the MCID image analysis software (Imaging Research, St. Catherine’s, ON). Baseline gray-scale
values were initially standardized by arbitrarily selecting
one shade of gray from a strip of film on a slide that had a
range of gray (black to white), which was subsequently
marked and identified as the reference point from which all
further illumination settings were to be established. This
procedure allowed us to establish a criterion illumination
level throughout the entire “blinded” analysis that was consistent and reproducible and that permitted the unbiased
measurement and comparisons between groups of gray-scale
values generated from muscle fibers of varying PAS staining
intensities.
Quantitation of CGRP⫹ motoneurons and motor end
plates. The quantitation of CGRP⫹ motoneurons and motor
end plates was completed by using a Leica DM/RB fluorescence microscope. Counts of motoneurons staining positively
for both CGRP and FluoroGold were obtained by selecting
only those somata with observable nuclei; profiles not containing a nucleus or nucleolus were not quantitated (cf., Ref.
46). The motor nuclei of the Sol, LG, pMG, and dMG were
topographically located in the medial-ventral region of the
spinal cord, beginning at the L4 ventral root entry zone and
extending rostrally ⬃2,000 ␮m (27). FluoroGold-labeled motoneurons exhibited cell bodies and dendrites filled with
granules of bright gold fluorescence (Fig. 2C). CGRP-Texas
red immunofluorescence was characterized as cytoplasmic
and punctate, with the fluorescent granules preferentially
located in the soma and in the proximal parts of the major
dendrites (Fig. 2D). All motoneurons with FluoroGold labeling were counted, and their CGRP⫹ or CGRP-negative
(CGRP⫺) status was determined. MG muscles injected with
Fluorogold were analyzed for Fluorogold content by examin-
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EXERCISE INCREASES CGRP AT FAST-TWITCH MOTOR END PLATES
Fig. 3. Profile of double-labeled FluoroGold and CGRP⫹ soleus (Sol), lateral gastrocnemius (LG), and MG motoneurons
72 h and 2 wk after downhill exercise
(time 0 ⫽ control). A: percentage of
CGRP⫹/FluoroGold-labeled Sol motoneurons. B: percentage of CGRP⫹/FluoroGold-labeled LG motoneurons. C: percentage of double-labeled CGRP⫹/FluoroGold
motoneurons in the MG motor pool (pMG
and dMG) in lumbar section L4 of the rat
spinal cord. * P ⬍ 0.05 (ANOVA; Kruskal
Wallis test). Double-labeled cells from the
Sol and LG were not identified in the same
cross sections.
ing the center of the muscle at the midline to determine
whether diffusion had occurred as a result of the injection
(27).
CGRP staining at the motor end plate was evaluated in 12
animals that were randomly divided into three groups: control and 72 h and 2 wk postexercise (Fig. 3). Motor end plates
were identified in transverse section by AChE staining.
Based on the results from a separate series of experiments in
which we evaluated AChE-stained longitudinal muscle sections taken from the belly of the MG (see MATERIALS AND
METHODS), we were able to determine that the average length
of a MG motor end plate was ⬃200 ␮m. This method permitted us to establish a sampling distance within the muscle
region that would not result in duplicate counts of motor end
plates that were evaluated for ␣-BuTx. In subsequent adjacent sections, motor end plates were identified with ␣-BuTx
fluorescence by using a blue filter (525 nm; 10⫻ PL FLUOTAR objective) for fluorescein detection, followed by a green
filter (625 nm; 100⫻/1.25 N Plan Oil) for detecting Texas Red
immunofluorescence, to colocalize the CGRP⫹ signal. CGRP
immunofluorescence was clearly detected as a punctate
staining pattern visible in regions of the junctional folds (see
Fig. 6D), overlapping the ␣-BuTx labeling that filled the
junctional area (see Fig. 6C). Approximately 140 end plates
were quantitated per muscle sample (Table 2). Slides with
CGRP⫹ motor end plates were then compared with adjacent
serial sections stained for myosin ATPase to determine the
fiber type of the identified neuromuscular junction.
Importantly, all tissue analyses were done blinded to the
experimental condition throughout all the procedures described in this study. The identity of the experimental groups
was subsequently decoded after completion of data collection
to permit statistical analysis. Statistical significance was
determined by Student’s t-test, ANOVA, and, where necessary, the Kruskal Wallis test for nonparametric distributions
(SigmaStat version 1.0, Jandel Scientific). Data are pre-
sented as means ⫾ SE. Differences were considered to be
significant at P ⬍ 0.05.
RESULTS
CGRP response in retrogradely labeled motoneurons.
FluoroGold-labeled motoneurons within the motor nuclei of the Sol, LG, pMG, and dMG were readily identified under ultraviolet light (Fig. 2). Double-labeled
Texas red-conjugated CGRP⫹ cells had a punctate, red
staining pattern (Fig. 2D), making this easily discernible from the more homogeneous, finely granular,
white FluoroGold signal (Fig. 2C). Motoneurons that
were labeled with FluoroGold and identified as
“CGRP⫺” were stunningly clear in their lack of CGRP
(Fig. 2B). Numbers of double-labeled motoneurons in
the identified Sol motor nucleus did not change after
downhill exercise over the experimental time period
(Fig. 3A). A similar observation was true for the double-labeled motoneurons of the LG motor nucleus (Fig.
3B).
In both regions of the MG, however, downhill exercise resulted in a significant increase in the number of
double-labeled CGRP⫹ motoneurons 72 h after exercise compared with control (pMG: P ⫽ 0.003; dMG: P ⫽
0.03; Fig. 3C). Significant differences in the number of
CGRP⫹ motoneurons were not observed between control and 2 wk postexercise groups in pMG or dMG (P ⬎
0.05), indicating that CGRP expression in these motoneurons had returned to baseline levels by this time
and confirming our previous results (27).
Glycogen depletion experiments. Based on our observation of the enhanced expression of CGRP in MG
Table 2. Total number of motor end plates quantified in the proximal and distal regions
of the MG in the control and experimental groups
Region of MG
Proximal
Distal
No. of
Animals
Total No. of
FITC-␣-BuTx
End Plates
Total No. of
CGRP⫹ Motor
End Plates
ST
FOG
FG
12
12
1,700
1,700
150
400
1
0
1
0
148
400
Fiber Type of CGRP⫹ End Plates
FITC-␣-BuTx, fluorescein-conjugated ␣-bungarotoxin; CGRP⫹, calcitonin gene-related peptide positive.
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EXERCISE INCREASES CGRP AT FAST-TWITCH MOTOR END PLATES
Fig. 4. Photomicrographs of myosin
ATPase and periodic acid Schiff (PAS)stained muscle fibers from the proximal region of the MG describing the
glycogen depletion pattern before and
after downhill exercise. Frozen serial
cross sections of the MG stained for
myosin ATPase (A and C) and PAS (B
and D) in the control condition (B) and
20 min after downhill running exercise
(A, C, and D). ST, slow-twitch fibers.
Calibration bars, 50 ␮m.
motoneurons, it was of interest to ascertain whether
both pMG and dMG were equally recruited by the
downhill running protocol. Differences in the activity
patterns of glycogen depletion would permit us to qualitatively interpret the physiological state of the muscle.
Therefore, the glycogen content of both pMG and dMG
was determined by PAS histochemistry (Fig. 4). Relative optical density measurements (Fig. 5) showed that
both regions and all fiber types in the pMG (SO, P ⫽
0.00002; FOG, P ⫽ 0.0002; FG, P ⫽ 0.0005) and dMG
(FG, P ⫽ 0.0006) were significantly depleted of glycogen stores after this eccentric exercise paradigm. While
indicating that both pMG and dMG were active, these
results did not allow us to quantitate the relative
amounts of glycogen breakdown in the different muscle
fiber types.
CGRP response at motor end plates in the pMG and
dMG after eccentric exercise. When viewed in transverse section, the motor end plates in the MG were
easily identified by FITC-␣-BuTx staining as they
followed a visible pattern throughout the belly of the
muscle. The junctional folds of the end plate region
Fig. 5. Glycogen depletion profiles of ST, FOG, and FG
muscle fiber types in the pMG (A) and dMG (B) 20 min
postexercise, as indicated by the relative optical density
(ROD) measurement. * Significant difference compared
with control, P ⬍ 0.001 (t-test).
were consistently and clearly labeled with bright
green FITC-␣-BuTx (Fig. 6). Texas red CGRP immunofluorescence staining was similar to that observed
in the motoneuron, presenting with an irregular,
punctate distribution that overlapped portions of
FITC-␣-BuTx immunoreactivity in the junctional
folds (Fig. 6C). This distinction in staining pattern
made it easy to distinguish between CGRP⫹ and
CGRP⫺ end plates and to identify end plates that
were not CGRP⫹ as being affected by bleed through
of the ␣-BuTx signal. Interestingly, CGRP⫹ end
plates were observed most often in regions in which
clusters of neuromuscular junctions were found, although not all neuromuscular junctions within a
cluster were CGRP⫹ (Fig. 6D), with the majority
being CGRP⫺ (Fig. 6B).
Elevated numbers of immunofluorescent CGRP⫹
motor end plates were observed in both pMG and dMG
after downhill exercise. In pMG, a mixed fiber type
region, a significant increase in the numbers of CGRPimmunoreactive neuromuscular junctions was observed at 72 h postexercise (10%; P ⫽ 0.03) and re-
EXERCISE INCREASES CGRP AT FAST-TWITCH MOTOR END PLATES
1933
Fig. 6. Photomicrographs of the motor
end plates in the pMG double-labeled
with FITC-conjugated ␣-bungarotoxin
(␣-BuTx) and Texas red (TR)-conjugated CGRP 2 wk after 1 bout of downhill exercise. FITC-␣-BuTx identified
motor end plate (A) colocalized with
TR-CGRP⫺ MG motor end plate (B).
Nonspecific bleed through of FITC signal completely overlaps the same area
and outlines the morphology of the
neuromuscular junction specifically
stained for ␣-BuTx in A. C: FITC-␣BuTx identified pMG motor end plate.
Specific TR-CGRP immunoreactivity
colocalized with ␣-BuTx (D) overlaps
with, but is not identical to, neuromuscular junction morphology (C). Calibration bars, 10 ␮m.
mained significantly elevated 2 wk later (9.9%, P ⫽
0.03; Fig. 7A). In the dMG, composed entirely of FG
fibers, a 25% increase in the number of CGRP⫹ motor
end plates compared with control was observed at 72 h
postexercise (P ⫽ 0.002) (Fig. 7B). Interestingly, this
percentage continued to be significantly elevated in the
dMG compared with control, increasing to 33% by 2 wk
after exercise.
CGRP immunoreactivity and muscle fiber type. Analyses of serial cross sections of pMG and dMG stained
histochemically for myosin ATPase content showed
that CGRP⫹ motor end plates colocalized almost exclusively to FG muscle fibers (Fig. 8). Out of ⬃3,000
end plates examined in this study, CGRP⫹ end plates
at FOG muscle fibers and SO fibers were rarely observed (Table 2).
DISCUSSION
We previously reported (27) that one 30-min bout
of downhill running resulted in increased numbers of
CGRP⫹ motoneurons in hindlimb extensor but not
flexor motor nuclei. Those results were the first to
demonstrate increased CGRP levels as a result of
physiological neuromuscular activity rather than a
lack of activity, i.e., as induced by surgical or pharmacological paralysis or by hormone deprivation.
Our present report demonstrates that CGRP expression was exclusively elevated in MG motoneurons
and that, within the muscle, this response was specifically localized to motor end plates on FG muscle
fibers.
The increased CGRP levels in MG motoneurons, and
subsequently at motor end plates in the muscle, could
be due to a variety of factors. For example, the exercise
regime may result in frank histopathological damage
to the muscle, thereby initiating repair and regenerative mechanisms that would require new end plates on
de novo myofibrils. Alternately, unaccustomed exercise
may induce growth-related morphological alterations
within the individual myofibrils and subsequently at
their neuromuscular junctions, leading to growth of the
motor end plates. A more subtle process could be that
the particular demands of downhill exercise may differentially affect muscle fibers within a motor unit,
leading to remodeling events at the affected neuromuscular junctions.
Fig. 7. Increase in the percentage of CGRP⫹
motor end plates in the pMG and dMG after
downhill exercise. A: increased numbers of FITC␣-BuTx and TR-CGRP⫹ motor end plates in the
pMG 72 h and 2 wk postexercise compared with
control (time 0) (* P ⫽ 0.03, ANOVA; Kruskal
Wallis test). B: increased numbers of FITC-␣BuTx and TR-CGRP⫹ motor end plates in the
dMG 72 h and 2 wk postexercise compared with
control (* P ⫽ 0.002, ANOVA; Kruskal Wallis
test).
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EXERCISE INCREASES CGRP AT FAST-TWITCH MOTOR END PLATES
Fig. 8. CGRP immunoreactivity is present at motor end plates of type IIB muscle fibers in the MG after downhill
exercise. A: muscle cross section from the pMG identified as a type IIB muscle fiber by myofibrillar ATPase
histochemistry. Serial cross section identifying a FITC-␣-BuTx-labeled neuromuscular junction that is CGRP⫹ (B)
as detected by Texas red immunofluorescence (C). Calibration bars: 30 ␮m (A) and 10 ␮m (B and C).
Downhill running produces changes in MG motoneurons in the absence of muscle damage. Although a
detailed biomechanical analysis of downhill running in
rats has not yet appeared in the literature, in this
downhill running model the ankle flexors are assumed
to be performing concentric exercise (shortening while
loaded), whereas the ankle extensors are similarly
assumed to undergo eccentric contractions (lengthening while loaded). Previous studies that have used
chronic and/or extended bouts of downhill running
activity in rats report Sol muscle damage with indexes
of pathology clearly observed 3–5 days postexercise (1,
32). Although Smith et al. (42, 43) detected the formation of new muscle satellite cells in the rat Sol after
downhill running, followed by significant increases in
developmental myosin isoforms and the appearance of
new myofibrils (42), neither the MG nor the LG showed
any significant trends, thus making the need for de
novo end plates in the MG appear unlikely. Because
there is no significant histopathology or inflammation
in the MG, compared with the Sol, after either chronic
or acute eccentric exercise protocols (27, 42), it seems
unlikely that the changes in CGRP levels we reported
can be attributed to myofibrillar damage and repair
processes. In addition, the relative lack of baseline
CGRP immunoreactivity in Sol motoneurons and the
absence of change in CGRP levels in this slow-twitch
muscle after exercise (despite the histopathological
data) argue against the idea that CGRP may be related
to extensively used or easily recruited (e.g., Sol) motoneurons (4). Our results, however, do support the
view that motoneurons innervating FG fibers [e.g.,
larger and faster motor units (5, 25)] contain more
CGRP.
CGRP and sprouting at the neuromuscular junction.
Sprouting of the motor nerve terminal in the adult has
been correlated with changes in CGRP peptide and
mRNA expression in motoneurons after either a significant injury to the motor nerve or a substantial pharmacological interruption of neuromuscular connectivity (3, 36, 39, 45). In our study, however, no damage is
incurred by the MG motor nerve, and overt muscle
damage is absent. Furthermore, it is unlikely that the
intramuscular injection of FluoroGold produced a
nerve injury that initiated a sprouting response and an
upregulation in motoneuronal CGRP expression. Data
from our first study demonstrated elevated numbers of
CGRP⫹ motoneurons after eccentric exercise; these
muscles were not injected with FluoroGold (27). Other
investigators have also reported no changes in CGRP
immunoreactivity and ␣-CGRP mRNA in the bulbocavernosus muscle in sham and vehicle-treated groups
with the use of a multiple injection protocol (38).
Chronic exercise has been shown to effect morphological changes at the neuromuscular junction (12, 13,
48, 49), in motoneurons, and in axons (16). It seems
unlikely to us that a single 30-min bout of downhill
running would elicit a measurable sprouting response;
whether chronic exercise elicits a sustained CGRP response and growth or sprouting at the neuromuscular
junction remains to be examined.
A neuromodulatory role for CGRP at the motor end
plate after exercise. Perhaps the elevated expression of
CGRP in MG motoneurons and their end plates on FG
muscle fibers may be correlated with more subtle remodeling events at the neuromuscular junction. Apart
from its “fast” neurotransmitter-like actions on the
nicotinic acetylcholine receptor (AChR), CGRP also
exerts longer lasting trophic functions at the neuromuscular junction, mediated by specific CGRP receptors localized to the postsynaptic membrane (reviewed
in Ref. 4). In cultured chick myotubes, CGRP has been
shown to upregulate the appearance, number, and
insertion of nicotinic AChRs into the muscle membrane
(22, 31). Other in vitro studies have shown that CGRP
increases the mRNA coding for the stabilizing ␣-subunit in the AChR complex (21, 22, 33). Whether CGRP
elicits such changes at the adult neuromuscular junction has not yet been examined.
The modulation of synaptic efficiency may also involve changes in the relative proportions of AChE
enzymes. One of the subunits, the asymmetric form G4,
located at the neuromuscular junction, is involved in
the fast clearance of ACh at junctional receptors (20)
and is known to be upregulated by chronic treadmill
exercise (26, 28) with the greatest response observed in
fast-twitch muscles (18, 26). Mouse myotube cultures
treated with CGRP displayed a 2.5-fold increase both
in (G4) AChE and in AChR ␣-subunit mRNA, further
implicating CGRP as a trophic factor regulating the
gene expression of integral postsynaptic molecules at
the intact adult neuromuscular junction (7). Intramuscular injection of exogenous CGRP, however, has been
reported to reverse the exercise-induced increase in G4
EXERCISE INCREASES CGRP AT FAST-TWITCH MOTOR END PLATES
in rat gracilis muscle (19). Whether CGRP regulates G4
AChE and AChR subunit expression at the neuromuscular junction after physiological exercise is not
known.
Elevated CGRP expression as a function of motor
unit recruitment. Both the LG and MG motor nuclei
have equivalent baseline levels of CGRP: 60% of motoneurons are CGRP⫹ in the sedentary animal. The
fact that there are no changes in CGRP expression in
LG motor nuclei after downhill running may suggest a
subtle effect of this exercise paradigm on the MG that
is not elicited in the LG. There is evidence to suggest
that the LG is less active than the MG during locomotion (15, 44). Studies by Duysens et al. (15) describe the
reduced activation of the LG in humans during walking or running, whereas Smith and Carlson-Kuhta (44)
observed a similar lack of LG activation in felines
during slope walking. The functional or biomechanical
implications of our findings need to be more rigorously
investigated using electrophysiological techniques.
The rat MG is a highly compartmentalized muscle
with distinct fiber type distributions. Morphologically,
the pMG, innervated by the proximal and lateral
branches of the MG nerve, is a mixture of slow- and
fast-twitch fibers (SO, FOG, FG), whereas the distal
region innervated by the distal branch of the MG nerve
is exclusively FG (9, 47). These compartments appear
to be preferentially activated in the performance of
different motor tasks (11, 47). There is growing evidence to suggest that the complex morphological structure (i.e., compartmentalization or regionalization) of a
muscle influences muscle fiber properties and intramuscular activity (reviewed in Ref. 29). Our PAS staining describes glycogenolysis in all fiber types of both
MG regions, suggesting that the expression of CGRP in
FG fibers reflects a difference in the activity response
of this fiber type within the MG after downhill running.
Perhaps the unaccustomed activity in our model preferentially perturbs neuromuscular junctions in FG fibers because of their higher susceptibility to transmission failure (41). This altered use may then initiate
morphological adaptation and repair of the affected
neuromuscular junctions for which CGRP is required
(39).
Based on human studies, motor control strategies for
eccentric work (e.g., downhill running) are suggested to
differ from those of concentric work (e.g., predominantly uphill running) in that fast- rather than slowtwitch motor units are recruited first (17, 30). To date,
motor unit recruitment strategies for downhill running
have not yet been addressed in animal models. To
determine whether enhanced CGRP expression at FG
motor end plates is associated with selective recruitment of fast-fatiguable or fast-fatigue-resistant motor
units, intracellular recordings from identified motoneurons (cf., Refs. 10, 40) will need to be done.
In conclusion, we have shown that, after downhill
running in the rat, CGRP expression is elevated in MG
motoneurons and at MG motor end plates on FG muscle fibers. Our results may indicate a preferential response of FG fibers after unaccustomed exercise, re-
1935
sulting in synaptic reorganization. This model provides
a novel system in which to further investigate whether
physiological exercise, specifically downhill exercise,
preferentially recruits FG motor units and whether
exercise-induced increases in CGRP expression play a
role in the remodeling of the postsynaptic junction in
the intact, adult neuromuscular system.
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