WEHRWEIN (2002) GDNF is Regulated in an Activity-Dependent

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ABSTRACT: Glial cell linederived neurotrophic factor (GDNF) is pro-duced by skeletal muscle and affects peripheral motor neurons. Elevatedexpression 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 demonstratedthat 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 neuromuscularactivity cause altered GDNF content in rat skeletal muscle. Following 4weeks of walk-training on a treadmill, or 2 weeks of hindlimb unloading,soleus, gastrocnemius, and pectoralis major were removed and analyzed forGDNF content by enzyme-linked immunosorbant assay. Results indicatedthat walk-training is associated with increased GDNF content. Skeletalmuscle from hindlimb-unloaded animals showed a decrease in GDNF insoleus and gastrocnemius, and an increase in pectoralis major. The alteredproduction of GDNF may be responsible for activity-dependent remodelingof the neuromuscular junction and may aid in recovery from injury and dis-ease.

2002 Wiley Periodicals, Inc. Muscle Nerve 26: 206211, 2002

GDNF IS REGULATED IN AN ACTIVITY-DEPENDENTMANNER 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

M ost 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 andfunction. Although a variety of neurotrophic factorshave been identified that exert effects on motor neu-rons, 16 the findings that glial cell linederived neu-rotrophic factor (GDNF) is present in mature skel-etal muscle 23 and alters structure and function inmature motor neurons, 31 makes GDNF an excellent candidate as a neurotrophic molecule controllingmotor neuron plasticity in adult organisms.

Although several recent studies have examinedthe regulation of GDNF expression in neurons andglial cells,2,16,29 very little is known about processesinvolved in the regulation of GDNF expression inmuscle. 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 mechanicalstretch. 19 If alterations in mechanical activity affect skeletal muscle GDNF expression, then changes inphysical activity could affect motor neuron structureand function indirectly through changes in GDNFexpression.

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 increasessize and density of pre- and post-synaptic membranespecializations. 25,27 There are differential effects onsoleus neuromuscular junction morphology basedon intensity of training. 10 Animals trained at eitherhigh or low intensity demonstrated neuromuscular junction hypertrophy; however, high-intensity trained animals had more dispersed synapses,

Abbreviations: BSA, bovine serum albumin; EDTA, ethylenediamine-tetra-acetic acid; ELISA, enzyme-linked immunosorbant assay; GDNF,glial cell linederived neurotrophic factor; PBS, phosphate-buffered salineKey words: exercise; GDNF; neuromuscular junction; neurotrophic fac-tor; plasticityCorrespondence to: J. Spitsbergen; e-mail: [email protected]

2002 Wiley Periodicals, Inc.Publ ished onl ine 10 June 2002 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mus.10179

206 Activity Alters GDNF in Muscle MUSCLE & NERVE August 2002


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greater total length of branching, higher averagelength per branch, and greater number of secondary branches than the low-intensity group. 10

Hindlimb unloading and space flight lead tomuscle 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 orspace 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 beeninterpreted as the first stage in axonal degenera-tion. 4 It has been demonstrated that muscle activity,both frequency and amplitude, are decreased duringhindlimb unloading. 5

We tested the hypothesis that GDNF expressionin 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 adecrease in GDNF expression. If GDNF expression iscontrolled by the level of neuromuscular activity,then the alterations in motor neuron structure andfunction seen with changes in physical activity may be driven by changes in GDNF expression.

MATERIALS AND METHODS

Subjects. All animal experiments were performedin accordance with the Guide for the Care and Us-age of Laboratory Animals (National ResearchCouncil) 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) wererandomly 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 werehoused one or two per cage in Nalgene cages withaccess to food and water ad libitum. Animals werehoused 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 inmodified rabbit cages. Hindlimb suspension was ac-complished by taping the tail of the rat to the ceilingof 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 wasassured. Animals were kept on a 12:12 h light-darkcycle in a room with regulated temperature (22 24C).

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 21.3m/min, 5 days/week for 4 weeks. This protocol wasadapted 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 asdescribed below.

The 2-week time course was selected based ondata indicating that 14 days of unweighting resultedin maximal atrophy of soleus and gastrocnemius andthat neuromuscular junction modifications were alsonoted at this time point. 20 During this time, animalsremained 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 (0.2mph) on the treadmill prior to beginning the exer-cise. Training was performed using a simple escapecontingency. Water served as the punishment in theescape contingency when subjects failed to performat a satisfactory level. When a subject stopped walk-ing, it was sprayed with water. Animals were sacri-ficed by CO2-euthanization prior to thoracotomy 24h after the final bout of exercise. Age-matched con-trol animals were sacrificed on the same day.

Tissue Collection and Processing. Selected skeletalmuscles (soleus, gastrocnemius, and pectoralis major) 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 80C.Muscles were removed bilaterally. The left-sidedmuscles were used for protein assay and the right-sided muscles for immunocytochemistry. Just priorto processing, frozen muscle samples were dippedinto liquid nitrogen and then pulverized on a metal

block chilled on dry ice. The pulverized muscle wasthen suspended in 0.1 M phosphate-buffered saline(PBS: 0.225 M NaCl, 0.02 M NaH 2 PO 4, 0.08 MNa2HPO 4) containing 0.1% Tween-20, 0.05% bovineserum 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

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variable speed Tissue Tearor (Biospec Products,Inc., Bartlesville, Oklahoma). Homogenate was cen-trifuged at 13,000 g and the supernatant was analyzedusing 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 amonoclonal antibody raised against GDNF (R&DSystems, 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 addedto each well. All samples and standards were run inquadruplicate. GDNF standard (2 g/ml) was di-luted in sample buffer to prepare a standard curvebetween 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 washedand 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 timesand 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 roomtemperature. The reaction was stopped with 0.1 Mphosphoric acid, and plates were read at 450 nmusing a microplate spectrophotometer. Standardcurves 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 andimmediately frozen in 2-methyl-butane chilled ondry ice. Tissue was stored at 80C until sectioned. Whole muscle was divided into three sections. Sec-tions for imaging were taken from the middle thirdof the whole muscle. Cryostat sections (50 m; 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 temperaturefor 15 min. Samples were blocked using 1% BSA inPBS for 20 min then rinsed for 10 min in washbuffer. Biotinylated primary polyclonal antibody raised against GDNF (R&D Systems) was used in a1:500 dilution in PBS. A 15-min PBS wash followed a

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 wavelengthof 495 nm and emission wavelength of 519 nm. Sec-

tions were viewed in 2 m 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 describedabove and then stained with 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine perchlorate/DiIC18 (Di-I) (Molecular Probes). A stock Di-I solu-t ion o f 10 mg/ml was p repa red in 50 :50ethanol:dimethyl sulfoxide. A working concentra-tion of 1:100 was prepared from the stock and di-luted in PBS. Dilute Di-I (100 l) was placed directly on the slide and cover slipped. The slide was allowedto incubate at room temperature for 15 min. Follow-ing incubation, the sections were rinsed three timesfor 5 min in PBS. Sections labeled with anti-GDNFand Di-I were visualized using confocal microscopy as described above, at an excitation wavelength of 535 nm and an emission wavelength of 590 nm forDi-I visualization.

Statistical Analysis. Comparisons between controland treatment were made using a Student s t -test.

For all tests, significance was set to P 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) andhindlimb-unloaded animals (414.86 6.9) were sig-nificantly lower ( P 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 showedpositive weight gain throughout the experiment.Measurements of individual muscle weights werealso 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).



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GDNF Content in Control Muscles. Immunocyto-chemical staining was performed on gastrocnemiusfrom 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. GDNFstaining in cross sections appeared to be localized to

the membrane (Fig. 1). Membrane localization wasconfirmed by demonstrating colocalization of GDNFand Di-I to the membrane of rat skeletal muscle. Thepattern 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. Imagesobtained in the absence of primary antibody showedlow levels of background staining.

Activity-Dependent Alterations in GDNF. In orderto study activity-dependent changes in trophic fac-tor, we selected two hindlimb locomotor muscles,soleus (type I) and gastrocnemius (mixed fibertype). 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 typemuscle, was selected as it continues to bear weight inthe hindlimb unloading studies. When GDNF con-tent of soleus, gastrocnemius, and pectoralis majorfrom walk-trained animals was measured using anELISA, we found an activity-dependent, significant increase in GDNF ( P < 0.05) compared to controls

(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-suspendedanimals showed a significant decrease in GDNF insoleus and gastrocnemius, and an increase in GDNFin 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 followingincreased motor activity.

GDNF is a survival factor for peripheral nervesand muscle. 12 It is important in motor and sensory neuron development, 30 but its role in adult rats isunclear. Results of a previous study 6 showed that skeletal muscle from rat contains low levels of GDNFmRNA, which led others to suggest a limited role forGDNF in adult skeletal muscle. We have demon-strated immunocytochemically and utilizing ELISA that GDNF protein is indeed present in measurableamounts in adult rat skeletal muscle. Using whole

FIGURE 1. Localization of GDNF protein in rat skeletal muscle from control animals . Left panel shows staining in the presence ofanti-GDNF primary antibody that appears to be localized to the membrane. (bar = 50 m). Right panel shows colocalization of GDNF andDi-I staining to the membrane of skeletal muscle cells (bar = 50 m).



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muscle homogenate, GDNF released from skeletalmuscle cannot be distinguished from GDNF releasedfrom adherent Schwann cells. However, the findingsfrom the L6 skeletal muscle cell line in culture 22 andof 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 neurotransmitterrelease. Mechanical activity, in the form of stretch,appears to be a positive stimulus for trophic factorproduction in vascular and bladder smooth musclecells. Cyclic and static stretch increase productionand release of nerve growth factor in both bladderand vascular smooth muscle cells of rats 8 by activa-tion of protein kinase C signaling pathways. 19 Me-chanical stimulation increases the expression of ace-

tylcholinesterase in cultured myotubes. 13 Nifedipinetreatment partially blocks the effects of stretch onacetylcholinesterase expression, indicating that stretch induces calcium influx through voltage-gatedcalcium channels. 13 Thus, increased mechanical ac-tivity may be responsible for some of the increasedGDNF 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 modulateneurotrophic factor expression comes from studiesin vascular and bladder smooth muscle cells in cul-ture. In these studies, neurotransmitters from sym-pathetic neurons alter production of nerve growthfactor 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. Oppenheimet al.17 demonstrated that blockade of nicotinic ace-tylcholine receptors on skeletal muscle enhancedthe ability of skeletal muscle to provide trophic sup-port to innervating motor neurons. Direct evidencethat neurotransmitters exert a negative influence onGDNF expression in skeletal muscle has been ob-tained from studies demonstrating that denervatedskeletal muscle contains elevated levels of GDNFmRNA.14 Cell culture data from this laboratory 22 in-dicate that acetylcholine inhibits GDNF secretionfrom L6 skeletal muscle cells.

Hindlimb unloading leads to a decrease in neu-romuscular junction complexity 3,4,11,20 and we hy-pothesized that this may be due to a decrease introphic factor expression. Studies have shown that overexpression of GDNF leads to hyperinnervationof neuromuscular junctions. 15 It is possible that adecrease in GDNF expression leads to a decrease ininnervation. In our study, soleus and gastrocnemiusshowed a decrease in GDNF production followinghindlimb unloading. The increase in GDNF seen inthe pectoralis major may be due to an increased loadon the muscle from the altered body positionachieved with hindlimb unloading.

Activity-dependent regulation of trophic factorshas far-reaching implications. If trophic factors areregulated in an activity-dependent manner, thenconditions such as sedentary lifestyle, immobiliza-tion due to injury or illness, age-related decreases inactivity, hindlimb suspension, neuromuscular injury or illness, and exposure to microgravity may lead todramatic changes in trophic factor expression and

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 representcontrol and black bars represent walk-training. (B) Soleus andgastrocnemius showed a significant decrease in GDNF contentas compared to matched muscle from sedentary controls,whereas pectoralis major showed a significant increase in GDNFas compared to sedentary control. White bars represent controland 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 0.05).



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subsequent neuromuscular junction remodeling.Trophic factor expression may play a prominent rolein mediating these changes.This work was supported by NIH grant 1 R15 HL60240-01, TheFaculty 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 theIntegrative Biology of Exercise Conference, Portland, Maine, Sep-tember 2000. The authors acknowledge the advice and assistanceof Drs. Christine Byrd, John Jellies, and William Jackson, WesternMichigan 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 intissue processing, and Drs. Rob Eversole and John Stout in theBiological Imaging Center for assistance with confocal imaging.

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