HOMONKO, Darlene; TheRIAULT, Elizabeth 2000 - Downhill Running Preferentially Increases CGRP in Fast...

7/29/2019 HOMONKO, Darlene; TheRIAULT, Elizabeth 2000 - Downhill Running Preferentially Increases CGRP in Fast Glycolyti

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Downhill running preferentially increases CGRP in fastglycolytic 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 fastglycolytic muscle fibers. J Appl Physiol 89: 19281936,2000.Calcitonin gene-related peptide (CGRP) is present insome spinal cord motoneurons and at neuromuscular junc-tions in skeletal muscle. We previously reported increasednumbers of CGRP-positive (CGRP) motoneurons supplyinghindlimb extensors after downhill exercise (Homonko DAand Theriault E, Inter J Sport Med 18: 17, 1997). Thepresent study identifies the responding population with re-spect to muscle and motoneuron pool and correlates changes

in CGRP with muscle fiber type-identified end plates. Twenty-seven rats were divided into the following groups: control and72 h and 2 wk postexercise. FluoroGold was injected into thesoleus, lateral gastrocnemius, and the proximal (mixed fibertype) or distal (fast-twitch glycolytic) regions of the medialgastrocnemius (MG). Untrained animals ran downhill on atreadmill for 30 min. The number of FluoroGold/CGRPmotoneurons within proximal and distal MG increased by72 h postexercise (P 0.05). No significant changes wereobserved in soleus or lateral gastrocnemius motoneuronspostexercise. The number of-bungarotoxin/CGRP motorend plates in the MG increased exclusively at fast-twitchglycolytic muscle fibers 72 h and 2 wk postexercise (P 0.05).One interpretation of these results is that unaccustomedexercise preferentially activates fast-twitch glycolytic muscle

fibers in the MG.

neuropeptides; neuromuscular plasticity; activity; rat

IMMUNOCYTOCHEMICAL (23, 35) and in situ hybridization(6) studies in control animals have shown that mo-toneurons supplying fast-twitch muscles (e.g., extensordigitorum longus) show higher levels of calcitoningene-related peptide (CGRP) staining than do mo-toneurons innervating muscles of slow-twitch fibertype [e.g., soleus (Sol)]. A similar pattern of CGRPexpression is observed in the muscle, with CGRP foundpredominantly at the motor end plates of fast-twitch

muscle fibers (23, 24). However, none of these studieshas correlated CGRP expression patterns in identified(i.e., retrogradely labeled) motoneurons with motorend plates identified according to muscle fiber type.

Whereas the role of CGRP in the normal adult motorsystem is not entirely clear, the present framework ofevidence suggests that it is associated with presynaptic

sprouting and postsynaptic structural changes at theneuromuscular junction (21, 22, 33, 39, 45). Any formof experimental intervention that disrupts the connection between the motor nerve and the neuromuscularjunction, either surgically (3, 36) or pharmacologically(39, 45), results in an upregulation of CGRP peptideand/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 inthe 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 previouslydemonstrated that CGRP expression in rat hindlimbmotoneurons increased after an acute bout of downhilrunning exercise in sedentary animals (27). The resultsshowed that CGRP expression remained elevated overa 2-wk period, returning to baseline by 4 wk, in mo-toneurons of the knee extensors (triceps surae; e.g.muscles performing mostly lengthening contractionswhile loading) but not in the knee flexors (anteriocrural; e.g., muscles performing mostly shortening contractions).

We now identify the responding motoneurons, theirfiber type association, and the time course of change inCGRP expression after unaccustomed downhill exercise. Intramuscular injections of FluoroGold were usedto retrogradely identify motoneurons supplying theSol, lateral gastrocnemius (LG), and the proxima(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 theMG as the muscle within the triceps surae with asignificant increase in the numbers of CGRP-positive(CGRP) motoneurons after exercise. Interestingly, inthe MG muscle, there was a significant elevation in

CGRP levels at fast-twitch glycolytic (FG) motor endplates exclusively.

MATERIALS AND METHODS

Retrograde labeling of motoneurons. To identify the responding population(s) of motoneurons, we used a total of 34female Wistar rats (250275 g) for this study. All animals

Address for reprint requests and other correspondence: E. Theri-ault, Dean, School of Science and Technology, Sheridan College, 7899McLaughlin Road, Brampton, Ontario, Canada L6V 1G6 (E-mail:[email protected]).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

J Appl Physiol89: 19281936, 2000.

8750-7587/00 $5.00 Copyright 2000 the American Physiological Society http://www.jap.or1928


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with the exception of the animals used in the glycogen deple-tion study, were given intramuscular injections of 4%FluoroGold (Fluorochrome, Inglewood, CA). Identification ofthe three-dimensional topographic locations of the Sol, LG,and MG motor nuclei in the spinal cord was completed in aseries of retrograde labeling experiments, which have beendescribed previously (27). Briefly, FluoroGold (10 l) wasinjected into the belly of the left Sol muscle of 11 animals andinto the right LG muscle (15 l; belly portion) of 9 animals.

The pMG contains a mixture of fiber types [10% slow-twitchoxidative (SO), 10% fast-twitch oxidative glycolytic (FOG),35% FG; cf. Ref. 10], whereas the dMG is composed of FGmuscle fibers (80% FG; Ref. 10; Fig. 1). Therefore, in 14animals, the proximal-medial region of the MG (pMG) of theleft leg and the distal-medial region of the MG (dMG) of theright were injected with 15 l of FluoroGold (Fig. 1). A singleinjection of tracer was delivered into each muscle with theuse of an adapted 100-l Hamilton syringe (Fisher, Missis-sauga, ON). PE-20 Silastic tubing was placed onto the sy-ringe needle with the other end of the tubing supporting a30G1/2-gauge needle (Baxter Canlab, Mississauga, ON) at-tached to a micromanipulator. Care was taken during theseinjections to prevent leakage of tracer into other muscles byusing a localized microsurgical approach and by using petro-

leum jelly and tiny gauze pads to isolate each muscle (27).The injection site was then sealed with a drop of cyanoacry-late glue (Baxter Canlab). Three days after the muscle injec-tions, animals were exercised. All experimental procedureswere completed according to the Canadian Council on Ani-mal Care Guidelines on the Use of Animals in Research, withethics approval granted by the Toronto Hospital Animal CareCommittee.

Exercise protocol and tissue processing. Animals were ran-domly placed into three groups: control (nonexercise) and

72 h and 2 wk postexercise. These time points were selectedbased on the results of our previous study (27). Untrained(i.e., sedentary) animals ran continuously on a motor-driventreadmill at a speed of 12 m/min for one 30-min period on a20 slope. After exercise, runners from each group werereturned to their cages where they were given food and waterad libitum.

At the time of death, animals were administered an overdose of pentobarbital sodium (1.0 ml; 60 mg/ml). Befor

fixation perfusion and under anesthesia, the MG was quicklyexcised, and the pMG and dMG were separated, sliced into1-mm-thick cross sections, mounted in optimum cutting temperature embedding media on cardboard, and then snapfrozen in a 2-methylbutane (Baxter Canlab) bath immersedin liquid nitrogen. The spinal cord was harvested aftetranscardial perfusion with 500 ml of 4% paraformaldehyde(pH 7.357.45; BDH, Oakville, ON). The lumbar region of thespinal cord (L

3L

5) was removed intact, postfixed in 4%

paraformaldehyde for 18 h, and cryoprotected overnight in20% sucrose in 0.1 M phosphate buffer, pH 7.2 (BDH). Tissues were frozen in 2-methylbutane at 80C.

Immunocytochemistry of the spinal cord. The immunocytochemical methodologies have been previously reported (27and are briefly described here. Frozen spinal cord lumbar

regions (L3L4) were serially sectioned at 10 m. In anattempt to reduce interexperimental variability, spinal cordcross sections from each experimental group were placed onthe same slide (i.e., control, 72 h, 2 wk). Sections were thenwashed in 0.1 M PBS, pH 7.1, blocked with 10% normal goatserum (Gibco, Baxter Canlab), and then incubated overnightat 4C with a polyclonal antibody to CGRP [rabbit anti-CGRP(Rat, 137); 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 incubationwith avidin-conjugated Texas red fluorophore (Vector). Slideswere coverslipped with Mowiol.

Acetylcholinesterase histochemistry and immunocytochemistry of muscle tissue. Motor end plates were identified by acetyl

cholinesterase (AChE) histochemistry to determine the innervation pattern and location of neuromuscular junctions in thetwo regions of MG. Frozen, unfixed muscle tissue was seriallysectioned at 12m. Three series of samples were collected every200 m and placed directly onto slides. The first series wasprocessed 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), crosssections on slides were incubated in 20% sodium sulfate (BDHfor 3 min followed by a wash in deionized water, incubated inreaction solution [pH 7.2; 5-bromoindoxyl acetate, ethanolK3Fe(CN)6, K4Fe(CN)63H2O, TrisHCl, Tris base, and CaCl2Sigma Chemical] for 15 min, washed in deionized waterquickly dipped in eosin (Sigma Chemical), and then defatted

and coverslipped with Entellan (BDH).For immunocytochemistry, frozen serial sections from un

fixed muscle tissue were collected on slides as describedabove. Slides were washed in 0.1 M PBS, pH 7.1, and immersed in 4% paraformaldehyde fixative (pH 7.4; BDH) for30 min. Slides were then washed in 0.1 M PBS, pH 7.1blocked with 10% normal goat serum (Gibco, Baxter Canlab)and incubated overnight at 4C with the CGRP antisera at a1:1,000 dilution. Tissue sections were processed by using theavidin-biotin complex kit with a goat anti-rabbit secondaryantibody (Vectastain, Vector) followed by incubation withavidin-conjugated Texas red (Vector). After a washing in 0.1M PBS, the tissues were incubated overnight in fluorescein

Fig. 1. Schematic representation of the rat hindlimbs [left and rightmedial gastrocnemius (MG)] as viewed from the dorsal surface.Speckled areas indicate FluoroGold injection sites into the proximalregion of the left MG (pMG) (speckled region overlaying light grayarea illustrating the mixed-fiber-type composition of the region) andthe distal region of the right MG (dMG) [speckled region overlayingwhite area illustrating exclusive fast-twitch glycolytic (FG) compo-sition]. Muscle fiber type is indicated by the white regions thatcontain FG fibers, and the light gray indicates mixed-fiber-typeregion containing fast-twitch glycolytic, fast-twitch oxidative glyco-lytic, and slow-oxidative (FG, FOG, SO).

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conjugated (FITC) -bungarotoxin (-BuTx) (FITC--BuTx;1:1,500; Sigma Chemical). Slides were then washed andcoverslipped with Mowiol as described above.

Glycogen study: tissue sampling and analysis. To evaluatewhether the exercise protocol may have preferentially acti-

vated the pMG or the dMG, we examined the pattern ofglycogen depletion in both regions of the muscle after down-hill running. Two groups of animals were divided into control

(n 4) and downhill runners (n 4). The running groupcompleted the exercise protocol and was killed 20 min later.The MG (2 per animal) were quickly dissected out and snapfrozen as described above. Frozen control and exercise pMGand dMG muscle were serially sectioned, placed on the sameslide, histochemically analyzed for glycogen content usingthe periodic acid Schiff (PAS) stain (14), and placed on sep-arate slides for myosin ATPase.

Myofibrillar ATPase histochemistry. Muscle fibers wereidentified and classified as SO, FOG, and FG from the

ATPase stain (8). Briefly, frozen muscle sections mounted onslides were placed in coplin jars containing acid medium(NaC2H3O2 and KCl, pH 4.6; BDH) for 4 min, washed in basicmedium (C

2H5

NO2

, CaCl2

, NaCl, and NaOH, pH 9.4; BDH)for 30 s, and placed in incubation medium (basic medium

ATP, pH 9.4, 30 min, 37C; Sigma Chemical), followed by aseries of washes in CaCl2 (BDH), rinses in CoCl2 (Fisher),and rinses in distilled H2O, ending with a 1-min incubationin 20% (NH

4)2

(Fisher). Slides were then cleared and cover-slipped with Entellan (BDH).

Fiber populations were quantified by counting and categorizing 100120 fibers in pMG and 500600 fibers in dMGfrom each sample in randomly chosen fascicles distributedthroughout the entire cross-sectional area of the sample (seeTable 1). These values represent 10% of the muscle fibers inthe proximal compartment (300/2,900) and 12.5% of the totanumber of muscle fibers in the distal compartment (5004,000) (42). With the use of bright-field settings with a LeicaDM/RB microscope, staining intensities for glycogen in the

fibers were automatically analyzed by relative optical densitymeasurement using the MCID image analysis software (Imaging Research, St. Catherines, ON). Baseline gray-scal

values were initially standardized by arbitrarily selectingone shade of gray from a strip of film on a slide that had arange of gray (black to white), which was subsequentlymarked and identified as the reference point from which alfurther illumination settings were to be established. Thiprocedure allowed us to establish a criterion illuminationlevel throughout the entire blinded analysis that was consistent and reproducible and that permitted the unbiasedmeasurement and comparisons between groups of gray-scale

values generated from muscle fibers of varying PAS stainingintensities.

Quantitation of CGRP motoneurons and motor endplates. The quantitation of CGRP motoneurons and motorend plates was completed by using a Leica DM/RB fluorescence microscope. Counts of motoneurons staining positivelyfor both CGRP and FluoroGold were obtained by selectingonly those somata with observable nuclei; profiles not containing a nucleus or nucleolus were not quantitated (cf., Ref46). The motor nuclei of the Sol, LG, pMG, and dMG weretopographically located in the medial-ventral region of thespinal cord, beginning at the L4 ventral root entry zone andextending rostrally 2,000 m (27). FluoroGold-labeled motoneurons exhibited cell bodies and dendrites filled withgranules of bright gold fluorescence (Fig. 2C). CGRP-Texasred immunofluorescence was characterized as cytoplasmiand punctate, with the fluorescent granules preferentially

located in the soma and in the proximal parts of the majordendrites (Fig. 2D). All motoneurons with FluoroGold labeling were counted, and their CGRP or CGRP-negative(CGRP) status was determined. MG muscles injected withFluorogold were analyzed for Fluorogold content by examin

Table 1. Number of muscle fibers analyzed per fibertype in the proximal and distal regions of MGfor glycogen depletion studies

Region of MGFiberType

No. ofAnimals

No. of FibersAnalyzed per Animal

Average No. ofFibers per Cross

Section

Proximal ST 8 100 2,900FOG 8 100 2,900

FG 8 100 2,900Distal FG 8 500 4,000

MG, medial gastrocnemius; ST, slow-twitch; FOG, fast-twitch ox-idative glycolytic; FG, fast-twitch glycolytic.

Fig. 2. Photomicrographs of double-la-beled pMG motoneurons 72 h afterdownhill exercise. A: FluoroGold-filled

motoneuron in rat lumbar (L4) spinalcord retrogradely labeled from thepMG that is calcitonin gene-relatedpeptide negative (CGRP) (B). Arrowindicates the location of the CGRPmotoneuron. C: FluoroGold-filled mo-toneuron retrogradely labeled from thepMG that is CGRP positive (CGRP)(D). Calibration bars, 30 m.

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ing the center of the muscle at the midline to determinewhether 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: con-trol and 72 h and 2 wk postexercise (Fig. 3). Motor end plateswere identified in transverse section by AChE staining.Based on the results from a separate series of experiments inwhich we evaluated AChE-stained longitudinal muscle sec-tions taken from the belly of the MG (see MATERIALS ANDMETHODS), we were able to determine that the average lengthof a MG motor end plate was200 m. This method permit-ted us to establish a sampling distance within the muscleregion that would not result in duplicate counts of motor endplates that were evaluated for -BuTx. In subsequent adja-cent sections, motor end plates were identified with -BuTxfluorescence by using a blue filter (525 nm; 10 PL FLUO-TAR objective) for fluorescein detection, followed by a greenfilter (625 nm; 100/1.25 N Plan Oil) for detecting Texas Red

immunofluorescence, to colocalize the CGRP signal. CGRPimmunofluorescence was clearly detected as a punctatestaining pattern visible in regions of the junctional folds (seeFig. 6D), overlapping the -BuTx labeling that filled the

junctional area (see Fig. 6C). Approximately 140 end plateswere quantitated per muscle sample (Table 2). Slides withCGRP motor end plates were then compared with adjacentserial sections stained for myosin ATPase to determine thefiber type of the identified neuromuscular junction.

Importantly, all tissue analyses were done blinded to theexperimental condition throughout all the procedures de-scribed in this study. The identity of the experimental groupswas subsequently decoded after completion of data collectionto permit statistical analysis. Statistical significance wasdetermined by Students t-test, ANOVA, and, where neces-

sary, the Kruskal Wallis test for nonparametric distributions(SigmaStat version 1.0, Jandel Scientific). Data are pre-

sented as means SE. Differences were considered to besignificant at P 0.05.

RESULTS

CGRP response in retrogradely labeled motoneuronsFluoroGold-labeled motoneurons within the motor nuclei of the Sol, LG, pMG, and dMG were readily iden-tified under ultraviolet light (Fig. 2). Double-labeledTexas red-conjugated CGRP cells had a punctate, redstaining pattern (Fig. 2D), making this easily discernible from the more homogeneous, finely granularwhite FluoroGold signal (Fig. 2C). Motoneurons thatwere labeled with FluoroGold and identified aCGRP were stunningly clear in their lack of CGRP(Fig. 2B). Numbers of double-labeled motoneurons inthe identified Sol motor nucleus did not change afterdownhill exercise over the experimental time period

(Fig. 3A). A similar observation was true for the double-labeled motoneurons of the LG motor nucleus (Fig3B).

In both regions of the MG, however, downhill exer-cise resulted in a significant increase in the number ofdouble-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 ofCGRP 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 timeand confirming our previous results (27).

Glycogen depletion experiments. Based on our observation of the enhanced expression of CGRP in MG

Fig. 3. Profile of double-labeled FluoroGold and CGRP soleus (Sol), lateral gastrocnemius (LG), and MG motoneuron72 h and 2 wk after downhill exercis(time 0 control). A: percentage oCGRP/FluoroGold-labeled Sol motoneurons. B: percentage of CGRP/Fluoro

Gold-labeled LG motoneurons. C: percentage of double-labeled CGRP/FluoroGoldmotoneurons in the MG motor pool (pMGand dMG) in lumbar section L4 of the raspinal cord. *P 0.05 (ANOVA; KruskaWallis test). Double-labeled cells from theSol and LG were not identified in the samecross sections.

Table 2. Total number of motor end plates quantified in the proximal and distal regionsof the MG in the control and experimental groups

Region of MGNo. of

Animals

Total No. ofFITC--BuTx

End Plates

Total No. ofCGRP Motor

End Plates

Fiber Type of CGRP End Plates

ST FOG FG

Proximal 12 1,700 150 1 1 148Distal 12 1,700 400 0 0 400

FITC--BuTx, fluorescein-conjugated -bungarotoxin; CGRP, calcitonin gene-related peptide positive.



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motoneurons, it was of interest to ascertain whetherboth pMG and dMG were equally recruited by thedownhill running protocol. Differences in the activitypatterns of glycogen depletion would permit us to qual-itatively interpret the physiological state of the muscle.Therefore, the glycogen content of both pMG and dMGwas determined by PAS histochemistry (Fig. 4). Rela-tive optical density measurements (Fig. 5) showed thatboth 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 glyco-gen stores after this eccentric exercise paradigm. Whileindicating that both pMG and dMG were active, theseresults did not allow us to quantitate the relativeamounts of glycogen breakdown in the different musclefiber types.

CGRP response at motor end plates in the pMG anddMG after eccentric exercise. When viewed in trans-verse section, the motor end plates in the MG wereeasily identified by FITC--BuTx staining as theyfollowed a visible pattern throughout the belly of themuscle. The junctional folds of the end plate region

were consistently and clearly labeled with brighgreen FITC--BuTx (Fig. 6). Texas red CGRP immunofluorescence staining was similar to that observedin the motoneuron, presenting with an irregularpunctate distribution that overlapped portions oFITC--BuTx immunoreactivity in the junctionafolds (Fig. 6C). This distinction in staining patternmade it easy to distinguish between CGRP andCGRP end plates and to identify end plates thatwere not CGRP as being affected by bleed throughof the -BuTx signal. Interestingly, CGRP endplates were observed most often in regions in whichclusters of neuromuscular junctions were found, although not all neuromuscular junctions within acluster were CGRP (Fig. 6D), with the majoritybeing CGRP (Fig. 6B).

Elevated numbers of immunofluorescent CGRPmotor end plates were observed in both pMG and dMGafter downhill exercise. In pMG, a mixed fiber typeregion, a significant increase in the numbers of CGRPimmunoreactive neuromuscular junctions was observed at 72 h postexercise (10%; P 0.03) and re

Fig. 4. Photomicrographs of myosinATPase and periodic acid Schiff (PAS)-

stained muscle fibers from the proxi-mal region of the MG describing theglycogen depletion pattern before andafter downhill exercise. Frozen serialcross sections of the MG stained formyosin ATPase (A and C) and PAS (Band D) in the control condition (B) and20 min after downhill running exercise(A, C, and D). ST, slow-twitch fibers.Calibration bars, 50 m.

Fig. 5. Glycogen depletion profiles of ST, FOG, and FGmuscle fiber types in the pMG (A) and dMG (B) 20 minpostexercise, as indicated by the relative optical density(ROD) measurement. *Significant difference comparedwith control, P 0.001 (t-test).



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mained significantly elevated 2 wk later (9.9%, P 0.03; Fig. 7A). In the dMG, composed entirely of FGfibers, a 25% increase in the number of CGRP motorend plates compared with control was observed at 72 hpostexercise (P 0.002) (Fig. 7B). Interestingly, thispercentage continued to be significantly elevated in thedMG compared with control, increasing to 33% by 2 wkafter exercise.

CGRP immunoreactivity and muscle fiber type. Anal-yses of serial cross sections of pMG and dMG stainedhistochemically for myosin ATPase content showedthat CGRP motor end plates colocalized almost ex-clusively to FG muscle fibers (Fig. 8). Out of3,000end plates examined in this study, CGRP end plates

at FOG muscle fibers and SO fibers were rarely ob-served (Table 2).

DISCUSSION

We previously reported (27) that one 30-min boutof downhill running resulted in increased numbers ofCGRP motoneurons in hindlimb extensor but notflexor motor nuclei. Those results were the first todemonstrate increased CGRP levels as a result of

physiological neuromuscular activity rather than alack of activity, i.e., as induced by surgical or pharmacological paralysis or by hormone deprivationOur present report demonstrates that CGRP expression was exclusively elevated in MG motoneuronsand that, within the muscle, this response was specifically localized to motor end plates on FG musclefibers.

The increased CGRP levels in MG motoneurons, andsubsequently at motor end plates in the muscle, couldbe due to a variety of factors. For example, the exerciseregime may result in frank histopathological damageto the muscle, thereby initiating repair and regenerative mechanisms that would require new end plates on

de novo myofibrils. Alternately, unaccustomed exercisemay induce growth-related morphological alterationswithin the individual myofibrils and subsequently attheir neuromuscular junctions, leading to growth of themotor end plates. A more subtle process could be thatthe particular demands of downhill exercise may dif-ferentially affect muscle fibers within a motor unitleading to remodeling events at the affected neuromuscular junctions.

Fig. 6. Photomicrographs of the motoend plates in the pMG double-labeledwith FITC-conjugated -bungarotoxin(-BuTx) and Texas red (TR)-conjugated CGRP 2 wk after 1 bout of downhill exercise. FITC--BuTx identifiedmotor end plate (A) colocalized with

TR-CGRP MG motor end plate (B)Nonspecific bleed through of FITC signal completely overlaps the same areaand outlines the morphology of thneuromuscular junction specificallystained for -BuTx in A. C: FITC-BuTx identified pMG motor end plateSpecific TR-CGRP immunoreactivitycolocalized with -BuTx (D) overlapwith, but is not identical to, neuromuscular junction morphology (C). Calibration bars, 10 m.

Fig. 7. Increase in the percentage of CGRPmotor end plates in the pMG and dMG aftedownhill exercise.A: increased numbers of FITC-BuTx and TR-CGRP motor end plates in thepMG 72 h and 2 wk postexercise compared withcontrol (time 0) (*P 0.03, ANOVA; KruskaWallis test). B: increased numbers of FITC-BuTx and TR-CGRP motor end plates in thedMG 72 h and 2 wk postexercise compared withcontrol (*P 0.002, ANOVA; Kruskal Wallitest).



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Downhill running produces changes in MG motoneu-rons in the absence of muscle damage. Although adetailed biomechanical analysis of downhill running inrats has not yet appeared in the literature, in thisdownhill running model the ankle flexors are assumedto be performing concentric exercise (shortening whileloaded), whereas the ankle extensors are similarlyassumed to undergo eccentric contractions (lengthen-

ing while loaded). Previous studies that have usedchronic and/or extended bouts of downhill runningactivity in rats report Sol muscle damage with indexesof pathology clearly observed 35 days postexercise (1,32). Although Smith et al. (42, 43) detected the forma-tion of new muscle satellite cells in the rat Sol afterdownhill running, followed by significant increases indevelopmental myosin isoforms and the appearance ofnew myofibrils (42), neither the MG nor the LG showedany significant trends, thus making the need for denovo end plates in the MG appear unlikely. Becausethere is no significant histopathology or inflammationin the MG, compared with the Sol, after either chronic

or acute eccentric exercise protocols (27, 42), it seemsunlikely that the changes in CGRP levels we reportedcan be attributed to myofibrillar damage and repairprocesses. In addition, the relative lack of baselineCGRP immunoreactivity in Sol motoneurons and theabsence of change in CGRP levels in this slow-twitchmuscle after exercise (despite the histopathologicaldata) argue against the idea that CGRP may be relatedto extensively used or easily recruited (e.g., Sol) mo-toneurons (4). Our results, however, do support theview that motoneurons innervating FG fibers [e.g.,larger and faster motor units (5, 25)] contain moreCGRP.

CGRP and sprouting at the neuromuscular junction.

Sprouting of the motor nerve terminal in the adult hasbeen correlated with changes in CGRP peptide andmRNA expression in motoneurons after either a signif-icant injury to the motor nerve or a substantial phar-macological interruption of neuromuscular connectiv-ity (3, 36, 39, 45). In our study, however, no damage isincurred by the MG motor nerve, and overt muscledamage is absent. Furthermore, it is unlikely that theintramuscular injection of FluoroGold produced anerve injury that initiated a sprouting response and anupregulation in motoneuronal CGRP expression. Datafrom our first study demonstrated elevated numbers of

CGRP motoneurons after eccentric exercise; thesemuscles were not injected with FluoroGold (27). Otherinvestigators have also reported no changes in CGRPimmunoreactivity and -CGRP mRNA in the bulbocavernosus muscle in sham and vehicle-treated groupswith 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 seemsunlikely to us that a single 30-min bout of downhilrunning would elicit a measurable sprouting responsewhether chronic exercise elicits a sustained CGRP re-sponse and growth or sprouting at the neuromuscularjunction remains to be examined.

A neuromodulatory role for CGRP at the motor endplate after exercise. Perhaps the elevated expression oCGRP in MG motoneurons and their end plates on FGmuscle fibers may be correlated with more subtle re-modeling events at the neuromuscular junction. Apartfrom its fast neurotransmitter-like actions on thenicotinic acetylcholine receptor (AChR), CGRP also

exerts longer lasting trophic functions at the neuro-muscular junction, mediated by specific CGRP receptors localized to the postsynaptic membrane (reviewedin Ref. 4). In cultured chick myotubes, CGRP has beenshown to upregulate the appearance, number, andinsertion of nicotinic AChRs into the muscle membrane(22, 31). Other in vitro studies have shown that CGRPincreases the mRNA coding for the stabilizing-subunit in the AChR complex (21, 22, 33). Whether CGRPelicits 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 AChEenzymes. One of the subunits, the asymmetric form G

4

located at the neuromuscular junction, is involved inthe fast clearance of ACh at junctional receptors (20and is known to be upregulated by chronic treadmilexercise (26, 28) with the greatest response observed infast-twitch muscles (18, 26). Mouse myotube culturestreated with CGRP displayed a 2.5-fold increase bothin (G

4) AChE and in AChR -subunit mRNA, further

implicating CGRP as a trophic factor regulating thegene expression of integral postsynaptic molecules atthe intact adult neuromuscular junction (7). Intramuscular injection of exogenous CGRP, however, has beenreported to reverse the exercise-induced increase in G

4

Fig. 8. CGRP immunoreactivity is present at motor end plates of type IIB muscle fibers in the MG after downhillexercise. A: muscle cross section from the pMG identified as a type IIB muscle fiber by myofibrillar ATPasehistochemistry. 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).



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in rat gracilis muscle (19). Whether CGRP regulates G4

AChE and AChR subunit expression at the neuromus-cular junction after physiological exercise is notknown.

Elevated CGRP expression as a function of motorunit recruitment. Both the LG and MG motor nucleihave equivalent baseline levels of CGRP: 60% of mo-toneurons are CGRP in the sedentary animal. The

fact that there are no changes in CGRP expression inLG motor nuclei after downhill running may suggest asubtle effect of this exercise paradigm on the MG thatis not elicited in the LG. There is evidence to suggestthat the LG is less active than the MG during locomo-tion (15, 44). Studies by Duysens et al. (15) describe thereduced activation of the LG in humans during walk-ing or running, whereas Smith and Carlson-Kuhta (44)observed a similar lack of LG activation in felinesduring slope walking. The functional or biomechanicalimplications of our findings need to be more rigorouslyinvestigated using electrophysiological techniques.

The rat MG is a highly compartmentalized musclewith distinct fiber type distributions. Morphologically,

the pMG, innervated by the proximal and lateralbranches of the MG nerve, is a mixture of slow- andfast-twitch fibers (SO, FOG, FG), whereas the distalregion innervated by the distal branch of the MG nerveis exclusively FG (9, 47). These compartments appearto be preferentially activated in the performance ofdifferent motor tasks (11, 47). There is growing evi-dence to suggest that the complex morphological struc-ture (i.e., compartmentalization or regionalization) of amuscle influences muscle fiber properties and intra-muscular activity (reviewed in Ref. 29). Our PAS stain-ing describes glycogenolysis in all fiber types of bothMG regions, suggesting that the expression of CGRP in

FG fibers reflects a difference in the activity responseof this fiber type within the MG after downhill running.Perhaps the unaccustomed activity in our model pref-erentially perturbs neuromuscular junctions in FG fi-bers because of their higher susceptibility to transmis-sion failure (41). This altered use may then initiatemorphological adaptation and repair of the affectedneuromuscular junctions for which CGRP is required(39).

Based on human studies, motor control strategies foreccentric work (e.g., downhill running) are suggested todiffer from those of concentric work (e.g., predomi-nantly uphill running) in that fast- rather than slow-twitch motor units are recruited first (17, 30). To date,

motor unit recruitment strategies for downhill runninghave not yet been addressed in animal models. Todetermine whether enhanced CGRP expression at FGmotor end plates is associated with selective recruit-ment of fast-fatiguable or fast-fatigue-resistant motorunits, intracellular recordings from identified mo-toneurons (cf., Refs. 10, 40) will need to be done.

In conclusion, we have shown that, after downhillrunning in the rat, CGRP expression is elevated in MGmotoneurons and at MG motor end plates on FG mus-cle fibers. Our results may indicate a preferential re-sponse of FG fibers after unaccustomed exercise, re-

sulting in synaptic reorganization. This model providesa novel system in which to further investigate whetherphysiological exercise, specifically downhill exercisepreferentially recruits FG motor units and whetherexercise-induced increases in CGRP expression play arole in the remodeling of the postsynaptic junction inthe intact, adult neuromuscular system.

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