ABSTRACT: Glial cell line–derived neurotrophic factor (GDNF) is pro-

duced by skeletal muscle and affects peripheral motor neurons. Elevated

expression of GDNF in skeletal muscle leads to hyperinnervation of neuro-
muscular junctions, whereas postnatal administration of GDNF causes syn-
aptic remodeling at the neuromuscular junction. Studies have demonstrated
that altered physical activity causes changes in the neuromuscular junction.
However, the role played by GDNF in this process in not known. The ob-
jective of this study was to determine whether changes in neuromuscular
activity cause altered GDNF content in rat skeletal muscle. Following 4
weeks of walk-training on a treadmill, or 2 weeks of hindlimb unloading,
soleus, gastrocnemius, and pectoralis major were removed and analyzed for
GDNF content by enzyme-linked immunosorbant assay. Results indicated
that walk-training is associated with increased GDNF content. Skeletal
muscle from hindlimb-unloaded animals showed a decrease in GDNF in
soleus and gastrocnemius, and an increase in pectoralis major. The altered
production of GDNF may be responsible for activity-dependent remodeling
of the neuromuscular junction and may aid in recovery from injury and dis-
ease.
© 2002 Wiley Periodicals, Inc. Muscle Nerve 26: 206–211, 2002

GDNF IS REGULATED IN AN ACTIVITY-DEPENDENT

MANNER IN RAT SKELETAL MUSCLE
ERICA A. WEHRWEIN, MS, ERIC M. ROSKELLEY, BS, and JOHN M. SPITSBERGEN, PhD

Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan 49008, USA

Accepted 27 March 2002

Most neurons require a constant supply of neuro-
trophic factors for growth, maintenance of cell phe-
notype, and possibly continued survival in adult
organisms. Alterations in neurotrophic factor pro-
duction, release, or biological effects may have pro-
nounced effects on nervous system structure and
function. Although a variety of neurotrophic factors
have been identified that exert effects on motor neu-
rons,16 the findings that glial cell line–derived neu-
rotrophic factor (GDNF) is present in mature skel-
etal muscle23 and alters structure and function in
mature motor neurons,31 makes GDNF an excellent
candidate as a neurotrophic molecule controlling
motor neuron plasticity in adult organisms.
Abbreviations: BSA, bovine serum albumin; EDTA, ethylenediamine-
tetra-acetic acid; ELISA, enzyme-linked immunosorbant assay; GDNF,
glial cell line–derived neurotrophic factor; PBS, phosphate-buffered saline
Key words: exercise; GDNF; neuromuscular junction; neurotrophic fac-
tor; plasticity
Correspondence to: J. Spitsbergen; e-mail: john.spitsbergen@wmich.
edu
© 2002 Wiley Periodicals, Inc.
Published online 10 June 2002 in Wiley InterScience (www.
interscience.wiley.com). DOI 10.1002/mus.10179
Although several recent studies have examined
the regulation of GDNF expression in neurons and
glial cells,2,16,29 very little is known about processes
involved in the regulation of GDNF expression in
muscle. Studies examining neurotrophic factor ex-
pression in smooth muscle have shown that a variety
of stimuli can influence its expression, including ex-
posure to neurotransmitters 22 and mechanical
stretch.19 If alterations in mechanical activity affect
skeletal muscle GDNF expression, then changes in
physical activity could affect motor neuron structure
and function indirectly through changes in GDNF
expression.
Increased physical activity increases size and de-
gree of branching of motor nerve terminals at neu-
romuscular junctions of type I fibers,1,10 increases
expression of acetylcholinesterase,24 increases ex-
pression of acetylcholine receptor,9 and increases
size and density of pre- and post-synaptic membrane
specializations.25,27 There are differential effects on
soleus neuromuscular junction morphology based
on intensity of training.10 Animals trained at either
high or low intensity demonstrated neuromuscular
junction hypertrophy; however, high-intensity
trained animals had more dispersed synapses,

206 Activity Alters GDNF in Muscle MUSCLE & NERVE August 2002
greater total length of branching, higher average
length per branch, and greater number of secondary
branches than the low-intensity group.10
Hindlimb unloading and space flight lead to
muscle atrophy,28 neuromuscular junction atrophy,3
and neuromuscular weakness.11 The atrophic re-
sponse of skeletal muscle in the soleus and gastroc-
nemius is maximal after 2 weeks of suspension or
space flight.20 Post-flight, there appears to be a de-
creased number of synaptic vesicles in the nerve ter-
minal and mitochondrial swelling, which has been
interpreted as the first stage in axonal degenera-
tion.4 It has been demonstrated that muscle activity,
both frequency and amplitude, are decreased during
hindlimb unloading.5
We tested the hypothesis that GDNF expression
in skeletal muscle is regulated by the level of neuro-
muscular activity. We predicted that increased physi-
cal activity would lead to an increase in GDNF ex-
pression, and decreased physical activity to a
decrease in GDNF expression. If GDNF expression is
controlled by the level of neuromuscular activity,
then the alterations in motor neuron structure and
function seen with changes in physical activity may
be driven by changes in GDNF expression.
MATERIALS AND METHODS
Subjects. All animal experiments were performed
in accordance with the “Guide for the Care and Us-
age of Laboratory Animals” (National Research
Council) and experiments were approved by our In-
stitutional Animal Care and Usage Committee.
Twenty-one healthy, adult (8 weeks) male Sprague-
Dawley rats (Charles River, Portage, Michigan) were
randomly assigned to sedentary control (n = 7), walk-
trained (n = 7), or hindlimb-unloaded (n = 7)
groups. Weights at the onset of the experiment
ranged from 230.1 g to 250.7 g with an average of
240.3 ± 1.7 g. Control and walk-trained animals were
housed one or two per cage in Nalgene cages with
access to food and water ad libitum. Animals were
housed with another animal in the same treatment
group unless there were no treatment-matched ani-
mals available, in which case the animal was housed
alone. Hindlimb-unloaded rats were housed singly in
modified rabbit cages. Hindlimb suspension was ac-
complished by taping the tail of the rat to the ceiling
of a rabbit cage, such that the hindlimbs of the rat
were elevated off the bottom of the cage.18 Animals
were suspended so that access to food and water was
assured. Animals were kept on a 12:12 h light-dark
cycle in a room with regulated temperature (22–
24°C).
Activity Alters GDNF in Muscle
All animals were monitored daily and body
weights were measured to ensure positive weight
gain. Throughout the 4-week experimental period,
control animals remained in their cages and partici-
pated in normal ambulation. Walk-trained animals
were treadmill-trained using the following param-
eters: 15 min/day at 0% incline (three 5-min inter-
vals with 8-min rest period between trials) at 21.3
m/min, 5 days/week for 4 weeks. This protocol was
adapted from Tomas et al.26 This low-intensity exer-
cise, with breaks between exercise periods, was de-
signed to utilize primarily low-threshold motor neu-
rons innervating type I skeletal muscle fibers.
Hindlimb-unloaded animals were subjected to hind-
limb unloading for 2 weeks and then sacrificed as
described below.
The 2-week time course was selected based on
data indicating that 14 days of unweighting resulted
in maximal atrophy of soleus and gastrocnemius and
that neuromuscular junction modifications were also
noted at this time point.20 During this time, animals
remained in their cages and were not allowed to rest
their hindlimbs on the floor of the cage.
Each training day, animals in the exercise group
were allowed to warm-up for 1 min at 5.3 m/min (0.2
mph) on the treadmill prior to beginning the exer-
cise. Training was performed using a simple escape
contingency. Water served as the punishment in the
escape contingency when subjects failed to perform
at a satisfactory level. When a subject stopped walk-
ing, it was sprayed with water. Animals were sacri-
ficed by CO2-euthanization prior to thoracotomy 24
h after the final bout of exercise. Age-matched con-
trol animals were sacrificed on the same day.
Tissue Collection and Processing. Selected skeletal
muscles (soleus, gastrocnemius, and pectoralis ma-
jor) were removed and flash frozen upon contact
with dry ice. Frozen tissue was cut into serial cross-
sections weighing 0.5 g or less and stored at −80°C.
Muscles were removed bilaterally. The left-sided
muscles were used for protein assay and the right-
sided muscles for immunocytochemistry. Just prior
to processing, frozen muscle samples were dipped
into liquid nitrogen and then pulverized on a metal
block chilled on dry ice. The pulverized muscle was
then suspended in 0.1 M phosphate-buffered saline
(PBS: 0.225 M NaCl, 0.02 M NaH2PO4, 0.08 M
Na2HPO4) containing 0.1% Tween-20, 0.05% bovine
serum albumin (BSA), aprotinin (Sigma, St. Louis,
Missouri), 0.2 mM benzamidine, 0.01 mM benzetho-
nium chloride, and 0.2 mM ethylenediaminetetra-
acetic acid (EDTA).7 The suspension was chilled on
wet ice while being homogenized for 30 s using a
MUSCLE & NERVE August 2002 207
variable speed Tissue Tearor (Biospec Products,
Inc., Bartlesville, Oklahoma). Homogenate was cen-
trifuged at 13,000g and the supernatant was analyzed
using an enzyme-linked immunosorbant assay
(ELISA) specific for GDNF.
Enzyme-Linked Immunosorbant Assay. Assay plates
(96-well NUNC-Immuno, Nalgene Nunc Interna-
tional, Rochester, New York) were incubated with a
monoclonal antibody raised against GDNF (R&D
Systems, Minneapolis, Minnesota) overnight in a hu-
midified chamber. Remaining sites were blocked
with BSA (1.0%) for 1 h at room temperature. Plates
were rinsed three times and muscle sample homog-
enate or GDNF standard (R&D Systems) was added
to each well. All samples and standards were run in
quadruplicate. GDNF standard (2 µg/ml) was di-
luted in sample buffer to prepare a standard curve
between 1,000 pg/ml and 2 pg/ml. Internal control
wells were used that contained only sample buffer.
Sample incubation was done in a humidified cham-
ber for 2 h at room temperature. Following incuba-
tion of the sample or standard, the wells were washed
and incubated with an anti-GNDF antibody conju-
gated to biotin (R&D Systems) for 2 h at room tem-
perature. The wells were then washed three times
and horseradish peroxidase conjugated to streptavi-
din was added for 20 min at room temperature. The
wells were washed three times, and 3,3⬘,5,5⬘-
tetramethylbenzidine substrate was added and al-
lowed to incubate approximately 30 min at room
temperature. The reaction was stopped with 0.1 M
phosphoric acid, and plates were read at 450 nm
using a microplate spectrophotometer. Standard
curves were derived from known GDNF samples.
Muscle extract data was reported as picograms of
GDNF per gram of tissue wet weight.
Immunocytochemistry. Skeletal muscle samples
were removed from the right side of the animal and
immediately frozen in 2-methyl-butane chilled on
dry ice. Tissue was stored at −80°C until sectioned.
Whole muscle was divided into three sections. Sec-
tions for imaging were taken from the middle third
of the whole muscle. Cryostat sections (50 µm; cross
section) were mounted on slides and then affixed by
placing slides in a vacuum-sealed container for 4 h.
Sections were subsequently fixed using 4% para-
formaldehyde diluted in PBS at room temperature
for 15 min. Samples were blocked using 1% BSA in
PBS for 20 min then rinsed for 10 min in wash
buffer. Biotinylated primary polyclonal antibody
raised against GDNF (R&D Systems) was used in a
1:500 dilution in PBS. A 15-min PBS wash followed a
208 Activity Alters GDNF in Muscle
1-h incubation in primary antibody at room tempera-
ture. Slides were then treated with a streptavidin-
linked probe (Alexa 488, Molecular Probes, Eugene,
Oregon) diluted in PBS, at 37°C, for 30 min. Images
were captured using confocal microscopy (Zeiss Co.,
Thornwood, New York) at an excitation wavelength
of 495 nm and emission wavelength of 519 nm. Sec-
tions were viewed in 2 µm optical slices at 20× mag-
nification. Negative control images were obtained by
omitting incubation with primary antibody and uti-
lizing a streptavidin-linked fluorescent probe.
GDNF and Di-I Colocalization. GDNF-stained skel-
etal muscle sections were prepared as described
above and then stained with 1,1⬘-dioctadecyl-
3,3,3⬘,3⬘-tetramethylindocarbocyanine perchlorate/
DiIC18 (Di-I) (Molecular Probes). A stock Di-I solu-
tion of 10 mg/ml was prepared in 50:50
ethanol:dimethyl sulfoxide. A working concentra-
tion of 1:100 was prepared from the stock and di-
luted in PBS. Dilute Di-I (100 µl) was placed directly
on the slide and cover slipped. The slide was allowed
to incubate at room temperature for 15 min. Follow-
ing incubation, the sections were rinsed three times
for 5 min in PBS. Sections labeled with anti-GDNF
and Di-I were visualized using confocal microscopy
as described above, at an excitation wavelength of
535 nm and an emission wavelength of 590 nm for
Di-I visualization.
Statistical Analysis. Comparisons between control
and treatment were made using a Student’s t-test.
For all tests, significance was set to P ⱕ 0.05. All data
values are reported as the mean ± standard error of
the mean.
RESULTS
Gross Observations. Animal body weights were not
significantly different at the onset of the experiment;
however, weights of walk-trained (407.0 ± 11.3) and
hindlimb-unloaded animals (414.86 ± 6.9) were sig-
nificantly lower (P ⱕ 0.05) than control animals
(439.98 ± 8.9) at the end of the 4-week training pe-
riod or 2-week unloading period. All animals showed
positive weight gain throughout the experiment.
Measurements of individual muscle weights were
also taken. The weights of soleus (0.70 ± 0.6 g), gas-
trocnemius (0.77 ± 0.16 g) and pectoralis major
(0.79 ± 0.1 g) from control rats were not different
from walk-trained rats (0.75 ± 0.06 g, 0.73 ± 0.16 g,
and 0.71 ± 0.1 g, respectively) or from hindlimb-
unloaded rats (0.63 ± 0.06 g, 0.61 ± 0.16 g, and 0.82
± 0.1 g, respectively).
MUSCLE & NERVE August 2002
GDNF Content in Control Muscles. Immunocyto-
chemical staining was performed on gastrocnemius
from control animals to ensure that there was a mea-
surable amount of GDNF in control skeletal muscle.
Immunoreactivity for GDNF was observed in the rat
gastrocnemius muscle from control animals. GDNF
staining in cross sections appeared to be localized to
the membrane (Fig. 1). Membrane localization was
confirmed by demonstrating colocalization of GDNF
and Di-I to the membrane of rat skeletal muscle. The
pattern of membrane-associated GDNF is consistent
with that previously described.23 Control sections
were stained with streptavidin-linked fluorescent
probe in the absence of primary antibody. Images
obtained in the absence of primary antibody showed
low levels of background staining.
Activity-Dependent Alterations in GDNF. In order
to study activity-dependent changes in trophic fac-
tor, we selected two hindlimb locomotor muscles,
soleus (type I) and gastrocnemius (mixed fiber
type). Soleus is a primary load-bearing muscle and
was used to examine the effects of hindlimb unload-
ing as well. Pectoralis major, a mixed fiber type
muscle, was selected as it continues to bear weight in
the hindlimb unloading studies. When GDNF con-
tent of soleus, gastrocnemius, and pectoralis major
from walk-trained animals was measured using an
ELISA, we found an activity-dependent, significant
increase in GDNF (P < 0.05) compared to controls
FIGURE 1. Localization of GDNF protein in rat skeletal muscle from control animals. Left panel shows staining in the presence of
anti-GDNF primary antibody that appears to be localized to the membrane. (bar = 50 µm). Right panel shows colocalization of GDNF and
Di-I staining to the membrane of skeletal muscle cells (bar = 50 µm).
Activity Alters GDNF in Muscle
(Fig. 2). Hindlimb unloading resulted in a signifi-
cant decrease in GDNF content in soleus and gas-
trocnemius compared to controls (P < 0.05) (Fig. 2).
By contrast, hindlimb unloading resulted in a signifi-
cant increase in GDNF content in pectoralis major
(P < 0.05) compared to control (Fig. 2).
DISCUSSION
Our experiments showed that GDNF content in skel-
etal muscle was increased following 4-weeks of walk-
training. Skeletal muscle from hindlimb-suspended
animals showed a significant decrease in GDNF in
soleus and gastrocnemius, and an increase in GDNF
in pectoralis major. These findings suggest that
GDNF is modulated by activity level in vivo. It re-
mains to be seen whether changes in GDNF expres-
sion with activity play a role in the changes in neu-
romuscular junction architecture observed following
increased motor activity.
GDNF is a survival factor for peripheral nerves
and muscle.12 It is important in motor and sensory
neuron development,30 but its role in adult rats is
unclear. Results of a previous study6 showed that
skeletal muscle from rat contains low levels of GDNF
mRNA, which led others to suggest a limited role for
GDNF in adult skeletal muscle. We have demon-
strated immunocytochemically and utilizing ELISA
that GDNF protein is indeed present in measurable
amounts in adult rat skeletal muscle. Using whole
MUSCLE & NERVE August 2002 209

FIGURE 2. Effect of hindlimb unloading (A) and walk-training (B)
on skeletal muscle GDNF content. (A) GDNF content is signifi-
cantly increased in skeletal muscle following 4 weeks of walk-
training as compared to sedentary control. White bars represent
control and black bars represent walk-training. (B) Soleus and
gastrocnemius showed a significant decrease in GDNF content
as compared to matched muscle from sedentary controls,
whereas pectoralis major showed a significant increase in GDNF
as compared to sedentary control. White bars represent control
and black bars represent hindlimb unloading. Values are means
± SEM of skeletal muscle from seven animals. The asterisk in-
dicates a value significantly different than control (P ⱕ 0.05).
muscle homogenate, GDNF released from skeletal
muscle cannot be distinguished from GDNF released
from adherent Schwann cells. However, the findings
from the L6 skeletal muscle cell line in culture22 and
of immunocytochemical studies 23 indicate that
GDNF is produced by skeletal muscle cells.
This study has shown differential effects on tro-
phic factor production based on activity level. Fac-
tors that may mediate trophic factor expression in-
clude mechanical activity and neurotransmitter
release. Mechanical activity, in the form of stretch,
appears to be a positive stimulus for trophic factor
production in vascular and bladder smooth muscle
cells. Cyclic and static stretch increase production
and release of nerve growth factor in both bladder
and vascular smooth muscle cells of rats8 by activa-
tion of protein kinase C signaling pathways.19 Me-
chanical stimulation increases the expression of ace-
210 Activity Alters GDNF in Muscle
tylcholinesterase in cultured myotubes.13 Nifedipine
treatment partially blocks the effects of stretch on
acetylcholinesterase expression, indicating that
stretch induces calcium influx through voltage-gated
calcium channels.13 Thus, increased mechanical ac-
tivity may be responsible for some of the increased
GDNF levels observed in all muscles from walk-
trained rats and pectoralis major from hindlimb-
unloaded rats, whereas decreased mechanical activ-
ity may mediate the decrease in GDNF content of
hindlimb muscle from hindlimb-unloaded rats.
Data indicating that neurotransmitters modulate
neurotrophic factor expression comes from studies
in vascular and bladder smooth muscle cells in cul-
ture. In these studies, neurotransmitters from sym-
pathetic neurons alter production of nerve growth
factor by smooth muscle cells in culture.8,21 Indirect
evidence that motor neurotransmitters exert a nega-
tive influence on trophic factor production by skel-
etal muscle comes from studies performed on devel-
oping chick neuromuscular junctions. Oppenheim
et al.17 demonstrated that blockade of nicotinic ace-
tylcholine receptors on skeletal muscle enhanced
the ability of skeletal muscle to provide trophic sup-
port to innervating motor neurons. Direct evidence
that neurotransmitters exert a negative influence on
GDNF expression in skeletal muscle has been ob-
tained from studies demonstrating that denervated
skeletal muscle contains elevated levels of GDNF
mRNA.14 Cell culture data from this laboratory22 in-
dicate that acetylcholine inhibits GDNF secretion
from L6 skeletal muscle cells.
Hindlimb unloading leads to a decrease in neu-
romuscular junction complexity3,4,11,20 and we hy-
pothesized that this may be due to a decrease in
trophic factor expression. Studies have shown that
overexpression of GDNF leads to hyperinnervation
of neuromuscular junctions.15 It is possible that a
decrease in GDNF expression leads to a decrease in
innervation. In our study, soleus and gastrocnemius
showed a decrease in GDNF production following
hindlimb unloading. The increase in GDNF seen in
the pectoralis major may be due to an increased load
on the muscle from the altered body position
achieved with hindlimb unloading.
Activity-dependent regulation of trophic factors
has far-reaching implications. If trophic factors are
regulated in an activity-dependent manner, then
conditions such as sedentary lifestyle, immobiliza-
tion due to injury or illness, age-related decreases in
activity, hindlimb suspension, neuromuscular injury
or illness, and exposure to microgravity may lead to
dramatic changes in trophic factor expression and
MUSCLE & NERVE August 2002
subsequent neuromuscular junction remodeling.
Trophic factor expression may play a prominent role
in mediating these changes.
This work was supported by NIH grant 1 R15 HL60240-01, The
Faculty Research and Support Fund of Western Michigan Univer-
sity, and a Grant-In-Aid from the American Heart Association
(Michigan Affiliate). Preliminary results were presented at the
Integrative Biology of Exercise Conference, Portland, Maine, Sep-
tember 2000. The authors acknowledge the advice and assistance
of Drs. Christine Byrd, John Jellies, and William Jackson, Western
Michigan University. We greatly appreciate the technical assis-
tance of Berta C.R. Cohen. We also thank Matthew P. DeVries,
Angela Lim, Marisa Hart, and Judy Martin for their assistance in
tissue processing, and Drs. Rob Eversole and John Stout in the
Biological Imaging Center for assistance with confocal imaging.
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