changes in the levels of calcitonin · thesis abstract calcitonin gene-reiated peptide (cgrp) is a...
TRANSCRIPT
DOWNHILL EXERCISE ELICITS DIF'FERENTIAL
CHANGES IN THE LEVELS OF CALCITONIN
GENE-RELATED PEPTIDE IN THE INTACT
ADULT RAT NEUROMUSCULAR SYSTEM
Darlene Alice Homonko
A thesis submitted in conformity with the requirernents for the degree of
Doctor of Philosophy in the Graduate Department of the Institute of Medical Science,
University of Toronto
O Copyright by Darlene A Homonko, 1999
National Library Bibliothèque nationale du Canada
Acquisitions and Acquisitions et Bibliographie Sewices services bibliographiques
395 Wellington Street 395. rue Welfington Ottawa ON K1A O N 4 OtoiwON K1A ON4 Canada Canada
The author has granted a non- L'auteur a accordé une licence non exclusive licence aIlowing the exclusive permettant à la National Libraq of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seU reproduire, prêter, distribuer ou copies of this thesis in microfonq vendre des copies de cette thèse sous paper or electronic formats. la fome de microfichelfilm, de
reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.
This work is dedicated to the memory of my grandmother,
Mrs. Eugenia Tkacz
(1907-1997)
Thesis Abstract
Calcitonin gene-reiated peptide (CGRP) is a 37 amino acid peptide synthesised in the
nervous system. Although the functional role of CGRP in the marnmalian motor system is
unclear, variable amounts are present in motoneurons, and in motor nerve terminals on
skeletal muscle fibres. Experimental pemirbations affecthg neuromuscular connectivity alter
CGRP expression in motoneurons. Eccentric exercise is known to produce muscle damage.
The studies in this thesis were designed to investigate whether exercise uivolvhg eccentnc
contractions elicits changes in CGRP expression in the adult neuromuscular system.
In the first study, we demonstrated that one 30 minute bout of downhill running
resulted in increased numbers of CGRP-positive triceps surae motoneurons within 48 hours
post-exercise, with values returning to control levels by 4 weeks. In contrast, the numbers of
CGRP-positive anterior crural motoneurons remained unchanged following downhill exercise.
These results provided the first evidence in the literature that muscle exercise activity
increases CGRP expression preferentially in motoneurons innervating extensor muscles
undergoing eccentric contractions.
In the second study, retrograde labelling of the triceps surae (TS) muscle group,
combined with co-localisation of CGRP immunoreactivity, identzed a distinct response in
CGRP expression in rnotoneurons i~ervating the medial gastrocnemius (MG) muscle.
Elevated numbers of CGRP-positive motoneurons were observed by 72 hours post-exercise,
with values retuming to baseiine by 2 weeks. No changes in CGRP expression were observed
in the soleus or in the lateral gastrocnemius motoneurons over the experirnental tirne period.
The number of CGRP-positive motor nerve terminais in the MG increased significantly within
iii
the fkst 72 hours post-exercise and remained elevated at 2 weeks. CGRP imrnunoreactivity
was exclusively found at motor endplates of type IIB, f& giycolytic muscle fibres.
The eccentnc exercise protocol may evoke post-synaptic changes at affected
neuromuscular junctions that require stabilisation or remodehg to maintain or enhance
synaptic efficiency. Given the dserential recmitment of the distal and proximal regions of the
MG, Our results suggest that unique motor commands may be required in the execution of
eccentric contractions. The protocol we descnie would be suitable to test aitemate theories
of motor control and investigate the dynamic interactions of neuropeptides at the
neuromuscular junction.
Acknowledpents
The successfùl completion of this work involved the contribution of many people to
whom 1 wili always be gratefÙI.
1 would Iike to thank my supervisor, Elizabeth Thenault, PhD for providing me with
guidance, direction and moral support. Mostly, thank you for teaching me the value of process
while challenging me to always extend my goals and never stop asking "why" or "how".
1 would like to thank ail of the members of my Program Cornmittee. A special thanks to
Dr. R Inman for his invaluable assistance, loyal support and reliabilty. Warm thanks to Dr. M.
Plyley for his ardent support, timely discussions and good humour. 1 would like to thank Dr. H.
Atwood for many thoughtfùl insights about this work and for continuaiiy chailenging me to think
beyond the obvious. As well, thanks to Dr-K.W. Marshall for his encouragement and tutelage.
1 would like to thank Dr. Catherine Whiteside for her amazing spirit and ability to secure
resources. 1 would like to thank Josie Chapman for always having a smile and for guiding me and
aii my examiners through the snowstorm of '99.
1 wouid like to thank Steve Mortin-Toth and Ruth Ann Seerattan, and Linda Lee for their
excellent technical assisstance and troubleshooting skills over the years.
Finally, 1 would like to thank my wonderfil fnends and devoted f d y for their constant,
never-ending love, support, and encouragement.
CHAPTER 1. INTRODUCTION
A. Calcitonin Gene-ReIated Peptide (CGRP) in the Neuromuscular System
1.1. Role of CGRP in Developing Neuromuscular Systems
1 -2. Role of CGRP in the Adult Neuromuscufar System
1.3. CGRP Expression in Motoneurons and at Motor Nerve Terminals of Nonnal Animals
1.4. CGRP Expression in Motoneurons and at Motor Nerve Terminals Following Experimental Manipulations
1.4.1. Spinal Cord Transection, Axotomy and Models of Pharmacologicai Intervention
1 -4.2. Target Factor Regdation of CGRP Levels
1 S. Downhill Running, Eccentric Contractions, and Muscle Darnage
1.6. S prouting and the Neuromuscular Junction
1.6.1 Activity and the Neuromuscular Junction
1.6.2. Sprouting
1.6.3. Morphological and Functiond Characteristics of the Neuromuscular Junction
1.7. CGRP and Exercise
B. Statement of the Hypotheses
C W T E R II. MATERIALS AND METHODS
2.1. General Methods Cornmon to Al1 Experiments
2. 1.1 Exercise Protocol
Page
i
1
2
3
6
6
7
8
11
11
14
17
18
20
23
23
2.1.2. Tissue Processing
2.1.3. Retrograde Labehg of Motoneurons
2.1 -4. Immunocytochernistry
2.1.5. Data Acquisition and Analyses
2.1-6 Statistical Methods
3.1. Introduction
3 -2. Three-Dimensional Reconstruction o f Soleus and Tibialis Anterior Muscles in the Rat Following Downhill Exercise
3 -2.1. Introduction
3 -2.2. Methods
3 -2.3. Results and Discussion
3.3. Silicone Rubber Microangiography of Soleus and Tibialis Anterior Muscles in the Rat
3 -3 -2. Methods
3 -3 -3. Resuits and Discussion
C W T E R N.
Study T : "CALCITONIN GENE-RELATED PEPTIDE IS INCREASED
IN HINDLZMB MOTONEURONS AFTER EXERCISE."
4.1. Abstract
4.2. Introduction
4.3. Materials and Methods
4.3.1. Identification of Motoneuron Pools in Non-exercised Anirnals
4.3 -2. Experimental Design and Tissue Processing
4.3 -3. Muscle Histology
4.3.4. Immunocytochernistry of Spinal Cord Tissues
4.3 -5 Quantitation of CGRP-positive Motoneurons
4.4. Results
4.4.1. TS and AC Motoneuron Pools in Non-Exercised Anirnals
4.4.2. CGRP in TS and AC Motoneurons Mer Downhill Exercise
4.4.3 .Histoiogical Damage M e r Exercise
4.5. Discussion
4.5.1. Identification of Motor Nuclei
4.5.2. CGRP and Muscle Fibre Type
4.5 -3. Neuromuscular Plasticity
4.5.4. Muscle Damage and Unaccustomed Activity
4.6. Conclusion
4.7. Celi-Size Distribution of CGRP- positive Motoneurons Following Downhill Exercise
CHAPTER V.
Study 2: "ECCENTRIC EXERCISE PREFERENTIALLY INCREASES
CGRP IN MOTONEURONS INNERVATING FAST GLYCOLYTIC
MUSCLE FIBRES-"
5 -2. Introduction
5.3 Materials and Methods
5 -3.1. Experimental Protocol and Tissue Processing
5.3 -2. Immunocytochemistry of the Spinal Cord
5 -3 -3. Acetylcholinesterase Histochemistry and Imrnunocytochemistry of Muscle Tissue
5.3 S. Glycogen Study: Tissue Sarnpling and Analyses
5 -3 -6. Quantitation of CGRP-positive Motoneurons and Motor Endplates
5.4. Results
5.4.1. CGRP Response in SOL, LG, and pMG and dMG Motoneurons
5.4.2. Glycogen Depletion Experiments
5.4.3. CGRP Response at Motor Endplates in the pMG and dMG M e r Eccentric Exercise
5.4.4. CGRP Immunoreactivity and Muscle Fibre Type
5.5. Discussion
5.5.1. Downhill Running Produces Changes in MG Motoneurons in the Absence of Muscle Darnage
5 - 5 2 CGRP and Sprouting at the Neurornuscular Junction
5.5.3. A Neuromodulatory RoIe for CGRP at the Motor Endplate After Exercise
5.5.4. Motoneuronal and Motor Nerve Terminal CGRP Expression as a Function of Motor Unit Recruitment
5.6. Conclusions
C W T E R VI. Experiments Designed to Investigate the Role of CGRP at the Neuromuscular Junction
6.1 Acetylcholinesterase (G4) Activity in the Medial Gastrocnemius Muscle Foilowing Downhill Exercise.
6.1.1. Introduction
6.1.2. Materials and Methods
6.1.3. Results and Discussion
6.1.4, Conclusions
6.2. GDNF Expression at MG Neurornuscular Junctions Foilowing Downhill Exercise.
6.2.1 Introduction
6.2.2. Methods
6.2.3. Results and Discussion
6.2.4. Conclusions
CHAPTER W. GENERAL DISCUSSION
7.1. Discussion of the Functional Role of CGRP at the Neuromuscular Junction
7.1.1. A Re-Assessment of the Hypotheses
7.2. CGRP as an Effector Peptide in Type IIB Motor Units
7.2.1. Suggestions for Future Experiments
7.3. Muence of Motor Unit Recruitment on the Regulation of CGRP Acivity in FG Motor Units
7.3.1. Suggestions for Future Experiments
7.3.2. Experiments with Neurotrophic Factors
7.3.2.1. Experiments with Neurotrophins and CGRP
7.3 -2.2. Experiments with Muscle Growth/Tumover Proteins and CGRP
7.4. Limitations of Experiments in Chapters III, IV, and V.
7.4.1. Limitations of Experiments in Chapter IV: The Exercise Model.
7.4.2. Limitations of Experiments in Chapter V.
7.4.2.1. Injection of Tracer into the Two Regions of MG.
7.4.2.2. Muscle Damage as a Result of the Intramuscular Injection Technique.
7.4.2.3. Limitations of the Giycogen Depletion Studies
CHAPTER W. Sumniary and Conclusions
Bibliograp hy
Appendix 1
ACh
AChR
AChE
AChE(G4)
aBuTx
BoTx
BDNF
CGRE'
CGRP-ir
aCGRP
PCGRP
CGW-ir
CGRP+ve
CGRP-ve
CNS
DAB
dMG
DOMS
EDL
List of Abbreviations and Giossaw of Terms
anterior crural (muscle group responsible for flexing the ankle: tibialis
antenor, extensor digitorum longus)
acetylcholine
acetylcholine receptor
acetyicholinesterase
acetyichohesterase tetrameric globular subunit
alpha bungarotoxin
bo tulinum t oxin
brain-derived neurotrophic factor
caicitonin gene-related peptide
calcitonin gene-related peptide immunoreactivity
calcitonin gene-related peptide alpha subunit
calcitonin gene-related peptide beta subunit
calcitonin gene-related peptide immunoreactivity
calcitonin gene-related peptide positive
calcitonin gene-related peptide negative
central nervous system
diarninobenzadine
distal medial gastrocnemius
delayed onset muscle soreness
extensor digitorum longus
ECM
FF
FGF
FITC
FOG
FT
GDNF
H&E
LG
MG
MHC
MATPase
NADZ-TR
NMJ
PAS
PGE2
extracellular matrk
fast fatiguable (based on electrophysiological twitch criteria and
c haracteristics)
fast glycolytic (based on metabolic/histochemical rnATPase staining
charact eristics)
fibroblast growth factor
fi uorescein
fast oxidative glycolytic (based on metabolic/hïstochemical mATPase staining
c haracteristics)
fatigue resistant (based on electrophysiological twitch criteria and
charactenstics)
fast twitch
glial-derived neurotrophic factor
haematoxylin and eosin
lateral gastrocnemius
mediai gastrocnemius
myosin heavy chah
rnyofibrillar adenosine triphosphatase
nicotinamide adenine dinucleotide tetrazolium reductase
neuromuscular junction
Penodic Acid S c h S
prostaglandin of the E senes
PKA
PMG
ROD
so
SOL
ST
SD
SEM
TA
TR
TS
TTX
VREZ
protein kinase A
proximal medial gastrocnernius
relative optical density
slow oxidative @ased on metabolic/histochemical rnATPase staining
characteristics)
soleus
slow twitch
standard deviation
standard error of the mean
tibialis antenor
Texas Red
triceps surae (muscle group responsible for extending the ankle: soleus, media1
and lateral gastrocnemius)
tetrodotoxin
ventral root entry zone
List of Tables
Page
Table 1 . Counts of retrogradely labeiied triceps surae (TS) and anterior ................. -47
Table 2 . Nurnber of muscle fibres analysed per fibre type in the proximal ................. -73
Table 3 . Total number of motor endplates quantified in the proximal and distal ......... -77
Table 4 . Specific activity measurements of acetylcholhesterase subunits .................. -92
Figure 1 .
Figure 2 .
Figure 3 .
Figure 4 .
Figure 5 .
Figure 6 .
Figure 7 .
Figure 8 .
Figure 9 .
Page
........................................... Schematic representation of motor nerve sprouting -15
........................... Three dimensional reconstruction of the rat SOL and TA -33
................................... Silicon rubber micro-angiography of the rat SOL and T A 37
Topographie localisation of FIuoroGold labelled TS and AC ............................. 43
StainLig patterns of TS and AC CGRP+ve motoneurons in control .................... 48
Staining patterns of TS and AC CGRP+ve motoneurons 48 hours .................... 49
Photomicrographs of H&E stained SOL and TA muscles 48 hours .................... -50
DEerentiai increase of motoneuronal CGRP in the TS and AC ......................... -51
Ceil size distribution of CGRP+ve motoneurons as a fùnction of time ............... -59
Figure 10 . Schematic representation of the rat hindlunbs .................................................. 63
.................................... Figure 1 1 . Photomicrographs of double-labelled pMG motoneurons 70
Figure 12 . Profüe of double-labelied FIuoroGold and CGRP+ve soleus (SOL) ..................... 72
Figure 13 . Photomicrographs of myosin ATPase and PAS-stained muscle .......................... -75
........................ Figure 14 . Glycogen depletion profiles of ST, FOG, and FG muscle fibres 76
... .............................. Figure 15 . Photomicrographs of the neuromuscular junctions in the .. 70
........... ............ . Figure 16 The increase in the percentage of CGRP+ve motor endplates .. 78
....................... Figure 17 . CGRP immunoreactivity is present at rnotor nerve terminais 70
................................................. Figure 18 . AChE subunit activity Ui the MG in the control 89
........................................ Figure 19 . AChE subunit activity in the MG 72 h post-exercise 90
........................................ Figure 20 . AChE subunit activity in the MG 2 wk post-exercise 91
.................. Figure 21 . The profile of CGRP+ve and GDNF+ve immunofluorescence 98
........................ . Figure 22 Photomicrographs of GDNWve irnmunofluorescent staining -99
. ....................... Figure 23 Schematic diagram of the known short-tem effects of CGRP -107
................................... Figure 24 . Schematic representation of a neuromuscular junction 109
................................... . Figure 25 Schematic diagram depictùig the sites of ongin -115
. ......................................... Figure 26 Schematic representation of the lefi rat MG -119
CHAPTER 1-
INTRODUCTION
A, Calcitonin Gene-Related Peptide in the Neuromuscular Svstem
C Jcitonin gene-related peptide (CGRP), a 37 amino acid peptide denved fiom the
alternative splicing of the calcitonin gene mosenfeld et al., 1983), is distnbuted throughout
the mammalian centrai and penpherai nervous systerns. CGRP knctions as a
neurotransmitter In penpherai and centra1 nociceptive pain pathways (Dubner and Ruda,
1992), and has been shown to be a potent vasodilator (Ohien et al., 1987; Yamada et al.,
1997). In the developing neuromuscular system, a neurotrophic or neuromodulatory role for
CGRP has been proposed, based on in v&o studies of CGRP effects on acetylcholine
receptor activity (Fontaine et al., 1987; Miles et al., 1989; Changeux et al., 1992) and on the
post-natal development of neurornuscular junctions (New and Mudge, 1986; Matteoli et al.,
1990; Lu et al., 1993). The roIe of CGRP in the normal adult motor system is stdi unclear.
However, the evidence suggests that it is associated with plasticity and sprouting events at
the neuromuscular junction ( Sala et al., 1995; Tarabal et aI., 1 996b).
1.1. Role of CGRP in developing nezrromuscuhr qsterns
Experiments using Xenopus nerve-muscle cultures have demonstrated that CGRP
potentiates spontaneous synaptic currents and enhances acetylchohe channel open time in
developing myocytes (Lu and Fu, 1995) via a CAMP-dependent protein kinase A.,
intracellular s ignahg pathway &iou and Fu, 1995). CGRP also increases the desensitisation
rate of the acetylcholine receptor in developing mouse myocytes (Mulle et al., 1988) and
enhances phosphorylation of the acetylchohe receptor in cultured rat myotubes (Miles et al.,
1989). In cultured chick myotubes, CGRP activates adenylate cyclase (Laufer and Changeux,
1987) and increases the rate of synthesis and accumulation of acetylcholine receptors on the
muscle membrane (New and Mudge, 1986) by stimulating transcription of the A C b
subunit (Fontaine et d., 1986, 1987; Osterlund et al., 1989). In neonatal rat rnotoneurons,
maximal levels of CGRP expression are observed eariy in the development of the
neurornuscular junction (Li and Dahlstrom, 1992) implicating a functional role for CGRP in
synaptogenesis. In embryonic chick muscles, CGRP binding sites occur in a ratio of 1 for
every 10 a-bungarotoxin binduig sites, with the highest specinc aaivity of the peptide being
found in the membranes of 11-14 day old embryos (Roa and Changeux, 1991). Following
denervation in the chick, the number of CGRP binding sites does not increase in direct
cornparison to the large increases observed in AChR binding sites (Roa and Changeux, 199 1)
indicating that the CGRP response at the neuromuscular junction may be more favourably
linked with changes in pre-synaptic activity. Hence, this evidence collectively suggests that
CGRP may play a role in both short-terrn and long-term events at the developing
neuromuscular junction.
1.2. Role o f CGRP in the addi nez~romusarlar system:
CGRP is localised to dense core vesicles in both immature arnphibian (Matteoii et al.,
1990) and mature mammalian motor newe terrninals (Takarni et al., 1985; Matteoli et al.,
1988; Kashihara et al., 1989). In adult rat nerve-muscle preparations, electrical stimulation of
the motor nerve, or muscle depolarisation with elevated extracellular K', produces a release
of the peptide in a ca2'-dependent manner (LTchida et al., 1990; Sakaguchi et al., 1991) that
enhances muscle contraction (Takami et al., 1985a) via a cyciic AMP rnediated pathway
(Takarni et al., 1986). In the isolated rat soleus muscle, exogenous CGRP stimulates Na'K
pump activiw as weii as reducing intraceliular Na' concentrations by 50% over a 20 minute
experimental incubation (Andersen and Clausen, 1993). The current evidence indicates that
the release of CGRP at the NMJ enhances neuromuscular transmission. Evidence in the ffog
sartorius muscle Ni situ demonstrates that exogenously applied CGRP increases quantal size,
output and miniature endplate potentials via PKA activation (Van der Kloot et al., 1998). In
addition, there is evidence that it may act as a trophic factor since CGRP also contributes to
rnotor endplate morphogenesis by maintaining a high density of acetyfcholine receptors at the
motor nerve tenninal (Matteoli et al., 1990).
1.3. CGRP eqression in motoneurom and ut motor nerve teminah of normal animals:
In the central nervous system (CNS), CGRP is detected in subpopulations of spinal
cord motoneurons in many vertebrate species, including mamals (Gibson et al., 1984),
where it CO-localises with acetylcholine (Takami et al., 1985b). There are two forms of
CGRP, aCGRP and PCGRP, that are expressed by motoneurons in the ventrai horn of the
spinal cor& with aCGRP being more abundant. Both forms of the peptide appear to be
dserentially regulated in the spinal cord following vanous experimental manipulations
(Noguchi et al., 1990b; Arvidsson et al., 1993b; Piehl et al., 1995b, 1998b). Evidence from
nerve transection studies suggested that aCGRP may play a role in regeneration-associated
activities (Piehl et al., 1998a). In contrast, in vitro studies (Fontaine et al., 1986, 1987; Mulle
et al., 1988; Osterlund et al., 1989; Changeux, 1991; Changeux et al., 1992) suggest that
PCGRP may have neurotransmitter-associated functions, since it is regulated similarly to
acetylcholine (Arvidsson et al., 1993).
Immunocytochemical (Forsgren et al., 1993; Piehl et al., 1993) and in situ
hybridisation (Blanco et al., 1997) studies in control animais have described contrasting
levels of motoneurond CGRP as a fiinction of muscle fibre type in the rodent. Piehl et ai.
(1993) have proposed that variations in motoneuronal CGRP stahing patterns are
representative of the muscle fibre type that is innervate4 based on their observation of
greater CGW expression in motoneuron pools innervating muscles composed predominantly
of fast twitch motor units as compared to those with slow twitch motor units. Blanco et al.
(1992) have shown a correlation between CGRP mRNA expression in adult rat motoneurons
and the percentage of MHC type IIi3 fibres in the innervated muscle. In a subsequent
investigation, using normal animals, the authors showed that mean aCGRP mRNA levels in
SOL and EDL motoneurons did not dzer significantly f?om each other (Blanco et al., 1997).
It has been noted that CGRP ùnmunoreactivity varies within a motor pool, with some cells
staining more bnghtly and intensely in cornparison to others (Piehl et ai., 1991). Thus,
dxerent Ievels of CGRP expression in motor nuclei and among motoneurons within one
motoneuron pool may imply that the functional significance of altered CGRP expression may
be associated with muscle activity. Presently, factors that influence the level of CGRP
expression in individual motoneurons are not known.
In the muscle, CGRP is localised predorninantly at motor nerve terminais of fast-
twitch muscle fibres (Forsgren et al., 1992, 1993). These authors reported the differential
distribution of CGRP at fast twitch motor nerve terminais using combined enzymatic and
imrnunofluorescence methodologies. Using NADH-TR as an index of muscle fibre type, the
authors showed that CGRP-ir was rarely found at neuromuscular junctions in the soleus
muscle (slow twitch; type I), and never seen in the deep (darkly stained NADH-TR-positive)
fast twitch-oxidative region of the tibiaiis anterior (TA) muscle. In this study of normal
animais, CGRP immunoreactivity was most eequentiy observed in association with fast-
twitch fibres which exhibited little NADH-TR, interpreted to be type IIB fibres, located in
the superficial portion of the TA (Forsgren et al., 1993). Therefore, in the unperturbed adult
neuromuscuIar system, these observations would suggest that CGRP is most highly
expressed in fast-twitch muscles. The fundonal implications of this are not known.
Presently, although substantial work has been directed at linking CGRP expression
with muscle fibre type characteristics, none of the studies to date have directiy corrdated
CGRP expression in identified motoneurons with muscle fibre type-identifled endplates using
a standard fibre typing protocol (i.e., myofibrillar ATPase histochemistry). NADH-TR
histochemistry has been used to determine the oxidative capacity of individuai muscle fibres
and is a general indicator of muscle fibre type. However, the fact that there are at least two
dflerent types of fast hvitch muscle fibres (fast oxidative glycolytic (FOG) and fast glycolytic
(FG); (Brooke and Kaiser, 1970) requires that a more sensitive indicator of muscle fibre type
be utilised (Le., mATPase). The inherent tiindamental differences that exist between the ST
and FT fibre types, as weU as within the FT muscle fibres, and their motor units (Burke,
198 1) may influence CGRP expression. Therefore, it stiii remains unclear whether there is a
preferential distribution of CGRP at FOG and FG motor endplates and in the corresponding
motoneurons.
1-4. CGW emtession in motonewons and af motor nerve fenninals folZowW experintentd
rnan~~~Ialions:
1.4.1 Spinal cord irrmsection, amtorny and modeZs of pharmoulogrgrcaZ intervention
Experirnental models that create a disruption in the comection between the motor
nerve and the neuromuscular junction 0, either surgicdy (Streit et al., 1989; Arvidsson
et al., 1990; Piehi et al., 1991) or pharmacologically (Sala et al., 1995b; TarabaI et al.,
1996b3, directly produce an up-regulation of CGRP peptide and/or CGRP rnRNA (Noguchi
et al., 1990; Piehl et al., 1995). Importantly, d these models involve severe disruptions of
the neuromuscular system, and result in either reduced activity or total inactivity (pardysis)
of the muscle. CGRP expression decreases in motoneurons following spinal cord transection
(Awidsson et al., 1989; Piehl et al., 1991), penpherd nerve section (Noguchi et al., 1990;
AMdsson et al., 1990; Piehl et al., 1991,1995) , nerve crush (Blake-Bruzzini et al., 1997),
and castration (Popper and Micevych, 1989; Popper et al., 1992). Axotomy of the sciatic
nerve produces an up-regdation of aCGRP mRNA in spinal cord motoneurons while
PCGRP rernains unchanged or slightly reduced (Noguchi et aI., 1990; Arvidsson et al., 1993;
Piehl et d., 1995). Sala et ai. (1995) pardysed the rat hindlirnb by either intramuscular
injection of botulinum toxin @oTx) or tetrodotoxin (TTX) infused via a sciatic cuff They
reported an accumulation of CGRP in extensor digitorum Iongus (EDL) motor nerve
tenninals, and an increased expression of the peptide in the lumbar spinal cord. Tarabal et al.
(1996) expanded this work and observed sprouting at EDL motor nerve t e d n d s 11 days
foUowing paralysis with BoTx (Tarabal et al., 1996b). InterestingIy, this effect could not be
blocked by exogenous application of CGRP (Tarabal et al., 1996b). In contrast, Tsujimoto
and Kuno (1 988) have shown that intrarnuscular injection of CGRP inhibits the sprouting of
soleus motor nerve terminais after TTX nerve treatment. Hence, the role that CGRP might
play at destabilïsed neuromuscular junctions as a sprouting or anti-sprouting agent remains
undetemiined. However, these reports collectively suggest that the motoneuronal CGRP
content is correlated with growth and/or activity-related events rit the neuromuscular
junction.
1.4.2. Target Factor ReguIafrion of CGRP levek
Popper and Mïcevych (1989) have shown that the exogenous administration of
testosterone to castrated adult male rats retums CGRP levels in bulbocavernosus
rnotoneurons to pre-castration values, indicating that hormonal influences can significantiy
alter motoneuronal CGRP content. Up-regdation of CGRP mRNA in the bulbocavemosi
motoneurons bas been shown to be due to soluble factors present within muscle extracts
obtained from the hormonally-deprived bulbocavernosus muscle Popper et al., 1992). Our
ernerging understanding of target-derived growth factors indicates that numerous factors
infiuence neuromuscular remodelling. Piehl et al. (1995) were able to show that
intraperitoneal injection of lpg bFGF reversed the effects of an axotomy, and induced an
increase in aCGRP and a decrease in PCGRP, while a sirnilar administration of BDNF had
no effect on motoneuronal CGRP expression. Still, Iittle is known about the interaction of
other muscle growth factors (e.g., insulin-like growth factor-1 and II, transforming growth
factor+, GDNF) and motoneuronal CGRP content. In the case of CGRP, it is likely that the
interaction is intluenced by one, or many, retrograde signals originating from the muscle or
the post-synaptic membrane.
1 -5. Downhill mnning eccenfrlc c o n ~ c t i o 1 1 ~ ~ and muscle damage
Downhill ninning is known to cause muscle pathology, primarily in those muscles
perfomiing eccentric lengthening contractions during the activity (Armstrong et al., 1983,
1991; Ogilvie et ai., 1988; Stauber 1989). The unaccustomed nature of this exercise
produces both rnicroscopic and macroscopic muscle damage in both animals (Armstrong et
ai., 1983; Ogilvie et al., 1988; Lieber et al., 1991; Friden and Lieber, 1998) and humans
(Newharn et al., 1983, 1988; Byrnes et ai., 1985; Stauber, 1989,1990; Kuipers, 1994;
Gibala et al., 1995). The mechanism responsible for the initiation of the damage remains a
focus of considerable debate and study, dthough evidence suggests that mechanical stress
produces darnage to the contractile apparatus (Lieber and Fnden, 1988; Stauber et al., 1990;
Lieber et al., 1991; Friden and Lieber, 1992; Warren et al., 1993; Ingalls et al., 1998),
resulting in calcium-mediated (Duan et al., 1990; Byrd, 1992; Balnave and M e n , 1995),
metabolic (Evans and Cannon, 199 l), and microcirculatory changes (Peeze Binkhorst et al.,
1989).
This comrnon form of muscle injury produces a sensation of pain and discornfort in
humans 2-3 days followlng activity, resulting in a condition descnbed as "delayed onset
muscle soreness" or DOMS (Newham, 1988; Jones et al., 1989; Stauber, 1989; Smith, 1991;
Kuipers, 1994). The origin of the pain associated with DOMS is most likely due to the
initiation of an acute infiammatory reaction within the injured muscle (Cannon et al., 1989,
199 1; Stauber et al., 1989, 1990; Smith, 199 1; Fielding et al., 1993; Kuipers, 1994; Tidbali,
1995), an effect that can be significantly reduced with training (Schwane and Armstrong,
1983; Byrnes et ai., 1985; Clarkson et al., 1992; Balnave and Thompson, 1993; Lynn et al.,
1998). Classical inflamrnatory events can be observed w i t h the muscle tissue 6-12 hours
foiIowing unaccustomed intense exercise (Ogilvie et al., 1988; Smith, 1991; Tidbd, 1995).
Large numbers of tissue bom monocytes and macrophages synthesising vast arnounts of
PGE2 are observed at 48-72 hours foIIowing muscle injury. By 72-96 hours post-injury, the
repair process had begun within the muscle concomitant with the dissipation of inflamrnatory
components (Smith, 1991).
Muscle fibre type composition has been suggested to be a predisposing factor for
eccentnc exercise-induced darnage (Lieber and Friden, 1988; Lieber et al., 199 1; Friden and
Lieber, 1992). The muscle most fiequently and thoroughly investigated in rodent eccentric
activity models has been the soleus (SOL; Armstrong et ai., 1983; Darr and Schultz, 1987;
Ogilvie et ai., 1988; Smith et al., 1997), a muscle composed predorninantly (95%) of slow-
twitch (ST) fibres (Armstrong and Phelps, 1984). However, based on obsexvations of human
vastus laterdis muscle following intense cycling activity, Friden et al. (1988) have proposed
that the wider 2-bands present in slow twitch fibres are less vulnerable to higher stresses than
the 2-bands of fast twitch fibres (Lieber and Friden, 1988; Friden and Lieber, 1992). Slow
motor units are preferentially recruited during sub-maximal work and are less fatiguable
(Etenneman and Olson, 1965). Warren et al. (1994) have suggested that fast-twitch muscles
may be more susceptible to injury due to their Sequent loading capacity and their lower
oxidative profile (Lieber and Friden, 1988; Lieber et al., 1991; Friden and Lieber, 1992),
rather than to their fibre type composition. It has been observed that muscle injury is a
function of the development of maximal force in electncally stimulated mouse EDL
performing lengthening contractions (McCully and Faulkner, 1 985, 1 986). The mechanical
disruption of muscle fibres as a result of high levels of force development within the muscle
has been proposed to be the initiai event in exercise-induced muscle darnage (McCuily and
Faulkner, 1985, 1986; Newham, 1988; Friden and Lieber, 1992). It is hypothesised that
eccentric lengthening contractions generate very high tension w i t b the myofibrils,
effectively mpturing and misaligning the actin-myosin cross-bridges, reducing muscle
contraction economy (Warren et al., 1996), and resulting in acute exercise-induced damage,
and if- occuning chronicaiiy, in muscle hypertrophy (Newham, 1988; Wong and Booth, 1990;
Booth and Kirby, 1992; Friden and Lieber, 1992).
Recent studies by Sorichter et al. (1997) and Lieber et al. (1994 1996) have been
able to identQ elements of the contractile apparatus and cytoskeletal network that undergo
changes foIIowing eccentric exercise. Sorichter et al. (1997) observed increased blood
plasma levels of ske1etal troponin 1, (a unique, exclusive skeletal muscle protein), in healthy,
untrained human subjects within 2-6 hours foiiowing downhill running. Lieber and his
colleagues (1994, 1996) found a reduction in the cytoskeletal protein desmin in rabbit EDL
muscle foiiowing cyclical electrically-induced eccentric contractions. These b food and muscle
tissue markers were found to be a more accurate indicators of eccentric exercise-induced
muscle darnage than creatine kinase (Sonchter et al., 1997) and provide more evidence that a
dismption in the contractile complex is most probably due to high force intolerance.
Eccentric exercise induces muscle fibre regeneration following muscle darnage
(Stauber, 1989), and de novo muscle protein synthesis (Wong and Booth, 1990; Booth and
Kkby, 1992; Lowe et al., 1995). Muscle satellite cells, the precursors of myoblasts, have
been shown to proliferate in the muscle within 24 hours following downhill running in the rat
soleus (Darr and Schultz, 1987). A swift augmentation of myofibrillar protein synthesis has
been observed in rodents (Wong and Booth, 1990; Booth and Kirby, 1992) and humans
Gowe et al., 1995) a e r acute or chronic downhill exercise, hdicating that changes in the
translational mechanisms of gene expression had been induced in the muscle in response to
the darnage. Whether regenerative changes within the muscle lead to an up-regulation in
CGRP expression in the motoneurons supplying regenerating or enlarging motor units has
not been examùied.
1 -6. Sproutinp and the NeuromusaiZar Juncfion
Changes occur at neuromuscular junctions following a variety of normal and
pathologicai conditions. The junctional region can be rebuilt following degeneration as a
result of injury or disease; it can be enlarged by end plate outgrowth due to sprouting, or due
to the establishment of new contact areas in response to the natural, active turnover of motor
endplates in normal adult muscle (Barker and Ip, 1966). The morphology of motor endplates
of various muscle fibre types is diverse, suggesting that a fùnctional signincance is asçociated
with the activity profile of the muscle. Hence, neuromuscular junctions are complex dynamic
structures undergoing constant adaptation according to the demands of the internai and
externai environment.
1 -6.1. Activity and the NeztrornuscuIar Juncfion:
Structural changes in neuromuscular morphology that are induced by activity and
visible at the iight microscopie level have not been thoroughly investigated in marnmals. The
effects of activity are complex and have produced contrasting results in vertebrate muscle
(reviewed in Wemïg et al., 1991b; Deschenes et al., 1994). Increases in activity produce
alterations in structure and function that are dependent on factors such as the amount and
pattern of activity. For example, an increase in the size of the mouse EDL motor endplate
following several months of voluntary wheel running exercise resulted in a slight elevation in
transrnitter release (Doriochter et ai., 1991). The opposite results were observed in the frog,
where a fast muscle was electricafly stimulated with a slow tonic pattern of electrical
impulses (Wernig et ai., 199 lb). In crustacean muscle, experiments using the same protocol
as Wemig et al. (1991b) resulted in an initial reduction in transmitter release which was
followed by "enhanced facilitation" and changes in morphology (Lnenicka and Atwood,
1985; Atwood and Lnenicka, 1987). Wernig et al. (1990) were also able to show a larger
significant change in synaptic transmission in the mouse soleus of occasional wheel runners
versus chronic runners, further suggesting that the pattern of activity is important. These
contIicting results may be due to differences in the imposed experimental paradigm. While
the electncal stimulation protocol may have been "non-physiological", abnormally stimulated
animals and normaliy moving animals demonstrated similar adaptative changes in
morphology and transmission parameters (Lnenicka and Atwood, 1985; Atwood and
Lnenicka, 1987; Atwood et al., 1991). In the mouse, the elevation in the animal's natural
activity pattern resulting nom wheel running may have led to an increased transmitter release
in "preferentially activated" synapses @orlochter et al., 1991), suggesting that muscle
activity above normal (Wernig et al., 1990, 1991) would be a determinant of hnctional
charactenstics.
Experimental work in the invertebrate has shown that structural changes are
associated with alterations in the speed of synaptic transm*ssion. The ultrastructure of
crustacean neuromuscular junctions innervated by fast axons undergoes a morphological
change toward the slow type following chronic, tonic stimulation (Lnenicka et al., 1986).
Wojtowicz et al. (1994) have shown that nerve terminais with a stimulation history dif5er
f?om unstimulated terminals in that they have more "active zones" (clumps of synaptic
vesicles which may represent areas of transmitter release). Furthemore, Cooper et al. (1995)
have shown that regional Merences in the location of muscle fibres, synapses, and their pre-
synaptic varicosities are factors in determining the response to tonic or phasic trains of
activity These investigators found that the 'quantity of zones" per nerve terminal
surface area and the number of synapses with multiple active zones (cornplex synapses) were
higher in "high-output" varicosities'. It may be that synapses with many "active zones" could
be directing trammitter discharge at low frequencies of stimulation, while synapses with less
"active zonesy' are required for more intense periods of mmulation. These authors suggest
that adaptations in synaptic structure and fùnction can occur in a relatively small time-frame
and quite likely involve an iderior proportion of the synapses available on a motor nerve
terminal (Cooper et al., 1995). It is important to note that the invertebrate neuromuscular
junction is innenrated by several endplates, while the vertebrate neuromuscular junction has
but one.
Activity in the form of exercise training results in morphological changes at the
vertebrate neuromuscuIar junction (Appell, 1984; Wemig et ai., 1990, 199 1; Waerhaug et al.,
1992; Deschenes et al., 1993). Both the area and length of motor nerve temiinds increases,
and the density of the nerve texminal varicosities is reduced in the 13 week old male rat EDL
following 6 weeks of t r e a d d exercise on a level grade (Waerhaug et al., 1992). Overload
training of the mouse diaphragm, as induced by anoxia for 7 or 14 days, resulted in an
enlarged motor end-plate region by seven days and stimulated an increase in motor endplate
number by 14 days (Appell, 1984). Wemig et al. (1991a) showed that in mice, a single or
repeat (3-5 days, daily) bout of treadrnill running produced axonal sprouting 3-21 days after
exercise . Substantiai enlargement and redistribution of motor units was observed with more
running episodes. Signs of stmcturai changes at the neuromuscu1ar junction (junctional
hypertrophy, total length of branching, average lengthhranch) were observed in the mouse
soleus &er 12 weeks of high intensity training without a change in the density of AChRs or
synaptic ACh vesicles, as indicated by SV-2 mouse monoclonal antibody immunostaining.
Hence, the authors concluded that there were no sigruficant alterations to synaptic fùnction,
dthough they did document structural adaptations (Deschenes et al., 1993). Therefore,
dthough contrasting reports exist, the amount of training andor the activity pattern of a
motoneuron are important factors in regulating the fiinctional and structural characteristics of
the neuromuscular junctions (DurIochter et al., 199 1).
1 -6 -2. Sprating
Peripheral nerve sprouting occurs at motor endplates during development and
continues into adulthood as a dynarnic process of neuromuscular renewal. The tenn
'sprouting' is used to describe a variety of outgrowth processes that occur at peripheral
nerve endings. Any form of blockage of neuromuscular activity either pre- or pust-
synaptically produces an enlargement of the motor nerve terminais by sprouting. One form of
sprouting is defined as "terminal sprouting", a process which occurs throughout Me and is
believed to be involved in the growth and reconstruction of motor endplates (Barker and Ip,
1966; Grimell and Herrera, 1981; Weniig and Herrera, 1986). Terminal sprouting is
characterised by the occurrence of tiny, fine, unrnyelinated outgrowths or sprouts that
onginate fiom the unmyelinated pretenninal or terminal nodes of the rnotor axon which
rnigrate toward and innervate the parent endplate (sarne muscle fibre). The result (see Fig.
lb) is an enlargement of the neurornuscular contact zone (Tuffery, 1971; Stebbins et al.,
1985; Brown et al., 1981; Appell, 1984; Barker and Ip, 1966). Terminal sprouting c m be
Figure 1 . Schematic representation of motor nerve sprouting. (a) Normal end-plate innervation; (b) terminal sprouting; (c) collateral sprouting; (d) nerveendplate remodelling. Established terminals (darkly filled) and new terminals (lightly filled). Modified fiom Appell, (1984).
stunulated by partial denervation and also by pharmacologie block (Grinneil, 1995; GrinneLi
and Herrera, 198 1; Pestronk and Drachrnan, 1978).
Along with many factors, increasing the functional workload of a muscle is suggested
to stimulate terminal sprouting at the stressed motor endplates (Barker and Ip, 2966; Grinneil
and Herrera, 198 1; Cardasis and Padykula, 198 1; GrinneIl, 1995). This observation may be
dependent on the age of the animal (Grinneii and Herrera, 198 1). For example, Stebbins et al.
(1985) evaluated terminal sprouting in aged rats by examinhg the percentage of endplates
with growth configurations following exercise. Chronic exercise was unable to initiate
terminal sprouting in the normal, distal gastrocnemius after 5 months of uphill ninning in
these aged (26 month old) rats.
Collateral or 'nodal' sprouting is characterised by the outgrowth of a new nerve
terminal from the node of Ranvier produchg a reinnervation and/or an innervation of two or
more muscle fibres. The nerve sprout establishes novel endplates on the parent and/or an
adjacent muscle fibre increasïng the innervation ratio (see Fig. lc; Tuffery, 1971; Brown,
1984; Appeil, 1984; Wernig and Herrera, 1986; Kawabuchi and Kosaka, 1993; Grimeil,
1995). This type of sprouting is stimulated by partial denervation where the initial stimuIus
appears to be inactivity. Hence, collateral and terminal sprouting are most tikely triggered by
different signais; the former is probably a response to one or many diffusible substances
onginating fiom denenrated muscle or degenerating axons, while the latter is denved fkom a
local signal such as a factor in the muscle membrane (Grinnell and Herrera, 1981; Grinnetl,
1995).
Nerve-endplate remodelling is a term that has been used to describe a general
response of the neuromuscular junction involvhg changes in both pre- and post-synaptic
morphology (see Fig. Id). The terminology encompasses terminal sprouting, synapse
formation and nerve retraction that arise under namal physiogicat conditions, such as
growth, altered use, and ageing. These conditions could produce a response whereby
sprouting or new synapse formation sthulates an enlargement in junctional size and an
associated increase in trammitter release (Wernig and Herrera, 1986). The factors that
initiate, promote and control remodehg are still being deciphered as are the role of
neuropeptides in this process. While the fünctiond consequences of remodehg may not be
immediately conspicuous, the capacity for adaptation is present and may be crucial for the
maintenance of neuromuscular efficacy. The use of the tenn 'remodelling' in this thesis wiil
foliow the standard definition as described above.
1 -6 -3 Morphological mdfunc fional cchmacteristtics of the narromusculm junction
Many dserences are found in the structure and composition of groups of muscles,
individual muscles, and within single muscle regions which reflect large variations in
functional demand. Hence, differences in the ultrastructure of muscle rnotor endplates may
also be correlated with fiinctional demand, If so, muscles could then be viewed as an
individual system with unique requirements and fûnctional needs (cc Zenker and
Scheidegger, 199 1).
Three types of neuromuscular junctions c m be disthguished at the ultrastructural
level in mammals (Padykula and Gauthier, 1970). On close observation, the motor endplates
of alpha motoneurons innervate dserent muscle fibre types and are rnorphologically diverse
with rnany dittèrences in ultrastructure. There is variety in both junctional shape and sue,
which suggests a correlation with ftnctional significance. For example, larger muscles are
innervated by bigger motor endplates (Burke, 198 1; Appell, 1984).
The post-synaptic membrane of motor endplates in slow twitch muscle fibres have
distinct, separate regions of contact with shallow, sparse, short branching junctional folds
that are hegular in their arrangement (Padykula and Gauthier, 1970; Henneman and
Mendell, 1981; Zenker and Scheidegger, 1991). In contrast, the post-synaptic membrane of
fast twitch muscle fibres have long, branching, tightly spaced junctional folds which provide a
large receiving area for neuromuscular transmission (Padykuia and Gauthier, 1970; Zenker
and Scheidegger, 1991). Axon terminais innervating fast twitch fibres are broad and flat, with
a large axoplasmic surface, and a large sarcolernrnal contact area. Fast-twitch oxidative
glycolytic muscle fibres are characteristically innervated by large axon tenninals. In contrast
to fast and slow twitch muscle fibres, the post-synaptic membrane of these fibres has
junctional folds which are longer, straighter, and unbranching, as weli as being the deepest
and the most widely spaced (Padykula and Gauthier, 1970; Henneman and Mendeli, 1981;
Zenker and Scheidegger, 199 1).
Functionally, the morphoiogica1 features of the post-synaptic membrane are positively
correlated with muscle fibre diarneter which in hrrn is correlated with the size of the motor
end-plate (Padykula and Gauthier, 1970; Hememan and Mendeii, 198 1) and neuromuscular
transmission. Simply, larger muscle fibres require greater amounts of transrnitter release for
signalling, hence, large fast twitch fibres need extensive synaptic surfaces. Conversely,
smaller junctions with fewer vesicles are sufficient for transmission in slow twitch fibres
(Hememan and Mendell, 298 1).
1 -7. CGRP and fiercise:
The use of muscle inactivity models has produced many insights into the possible role
of CGRP in the motor system (Tsujimoto and Kuno, 1988b; Popper et al., 1992b; Sala et al.,
1995b; Tarabal et al., 1996b). However, very little attention has been focused on the
relationship between CGRP and physical activity. Given the stirnulating effect of CGRP on
the Na+/K+ pump in isolated rat soleus muscle ceiis, it has been hypothesised that CGRP
may play a role in enhancing muscle performance (Andersen and Clausen, 1993; Schifter et
al., 1995). The effect may be regulated through variations in CGRP concentration or
aiterations in the number of CGRP receptors on muscle ce11 membranes that reflect individual
training status. Schifter et al. (1995) monitored changes in the concentration of plasma
CGRP in rniddle-aged, male endurance athletes at rest and following submaximd and
maximal running exercise. Significant increases in plasma CGRP concentrations were
observed foiiowing exercise; however, the investigators were unable to find a correlation
between CGRP and training status. They concluded that the secretion of CGRP d u ~ g
exercise and the training leveI of the individual were not related. However, the study did not
address the source of CGRP increase, Le., whether it was of sensory and/or motor origin.
Thus, the physiological significance of these observations remain to be clarified in terms of
their contribution to exercise performance.
B. STATEMENT of the HYPOTHESES:
Very little is known about the involvement of the nervous system in DOMS models.
The participation of neuropeptides in acute and chronic eccentnc exercise-induced muscular
activity is also poorly understood. There is evidence descnbing a functional role for CGRP at
the neuromuscular junction foiiowing perturbations to the neuromuscular system. Since
eccentric exercise has a damaging effect on muscle, it is suggested that this activiw may
induce changes in the neuromuscular system which are focused at the motor endplate. Again,
since CGRP is an effector neuropeptide at the neuromuscular junction, it would seem Orely
that changes in CGRP expression rnight be observed in the motor nuclei i~ervating the rat
ankle extensors following unaccustomed d o w M exercise.
In addition, the response of CGRP to enhanced motor activity in the form of exercise
in the peripheral nervous system is not known. Presently, novel theories have been proposed
to describe the neuromuscular commands involved in the performance of eccentric
contractions. Given the evidence of a role for CGRP in synaptic transmission and the
association of the peptide with a particular family of muscle fibre types, changes in CGRP
expression may reflect a novel motor unit recruitment order hypothesised for muscles
undergoing eccentric activity. Since these questions have not yet been addressed, it was the
purpose of this study to investigate whether exercise could affect CGRP expression in the
neuromuscular system. If so, we proposed to idente the responding populations with
respect to motoneuron pool and muscle fibre type in muscles performing eccentric
contractions during do& running. Based on previous demonstrations of histologicaliy
detectable darnage in eccentricaily contracthg muscles, we proposed that CGRP expression
may be changed in muscles displaying regeneration or repair processes.
aYPOTHESISk
DavnhiZI mmthg exercise caws an inmeme m CGRP q r e s h in motone~~om of
eccenhiudy conlractmg muscles.
In the fist part of this work, the objective was to investigate possible changes in
CGRP expression in motoneurons innervating two of the major muscle groups that perform
contrasting activity while downhiil ninning, and to descnbe the tirne course and magnitude of
those changes.
HYPOTEESIS lO[:
CGRP preferentially increares at the fast glycoiytic (FG) t p e IIB ertdplates and in theîr
motoneuruns f o l h i n g eccentric exercise.
In the second part of this shidy, the objective was to ident* the responding population of
motoneurons, and the target muscle fiom within the ankle extensor group, and then to determine
ifthe alterations in CGRP expression could be associateci with fibre type in our activity model.
HYPOTEESIS III:
Changes in CGRP ewpressïon are preceded by changes in GDNF expression at fast
glycolytic (FG) type IIB motor endplates following eccentnc exercise.
The objective of the last part of the thesis was to determine if the increase in CGRP
expression in motoneurons and at the motor nerve terminais on type IIB muscle fibres was a
result of a target-derived neurotrophic factor that has been irnplicated in remodeilhg events
at the neuromuscular junction in the adult mammal.
In the foiiowing chapters, the detaiis of the experiments designed to test these
hypotheses are presented. The Methods and Matenais cornmon to all experiments are
described in Chapter II. The fit part of this work, presented in Chapter ID, uicludes studies
describing the acute exercise rnodel. Chapter N, Study 1, presents the experimental results
examining the fust hypothesis. Based on the observations made in Chapter IV, part two of
the experimental work, Study 2, examines the second hypothesis, and is presented in Chapter
V. The preliminary investigations of the functional role of CGRP at the neuromuscular
junction are described at the end of Chapter V. A general discussion of the studies addressing
the major limitations of the research, the hypothetical finction of CGRP at the
neuromuscular junction, and suggestions for future experiment s are presented in Chapter
m.
CHAPTER II.
MATERIAL,S AND METHODS:
2.1. General Meth& Common to AZZ ExQeriments
The materials and methods used in the completion of the studies presented in this
thesis have many common elements. Some of the methods were particular to one set of
experirnents, and are more adequately addressed in subsequent sections, whereas other
techniques consisted of a standard protocol and are described below.
2-1.1. fiercise Protocol
Adult, female, Wistar rats, weighhg 250-325g, were used for all experiments.
Animals were randomly divided into groups for each experirnental series. Untrained anirnaIs
ran continuously on a motor-driven treadmill at a speed of 12 m-min-', for one 30 minute
period on a -20" incline. Generally, all animais were able to complete the exercise task with
relative eue. However, it was necessary to provide an occasional, brief, two minute rest
pei-iod for some of the runners ( ~ 3 ) . M e r the bout of exercise, animais Eom each
experimental group were returned to their cages, where they were given food and water ad
I i l j i fum until sacrifice. AU experimental procedures were completed according to the CCAC
Guidelines on the Use of Anbals in Research, with ethical approval granted by the Toronto
Hospital Animal Care Cornmittee.
2.1 -2. Tissue Processin~
Muscle and spinal cord tissues £iom each animal were removed and prepared for
analysis. At the t h e of sacrifice, the animals were adrninistered an overdose of sodium
pentobarbitol (1.0 ml; 60 mgmi-'). Prior to transcardiac perfusion and under anaesthesia,
several muscles (SOL, MG, LG, TA, EDL) were dissected fkee and placed into ice cold
saline. The muscles were quickly blotted on gauze, and a 1-2 mm thick cross section was
obtair..ed and mounted on 4 x 6 mm corkboard strips (0.5-1 mm thick) covered with OCT
Embedding Media (CaniablSaxter, Mississauga, ON). Mounted pieces of muscle tissue were
then submerged for 20 seconds in a 2-methylbutane bath cooled with liquid nitrogen, then
transferred to Eppendorf viais and stored at -80°C for subsequent imrnunocytochemistry and
histochemistry.
Spinal cord tissues were then obtained following transcardial perfusion with 500 ml
of 4% paraformaldehyde (23" C; pH 7.35-7.45; BDH, Mississauga, ON). The lumbar region
of the spinal cord (L2 to L5) was removed intact, post-fixed in 4% paraformaldehyde for 18
h at 4" C and cryoprotected overnight in 20% sucrose in 0.1M phosphate buffer pH 7.2
(BDH, Mississauga, ON). Tissues were then fiozen in 2-methylbutane cooled with CO2 and
stored at -80°C until processed for immunocytochemistry or immunofluorescent
quantification.
2.1 -3. Retroarade LubelZin~ of Motoneurons
Animals were placed on their ventral plane (belly d o m ) with the hindlimb held in a
dorsiflexed position (knee joint facing downwards) with masking tape so that the triceps
surae (TS) was clearly exposed. A neuroanatornical tracer was injected into each muscle
using an adapted 100 pl Hamilton Syringe (Fisher Scientific, CA) (see Chapter IV, section
4.3.1. and Chapter V, section 5.3.1). PE-20 silastic tubing was placed on to a syringe needle
with the other end of the tubing supporting a 1/2 inch 30 gauge needle (Baxter Canlab,
Mississauga, ON.). Tracer was taken up into the siiastic tubing and detivered in a single
injection to the mid-region of each muscle. The depth of injection ranged fiom 1.0 mm to 3.5
mm. A micro-manipulator was used to hold the needle in place during the injection. Care
was taken during these injections to prevent leakage of tracer into other muscles using a
localised microsurgical approach and by using petroleum jeliy and tiny gauze pads to isolate
each muscle. The injection site was then sealed with a drop of cyanoacrylate glue (Baxter
Canlab, Mississauga, ON). Three days after the muscle injections, the animals were
administered an overdose of sodium pentobarbitol and sacrificed by transcardial perfusion
with 500 ml of4% padorrnaldehyde (pH 7.35-7.45; BDEX, Mississauga, ON).
The purpose of these experiments was to eaablish a consistent retrograde l abehg
protocol that would label as much of the motoneuron pool as possible without producing
leakage of tracer into the general muscle compartment or contaminate neighbou~g muscles,
which might confound the specific identification of motoneuron pools. Therefore, the
rationale to overcome this problem was to restrict the sampling region to ensure no double
counts of motoneurons in overlapping motor pools. Therefore, the entire pool was not
labelled, and a potentiai underestimate of motoneurons must be acknowledged, although the
numbers we obtained are tight and closely correspond to those previously cited in the
literature (see section 4.3.1; Table 1 .).
Complications arising fiom the leakage of the label could have been addressed by
injecting each muscle in question followed by immediately transecting the nerves to adjacent
muscles in order to ensure that the motoneuron counts were representative of oniy one entire
muscle or muscle group at a time. Given the carefûl microsurgical approach developed for
these studies, this procedure was not employed in the experiments reported herein.
Irnrnunocytochemical techniques were used in this thesis to ident* CGRP in the
spinal cord or muscle tissues. In Study 1 (Chapter IV), single labelied motoneurons were
identified as CGRP (+ve) or (-ve), based on imrnunostaining procedures that used DAB as
the chromagen. In Study 2 (Chapter V), immunofluorescent techniques incorporating
retrograde tracers and fluorescent conjugated antibodies produced double-labelled
motoneurons and motor nerve tenninals for the identification of CGRP- Iabeiled profiles.
Briefly, &ed, fiozen tissues were serially cross-sectioned in a cryostat (Leiq Jung CM
3000). The sections of spinal cord were either allowed to fiee float in tissue culture dishes
(Falcon, CanlablSaxter, Mississauga, ON) or placed directly ont0 sIides. Sections were
washed in 0.1M phosphate buffered sahe (PBS; pH 7.1), blocked with 10% normal goat
serum (NGS; Gibco, Canlab/Baxter, Mississauga, ON), and incubated ovemight at 4°C with
a polyclonal antibody to CGRP (aCGRP (Rat [l-371) antibody, rabbit; Genosys, TX). The
tissues were then processed using an ABC kit with a goat anti-rabbit secondary antibody
(Vectastain, Vector Laboratorïes, Mississauga, ON). The eee-floating sections were
mounted on slides and coverslipped with Entellan (BDH, Mississauga, ON). Sections directly
rnounted ont0 slides were coverslipped with Mowiol (Hoescht, Montreal, PQ).
The technique of using antibodies to ident@ tissue and cellular markers is well
established. The generai peroxidase reaction routinely reveais endogenous peroxidases (Le.,
in red blood cells) making them a good control for the DAB-based reaction protocol. When
the diamino-be~dine @AB) reaction is allowed to proceed too long, it tums most parts of
the tissue a shade of brown due to the reactivity of the iipid components of the cellular
membrane. Therefore, the titration of the DAB reaction to balance the reaction product with
background st aining is crucial for successful staining.
It has been observed that formalin often causes a precipitate which adheres to par&
processed tissues sections following the DAB reaction. If this occuned during the
immunoreaction, artifiactual CGRP positivity woufd be observed. This potential problem was
avoided by using fiesh fiozen tissue and thoroughly washing sections in multiple washes of
0.1M PBS or 0.1M PB buffer solutions. Lady, to assess this possibility, negative controls
were run sequentidy in order to confirm primary antibody specificity, and to dserentiate
between the primary antibody signal and background non-specific staining. Ornitting the
primary in the negative control also provides information as to the specincity of the
secondary antibody. A non-spec5c secondary will produce background staining that
cornpikates the identification of the primary signal.
2.1 -5. Data Acwisition and AnuZvsis
The quantifkation of al1 CGRP-positive motoneurons, and the identification of their
topographie distributions within the lumbar spinal cord grey matter were completed on a
Micro Cornputer Imaging Device (MCID) Image Analysis System (Imaging Research Inc.,
Ste. Catharine's, ON). A Leica D m fluorescence microscope was used to visualise the
DAB stained motoneurons in bnght-field, and for the immunofiuorescent detection of single
and double-labelled motoneurons and motor endplates.
2.1 -6. Sraristical Me fhods:
Data are presented as the mean (x) k the standard error of the mean (SEM) unless
noted, where it is presented as rnean (x) f the standard deviation (SD). The statistical tests
used to determine the statistical significance between data sets are noted in the Methods
sections of each study. A surnmary of the tests used were: the Student's t-test, Mann Whitney
Rank Sum, one-way ANOVA and where necessary, the Kmskal Wallis Test for non-
parametnc distributions. AU statistics were completed using SigmaStat v1.0 statistical
software, (Jandel Scientific, USA). Differences between means were judged to be significant
at P<O.OS.
CHAPTER ID.
THE MODEL:
3-1. INTRODUCTION:
The pathological alterations in muscle morphology, which occur predorninantly d e r
unaccuçtomed eccentric exercise, in humans result in the familiar sensation of muscle pain
known as "delayed onset muscle soreness" (DOMS). The extensive dismption of stnictural
components in the muscle at the injury site (Tidball, 1995) (i.e., extraceUular matrix @CM)
disruption ) most likely mechanicaily generated, is accompanied by an autophagic response
that contributes substantially to muscle fibre darnage (Armstrong, 1990)- These "initial
catabolic events" are important as they promote regeneration and repair of the muscle
architecture relying on idammatory cells (e-g., mononucleated ceUs which appear at injury
sites) to remove cellular debris (Tidball, 1995). The time-course of these changes has been
well described (Smith, 199 1; Kuipers, 1994; Tidball, 1995).
Animal models used to study DOMS d s e r widely in terms of the exercise duration
(30-90 mk~) (Schwane and Armstrong, 1983; Armstrong et al., 1983; Ogilvie et al., 1988;
Duan et al., 1990; Balnave and Thompson, 1993; Smith et al., 1997), fkequency (1-7 times -
wk-') (Schwane and Armstrong, 1983; Armstrong et al., 1983; Ogilvie et al., 1988; Duan et
al., 1990; Smith et al., 19971, treadmill speed (10-100 m. min-') (Schwane and Armstrong,
1983; Armstrong et al., 1983; Ogrlvie et al., 1988; Duan et al., 1990; Balnave and
Thompson, 1993; Smith et al-, 1997) and inche (-16" to -25') (Schwane and Armstrong,
1983; Armstrong et al., 1983; Ogilvie et al., 1988; Duan et al., 1990; Balnave and
Thompson, 1993; Smith et al., 1997). Therefore, we selected a specific protocol(l2 m*mixïl,
-20°incline, 30 min.) and completed a series of experiments in an effort to document whether
inflammatory changes that take place in these muscles following this protocol are comparable
to those previously described, and to evaluate whether damage was related to some of the
microcirculatory anatomy of the SOL and TA muscles. The chapter has been divided into
two parts describing two separate experiments that provide a descriptive analysis of two
contrasting muscles (SOL and TA) which d s e r in fibre type composition and activity profile.
P a . 1.
3.2- Three Dimensional Reconstruction of Soleus and Tibialis Anterior Muscles in the Rat
Following Downhill Exercise.
3.2.1. Introduction:
Downhill running produces damage in muscles undergohg eccentric lengthenuig
contractions (Armstrong et al., 1983; Ogilvie et al., 1988). The ankle extensors (triceps
surae: SOL, LG, MG) perform eccentric contractions, wMe the ankie flexors (anterior
crural: EDL and TA) perform concentric contractions during downhill running exercise.
Morphological changes in the muscle folowing eccentric contractions have been more
fiequently investigated and described in the SOL (Armstrong et ai., 1983; Ogiivie et al.,
1988). The most cornmonly identified alterations in muscle architecture are focal dismptions
of the A-band, 2-line disintegration or streaming, and mitochondrial swelling and disarray
(Armstrong et al., 1983; Ogilvie et al., 1988). Although much is known about the
histopathology of eccentricaliy challenged muscles [Le., tirne-course of the onset of
pathology, the clearance of blood and tissue bom imrnuno-competent cells (Cannon et al.,
1989; Cannon et al., 1991; Evans and Cannon, 1991; Smith, 1991; Fielding et al., 1993;
Tidball, 1995)], the anatornical distribution of histopathological changes in the SOL has
never been thoroughiy described. There is some evidence to suggest that the ultrastructural
changes are not uniform throughout the muscle, but have a focal distribution within the
rnyofibres (Armstrong et al., 1983; Ogilvie et al., 1988; Byrd, 1992). Therefore, in order to
confirm that exercise-induced damage was present in the SOL a d o r TA foilowing our
ninning protocol, and to assess the relative extent of histopathology, three-dimensional
reconstructions of these muscles were completed 72 hours after exercise. Regions of ECM
disruption, identified as holes or spaces between muscle fibres andior muscle fascia and
rnononuclear cellular infiltration, were identified and considered to be macroscopic markers
of histopathological muscle damage.
3 -2.2- Methods:
3 -2 -2.1. Tissue process~hp - and pre~aration:
The SOL and TA were excised bilaterally fiom one a h a l 72 h &er completing the
downhill running protocol. The muscles were dissected, and serial cross-sectional pieces (3-4
mm) were mounted as described in Section 2.1.2. The SOL and TA were serially sectioned
(12 pm) in their entirety, and stained with haematoxyiin and eosin (Sigma, USA). The slides
were cleared and coverslipped as descnbed previously (Section 2.1.2).
3.2.2 -2. mree dimensional recons~c t im:
Camera lucida (Zeiss) drawings of every 40th muscle section were obtained for the
SOL and every 70th section for the TA (e.g., 320 pm and 560 Pm apart, respectively).
Drawings were digitised on a SummaSketch (Surnmagraphics Mode1 MM120 1, Fairfield,
CT) digitiser, and then entered into an IBM compatible 386 cornputer containing custorn
software for digital graphical reconstructions. Hard copy outputs of the completed graphics
were obtained on an HP 7475A plotter.
3.2.3. Results and Discussion:
Utilisation of the three dimensional reconstruction protocol provided a graphical
representation of the ECM disruption and the inflammatory response in the selected muscles.
In terms of the program that was used, data input was limited to 99 sections, therefore not all
muscle sections could be digitised which may have led to a misse3 observation or pattern.
One would require the input of a p a t e r number of sections, and hence, a more in depth
reconstruction, to more ngorously establish the extent of the ECM disruption and the focal
response of immuno-idammatory components in the muscle.
The SOL is a highly and thoroughly vascularised muscle. Most ofien, but not
exclusively, mononuclear infiltrates were observed near the vascular regions and areas of
ECM disruption. These indices of muscle pathology were found at random within the muscle
cross-section and throughout the entire length of the muscle. In contrast to previous reports,
there did not appear to be a regional concentration of histiocytes or ECM disniption. A
three-dimensional reconstruction of the SOL is presented in Fig2A.
The damage observed in the TA following downhill exercise was not nearly as
extensive, nor as evident throughout the muscle, as that seen in the SOL. When three-
dimensionally reconstmcted, the TA produced a slightly different protile than the SOL (Fig.
2B). As in the SOL, where present, mononuclear infiltrates were associated with vascular
regions and areas of the ECM that were randomiy situated in the muscle. A consistent,
regional pattern of mononuclear cellular infiltration was not observed in either the SOL or
the TA.
Mechanical stress, resulting fiom eccentnc exercise, is considered to be the major
contributing factor to muscle damage. This study conhned that the eccentric exercise
proximal
distal
tibialis anterior
proximal
w distal
Figurc 2. 'l'lircc diinciisioiinl rccoiistructioii o f ilic rat solcus niid tibialis antcrior rnusclcs 72 Iiours pasi-cxcrcisc, (A) soleus musclc and (il) tibialis aiiicrior inusclc. Opcri arrows (purplc)- tnonoiiuclcar iiifilinics, fillcd mows (green)= cxlroccllular matnx disniption, Omngc lincs = vascularisriiioii. Calibralion bar -. 2.5inin
protocol selected for this study produced detectable muscle histopathology in the SOL;
however, it was not as prevalent in the TA The 3D reconstruction analyses provided us with
a gross anatornical description of two characteristic indices of muscle pathology resulting
fiom eccentric exercise, Le., ECM disruption and infiammatory uifiltrates. Given the rather
homogeneous distribution in the SOL, quantification of the amount of ECM disruption and
infiarnrnatory ceils dong with cellular characterisation of the ùinammatory component was
not done.
In summary, ECM disniption and infiarnrnatory infiltrates were randondy distnbuted
in both the SOL and TA 72 hours after an acute bout of downhill running exercise with the
most intense response evident in the SOL rather than in the TA.
3 -3. Silicone Rubber Micromgiomaphv of Soleus and Tibialis Anterior Muscles in the Rat.
3.3.1, Introduction:
The pathophysiological basis of eccentric exercise-induced muscle darnage resulting
in subsequent muscle soreness is stiii unknown. Many hypotheses have been put forth
describing the cause of the initial damage (rwiewed in Stauber, 1989; Evans and Cannon,
1991; Kuipers, 1994). One hypothesis is 'a disruption in microcirculation'. This theory
suggests that the focal changes, which occur in muscle following damaging exercise, are a
result of rnicrocûculatory changes (Smith, 1991) that produce a dilation of the capillaries
and a sweIiing of the interstitial space (Peeze Binkhorst et al., 1989,1990). These factors, in
turn, may compromise the circulatory system and hasten the injury process (Smith, 199 1;
Evans and Cannon, 1991; Appel1 et al., 1992; Tidball, 1995; Kuipers, 1994).
The SOL and TA are two morphologicaliy and physiologically different muscles.
These differences (i.e., fibre type composition, force production, recmitment, etc.) and the
evidence indicating that downhill running preferentiaily causes damage in the SOL
(Armstrong et al., 1983; Ogilvie et al., 1988), and that submaximal uphiu exercise results in
dilation of SOL capillaries, suggested that the microcirculatory profile of the muscle may be
of importance in understanding the mechanism of eccentric exercise-induced damage.
Silicon rubber microangiography is a new, safe and easy technique that c m be used to
provide a clear and sensitive description of microcirculatory morphology. The technique has
been used successfufly in models of spinal cord injury (Koyanagi et al., 1993) and in muscle
(Plyley et ai., 1975, 1976). This study was undertaken in order to further describe the
microvascular anatomy of the rat SOL and TA muscles.
3.3 -2. Methods:
The method for perfùsion with silicone rubber Microfil has been described previously
(Koyanagi et al., 1993). Briefly, an arterial Iine was inserted into the femoral artery to
monitor infusion pressure. The silicone rubber (8 ml) (Mïcrofil; Flow Tek Inc., Boulder, CO)
was combined in proportion with a diluent (10 ml) and a catalysing agent (0.9 mi). Control,
non-exercised, animals (n=3) were anaesthetised with Somnotol (sodium pentobarbitol) and
prepared for transcardial perfusion, as described in General Methods (Chapter 2). Foflowing
injection of heparin into the lefi ventncle, Microfil was injected via a 20cc syringe with a 16
guage needle inserted into the left ventricle. A ligature was placed around the aorta and the
needle to secure the needle in place, and the right atrium was cut to allow for venous
drainage. The Microfil was then manualiy injected (18-20 ml per rat) at a pressure range of
100-130 rnmHg, which is approximately equivalent to normal rodent arterial blood pressure.
The animals were maintained at 4 ' ~ ovemight, and the muscles (SOL and TA) f?om
each h d l e g removed the following day. The muscles were placed in 10% formalin for one
week, and incubated for successive days in 25%, 50%, 75%, and 100% ethanol, then cleared
in methyl salicylate for 24 hours. The SOL and TA muscles were subsequently stored in
methyl salicylate until analysis.
3 -3 -3. Results and Discussion:
The technique provides a general protile of the vascular architecture, but as used in
these studies, would not be as useful for q u a n t m g changes in size or thickness of the
vessels, since there would be diiculty in measuring changes between control and
experimentai groups. However, this technique has been used to differentiate between very
small venous and artenal blood vessels on the basis of indentation of nuclei of endotheliai
cells in venous vessels (Koyanagi et al., 1993). If an increase in the branching of blood
vessels in muscles occurred following exercise, it may be possible to detect and measure such
changes using this technique in combination with standard morphological techniques used to
establish sprouting indices.
Employment of the method of Microfil pefision produced contrasting
microcirculatory morphologies between the SOL and the TA muscles in the rodent (Fig.3
AB). The rich, evenly and thîckly vascularised profile of the SOL muscles (Fig. 3A) was
readily apparent upon first observation, with many miniature branches feeding off larger
capillaries. In contrast, in al1 TA muscles, the microvasculature was clearly divided into two
distinct regions: one highly vascularised and one rninimally vascularised (Fig 2B). The
deeper, red portion of the muscle which attaches to the femur was populated with numerous
capillaries, branching into tiny vessels. In contrast, the white portion of the muscle was fed by
few capillaries and branching of microvessels appeared comparatively limited.
Figure 3. Silicon rubber micro-angiography of the rat SOL and the TA in the control condition A) Richly vascularized rat SOL. B) Vascuiarized region of the red TA and poorly vascularized white region of the TA White arrowheads denote fine branching of capillaries in A and 8, whiIe the black mow in (A) indicates an arterial vessel. Calibration bar = 8 mm.
At a finer level, complications may have been present with the collapse of a blood
vesse1 (e.g., due to obstruction or a change in pefision pressure). If so, one would expect to
see irregularly nUed patches of muscle in an otherwise homogeneously med area. We did not
observe this pattern in either SOL or T A In the TA a homogeneously filled area of greater
vascularisation was easily discernible and corresponded to the red, oxidative region of the
muscle; in contrast, the other haif of the muscle demonstrated a comparatively smaller
vascular tree, and corresponded to the white, largely glycolytic region of the muscle.
Patchiness of Microfil filling was not seen, ruling out the possibiiity that the observations
were the result of artifact, and therefore most Liely do reflect the actual fibre type pattern of
the muscle. 'Ihus, the Micronl technique is quite usenil in providing a qualitative assessment
of muscle vasculature.
The results of this experiment are in agreement with previous observations (Burke,
1981), and clearly indicate that the SOL is a highiy vascularised muscle, while the TA is
comparably vascularised only in its most medial portion. An in-depth and detailed
rnorphological assessment and analysis of changes in the rnicrocirculatory vessels before and
after eccentnc exercise would be required in order to detemine if a correlation between
microcirculatory change and darnage exists.
s m y 1:
CALCITONIN GENE-RELATED PEPTIDE IS INCREASED IN HINDLIMB
MOTONEURONS AFTER EXERCISE
4.1. ABSTRACT (modified fiom Int. J. Sports Med. (1997) 18: 1-7).
The purpose of this study was to investigate whether d o m running exercise,
which elicits muscle darnage and repak, also elicits changes in CGRP levels in hindlimb
motoneurons. Twenty, fernale, adult Wistar rats were divided hto £ive groups: control, 48
hour post-exercise (48 h), 72 hours (72 h), 2 weeks (2 wk) and 4 weeks (4 wk). The exercise
groups ran downhill for one 30 minute period. Histological examination of muscle fYom the
ankle extensors (triceps surae, TS) and flexors (anterior crural, AC) indicated the
characteristic presence of histiocytes by 48 h post-exercise in the TS, but not in the AC.
Paraforrnaldehyde-ked, 30 pm sections of the lumbar spinal cord (L2-L4) fiom the same
animais were incubated with polyclonal antisera to CGRP. The number of CGRP+ve TS
motoneurons increased significantly by 48 h after exercise (P = 0.001) versus control and
retumed to baseline values by 4 wk. In contrast, no significant changes were observed in the
AC motoneuron pool at any post-exercise interval. The temporal changes in numbers of
CGRP+ve TS motoneurons suggest that the expression of this neuropeptide may be
differentiaiiy regulated by exercise-induced changes in neuromuscular function, possibly as
related to muscle tissue damage/repair mechanisms, ancilor to remodeiiing at the
neuromuscular junction.
4.2. INTRODUCTION
CGRP is a 37 amino acid peptide locaiised to dense core vesicles in the presynaptic
motor nerve terminal (Matteoli et al., 1988), where it is released in a Ca2+ dependent manner
upon excitation of the rnotor nerve (Uchida et al., 1990), enhancing muscle contraction via a
cyciic AMP mediated pathway (Takami et al., 1986). Spinal cord transection (Piehl et al.,
19911, peripheral nerve section (Arvidsson et al., 1990; Piehl et al., 1991), castration (Popper
and Micevych, 1989; Popper et ai., 1992), and pharmacological blocks of neuromuscular
transmission (Sala et al., 1995; Tarabal et al., 1996b) are among the factors reported to
induce an increased CGRP expression in spinal motoneurons. While the role of CGRP in the
adult motor system is not entirely cIear, the weight of evidence suggests that it is involved in
the establishment and maintenance of the neuromuscular junction (Sala et al., 1995).
DownhÏll running is known to induce muscle pathology predominantly in those
muscles undergoing eccentnc lengthening contractions (Ogilvie et al., 1988). Chronic,
repetitive, eccentric exercise has been s h o w to result in an increased synthesis of muscle
proteins (Wong and Booth, 1990). The proposed mechanisms for muscle fibre adaptation
following downhill exercise include satellite ce11 proliferation (Dm and Schultz, 1987),
muscle fibre regeneration following muscle damage (Stauber, L989), and de novo protein
synthesis (Smith et al., 1997; Lowe et al., 1995; Booth and Kirby, 1992), processes which,
more than likely, will necessitate accompanying functiond a d o r morphological changes at
neuromuscular junctions. To date, the involvement of the nervous system in eccentric
exercise-induced muscular injus: and the subsequent repair processes have received scant
attention.
We wondered, therefore, whether unaccustorned exercise, leading to muscle
remodelling would result in an up-regulation in CGRP expression in the motoneurons
supplying the regenerating or remodeiiing rnotor units. To test this hypothesis, we used a rat
mode1 of eccentric exercise, consisting of a single 30 minute period of do& ninning.
Using intramuscular injections of fluorochromes to retrogradely label individual motoneurons
in non-exercised anirnals, we identifïed, and three-dimensionally reconstructed, the
topographic location of the motor nuclei imervating the rat ankle extensors and flexors. This
approach dowed us to demonstrate a differential increase in CGRP expression in the
extensor, but not the flexor, motor nuclei in the different groups of animals following a single
bout of downhill running. The results are discussed in terms of the physiological rnechanisrns
that may underlie exercise-induced changes in the expression of motoneuronal CGRP.
4.3 AdATERIALS AND METETODS:
4.3-1. Identification of Mofoneuron Pools in Non-fiercised Animals
In order to identfi the relative topographic locations of the triceps surae (TS) and
anterior crural (AC) motor nuclei in the spinal cord, a senes of prelirninary retrograde
labelhg experirnents was completed in control animais. In 6 animais, FluoroGold (4%; 10 p
1; Fluorochrome Inc., hglewood CA) was injected into the Ieft soleus (SOL) muscle and into
the contralateral (right) tibialis antenor (TA) muscle. In another 3 animals, the extensor
digitomm longus @DL) of both legs was injected with 5 pl of FluoroGold. The medial and
lateral gastrocnemii (MGLG) were labelled in a similar manner to the SOL and TA in 3
more animals (5% FluoroGold; 30~1; 5%, 30 pl fluorescein-conjugated Cholera Toxin b
subunit - CT; List Biological Laboratories, CA) (see Appendix 1). The tracer was injected
into each muscle as descnbed in Section 2.1.3. Three days after the muscle injections, the
animals were administered an overdose of sodium pentobarbitol and sacrificed by transcardial
perfusion.
The lumbar region of the spinal cord (L2 to LS) was removed intact with the dorsal
root ganglia (DRG) still attached and cryoprotected ovemight in 20% sucrose in 0.1M
phosphate buffer, pH 7.1 (BDH, Mississauga, ON). The ventral root entry zones (VREZ)
were identified by fïrst locating and matching the lumbar enlargement of the spinal cord at L4
with its correspondhg DRG, and then rnarking the spinal cord with a coloured waterproof
Pen. Each of the remaining lumbar segments (L2, L3) were sirnilarly identified, and the
rostrocaudal length of each segment was measured. Tissues were then fiozen in 2-
methylbutane at -80" C. Transverse serial sections (10 pm) of the spinal cord were mounted
on slides, coverslipped with Mowiol (Hoescht, Montreal, PQ.), and anaiysed for
fluorochrome-labelied motoneurons (cc TheriauIt and Tator, 1994). The rostrocaudal extent
and dorsoventraYrnediolateral positions of these individual motor nuclei were three-
dimensionally reconstructed using a computerised image analysis system (MCID, Imaging
Research Fnc,, Ste. Catherine's, ON) (Fig. 4).
Restrkting the sampling region of the lumber spinal cord for both groups (AC and
TS) established a clear, strict, identinable region of data sampling. The motoneuron pools of
the AC and TS extended slightly rostrally and caudally to this defined area. Hence, with our
restricted method, an underestimate of the counts was likely (see Section 4.3 S). Note that in
these studies, non-serial thick sections were analyzed, and that great care and strict criteria
were appiied to exclude the possibility of double counts being included in the data set.
VREZ
VREZ
VREZ
Figure 4. Topographie localisation of FluoroGold labelled TS and AC motoneurons. A) 3-Dimensional reconstmction of the L2L4 segment of the rat lumbar spinal cord outlining the sampling regions and the different topographie locations of the TS and AC motoneuron pools in the ventral hom after retrograde labelling experiments. The region of rostrocaudal overlap is the area between the arrows, Arrowheads denote the level of the ventd root entry zone (VREZ) for each lurnbar segment. Calibration bar = lrnm2. B) FluoroGold labelled AC motoneurons fiom retrograde labelling studies. C) F'luoroGold labelled TS rnotoneurons fiom retrograde labelling studies. (Note that in these studies, fluorochrome and CGRP-positive motoneurons were not identified in the same cross-sections; B and C; Calibration
bar= 100pm).
4.3 -2. hen'mentul Desim and Tissue Processing
Twenty, female, adult Wistar rats, 300-325g, were randomly placed into five groups:
control (non-exercise), 48 hours, 72 hours, 2 weeks, and 4 weeks post-exercise. In order tu
moid any potential damage associated with the intrmuscular injections of FluoroGold, we
did not retrograde& label the motonetirons in the grarps of exercised mimals in this shrdy.
The right SOL, MG, LG, and TA were quickly excised and snap fiozen in Zmethylbutane
(Canlab/Baxter, Mississauga, ON) and stored at -80°C for subsequent muscle histochemistry.
FoIlowing aldehyde perflsion, the corresponding muscles were removed fiom the
contralateral lefi leg and placed in 10% neutrai buffered formalin (BDH, Mississauga, ON)
for par& embedding and subsequent staining with haematoxylin and eosin (H&E, Sigma,
USA).
4.3 -3. Muscle Histolom:
The SOL, a slow twitch ankle extensor (Armstrong et aI., 1983), has been extensively
investigated in rodent models of eccentricdy biased exercise, and has been reported to
develop marked stnictural damage dunng downhill running (Smith et al., 1997). In contrast,
the Tq a fast twitch ankle flexor, has not been reported to develop structurai damage during
downhill mnning (Ogiivie et al., 1988). The extensor muscles have been reported to be
dflerentially afYected (Ogdvie et al., 1988; Armstrong et al., 1983), although the issue is
complicated by differences between species and exercise paradigms, and by divergent cnteria
used for assessing the degree of baseline and post-exercise darnage (Stauber. 1989; Smith et
al., 1997). Therefore, in our study, the pefision-fked SOL musdes were paraffin embedded,
serially cut into 6 prn sections, and stained with H&E to evaluate the extent of the
histological darnage. Likewise, the TA muscles were serially sectioned (20 pm) in a cryostat
(JUNG CM 3000, LEICq Canada) and stained with H&E for similar histopathological
analysis.
4.3 -4. Imunocvtochemish> of SjpinaI C o d Tissue
The generd immunocytochemicai methodologies have been descnbed in Section
2.1.4. Briefly, fiozen spinal cord lumbar regions (L2-LS) were senally sectioned at 30 Pm.
Free floating sections were washed in 0.1M phosphate buffered saline (PBS), pH 7.1, and
blocked with 10% normal goat serum (Gibco, CanlabBaxter, Mississauga, ON); they were
then uicubated overnight at 4°C with a polyclonal antibody to CGRP (rabbit aCGRP Rat [l-
371) antibody; Genosys, TX) at a 1 :3 500 titer. The sections were then processed using the
ABC kit with a goat anti-rabbit secondary antibody (Vector Laboratories, Mississauga, ON)
with subsequent visualisation using di-amino-benzidine @AB; Sigma, USA) as the
chromagen. Finally, the sections were mounted on slides and coverslipped with Entellan
(BDH, Mississauga, ON).
4.3 S. ~uantifation of CGRP- osi if ive Motonewons
The topographic locations of the TS and TA motor nuclei deterrnined fkom the
results in Section (4.3.1) above allowed us to select two separate, non-overlapping
rostrocaudal regions of the lurnbar spinal cord for subsequent quantification of the CGRP+ve
motoneurons. The standardised region selected for quantification of the TS motor nuclei
began at the L4 VREZ and extended rostrally 1800 pm hto lumbar segment L4 (Fig. 4A).
The AC motoneurons were found to be located in lumbar segment L3, and were quantified
beginning at the L2 ventral root, extending caudaiiy 2300 p m hto the L3 lumbar segment
(Fig. 4A)). A region of the L3 segment (approximately 500 pm) located at the very end of the
L4 segment and the very beginning of the L3 segment contained some motoneurons fiom
both the AC and TS motor pools. While the TS and AC motor pools occupied distinct
donoventrd and mediolateral positions in the transverse sections of the cord, this 500 p
region was not anaiysed in order to avoid any potential rniscounts. The quantification of the
CGRP+ve motoneurons, and the identification of their topographic distributions within the
grey matter, were completed on the MCID Image Analysis System (see Section 2.1.5). The
counts of motoneurons staining positiveiy for CGRP were obtained by selecting only those
somata with observable nuclei; profiles not containing a nucleus/nucleolus were not
quantitated (cf, Theriault and Diarnond, 198 8a).
4.3.6. Statisfical Analvsis:
The statistical analyses, and software have been described in Section 2.1.6. Bnefly,
for this data set, a one-way ANOVA was used to detennine statistical signifieance between
groups. An alpha level of 0.05 was accepted as significant. AU data determined to be
significant were marked with an asterisk.
4.4. RESULTS
4.4.1. TS and AC Motoneuron Pools in Non-Exercised Animals
Preliminary retrograde tracing experiment s with FluoroGold localised the AC
motoneuron pool to the extreme dorso-lateral edge of the ventral hom (Figs. 4,5,6), and
revealed that it extended 2300 pm into the L3 segment rostrocaudally fiom VREZ L2 to
VREZ L3 (Fig. 4A). The TS motoneurons were also positioned at the lateral edge of the
ventral hom, but were placed more medially and ventrally than the AC motoneuron pool
(Figs. 4,5,6), and extended 1800pm in the rostral-caudal direction corn VREZ L4 to VREZ
tibialis anterior extensor digitorurn longus
triceps surae: soleus
l lateral gastrocnernius media1 gastrocnemius
lotal # of HRP-Zabelled rnotonertrons previoicsly reported
(x 3 SEM)
Table 1. Counts of retrogradely labelled triceps surae (TS) and anterior crural (AC) motoneurons in the lumbar P -4 spinal cord. Quantification in the L4 segment was restricted to a distance of 1800pm beginning caudally at the
ventral root entry zone (VREZ) of M. Quantification of FluoroGold-labelled motoneurons in the L3 segment was
restricted to a distance of 2300ym beginning rostrally at the VREZ of L2 and extending caudally towards the
VREZ L3. (HRP=horseradish peroxidase; n=animal; x = mean; SD = standard deviation; SEM = standard error of
the mean). = Nicolopoulos-Stournaras and lles (1983) highest count reported from n=23 muscles, = Swett et al.
Figure 5. Staining patterns of TS and AC CGRP+ve motoneurons in control, non-exercised sedentary animais. A) Lmbar section (L4) of rat spinal cord indicating the TS motoneuron pool; dashed circles indicate the location of the TS motor nucleus in transverse section. B) Immunostained CGRP+ve motoneurons within the region descnbed in A. C) Lumbar section (L3) of rat spinal cord; dashed circles indicate the the location of the AC motor nucleus in transverse section. D) Immunostained CGRP- +ve motoneurons in the AC within the region descnbed in C. Arrowheads in (B) and (D) indicate ceils selected for quantitation. (A and C,
calibration bars=400prn; (B and D, calibration bars=lOOpm).
Figure 6. Staining patterns of TS and AC CGRP+ve motoneurons 48 hours foliowing
downhill exercise. (A) Lumbar section (L4) of rat spinal cord indicating the TS
motoneuron pool; dashed circles indicate the location of the TS motor nucleus in
transverse section. (B) Lumbar section (L3) of rat spinal cord following downhill
running; dashed circles indicate the location of the AC motor nucleus in transverse
section. (A and B; calibration bar, 500pm). (C) CGRP+ve motoneurons in the TS
within the region described in A. (D) CGRP+ve motoneurons in the AC within the
region described in B (C and D; calibration bar, 50pm).
Figure 7. Photomicrographs of H & E stained SOL and TA muscles 48 hours after downhill exercise. Paraffh-processed transverse sections of SOL in the control condition (A) and following one bout of downhill exercise (B). Frozen-sectioned TA muscle cross-section in the control condition (C) and following one bout of downhill exercise @). Filled white arrows indicate inflammatoty cells, open white triangles mark regions of extracellular matrix disnxption. Calibration bars = 50 prn
time following exercise
Fimire 8 . DiEerential increase of motoneuronal CGRP in the TS and AC motor nudei after downhill
exercise. (A) Increased numbers of CGRPtve TS motoneurons were observed in the 48 hour and 72
hour groups as compared to control (* denotes significant Merence as cornpared to control at the
P<O.OOl level; ANOVA). (B) Similar changes were not observed between control and experimental
conditions in the AC motor nucIei.
L3 contained in lumbar segment L4 mg. 4A). The numbers o f retrogradely labelled
motoneurons identified in the AC and TS motor pools are given in Table 1. Generaiiy, the
fluorochrome-labelled ce11 bodies in each nucleus were easily recognised, and were
compacted together with dendritic processes extending rostro-caudally throughout the
serially sectioned tissue Fig. 4B,C). Thus, in a transverse section, the relative positions of
the extensor and flexor motor nuclei were visudy and topographicaily distinct, which
ensured that in the unlabeZZed non-exercise and exercise groups, the CGRP+ve motoneurons
could easily be associated with either the AC or the TS motor nucleus (Fig. 5,6).
4.4.2, CG@ in TS and AC Motoneurons Af?er DownhiZZ Ekercise
Using retrograde labelling to identfi the locations of the TS and AC motor nuclei,
the numbers of CGRP+ve motoneurons in the difEerent groups of experimental animals were
then quantitated (Fig. 5A,C and 64B). In non-exercised animais, approxhatefy 50 TS
motoneurons stained positive for CGRP, while 150 AC motoneurons were CGRP+ve (Fig.
5A,B). The number of CGRP-positive TS motoneurons was found to increase f?om 50 to
150 (P <0.001) by 48 h d e r downhiii running. The counts retumed to control values by 4
wk (Fig.8A). In contrast, significant changes in the number of CGRP+ve motoneurons were
not observed in the AC motor nuclei after downhill mnning when compared to controls at
any tirne-point following exercise (l? >0.05; Fig. 8B).
4.4 -3 . HLstol~cal Dantape A fier Ekercise
The histologicai profiies of the TS and AC muscles 48 h after downhill exercise are
illustrated in Figure 7. Although not quantitated, at this post-exercise interval, charactenstic
idammatory celis were evident within the TS' (SOL), including mononuclear cells, such as
histiocytes and polymorphonuclear granulocytes. Connective tissue disruption, resulting in an
increased intrafascicular space, was readily observed in the SOL muscle sections 48 h
following downhiil exercise (Fig. 7B). Comparable pathologicai indices were not observed in
the AC at 48 h post-exercise (Fig. 7D).
4.5- DISCUSSION
In this study, we provide new evidence that physiologicaUy reasonable exercise
affects neuropeptide levels in motoneurons. Our results demonstrate that one 30 minute bout
of downhill mnning in sedentary animais results in a signi6cant and prolonged hcrease in the
numbers of CGRP+ve motoneurons innervating the ankle extensor muscles, but not the anlde
flexors,
4.5- 1. Identification of Mofor Nuclei
As a result of the retrograde tracing experirnents, two non-overlapping samplïng
regions were selected in the rat lumbar spinal cord; these regions contained the motor nuclei
of the extensor TS and flexor AC muscle groups. The counts of fluorochrome-labelled
motoneurons in the identified motoneuron pools (Table 1) correlated well with the
horseradish peroxidase @XP) data of previous investigators (Table l), but Our counts were
consistently lower than those previously reported due to both a more restrictive sarnpling
region and a more conservative labeiling technique. Since spinal cord tissues were cut at 30
pm thick sections, this could have contributed to missed counts of smalI neurons if the
nuc1euslnucleolus was not visible in the section being analyzed. ln addition, since effective
antibody penetration into the tissue sections was in the order of 5-7 gm, ce11 bodies located
in the centre of the slice may not have been adequately detected. Importantly, the TA and
EDL have sirnilar fast-twitch fibre type pronles (Armstrong and Phelps, 1984), and did not
display exercise-induced muscle injury as a result of this exercise protocol (Wemig et al.,
i99ia).
4.5.2. CGRP andMuscle Fibre Tm
Substantial immunocytochemical evidence indicates that the motoneurons supplying
fast-twitch muscles (e-g., EDL, LG) show higher levels of CGRP staining than the
motoneurons innervating muscles composed primarily with slow-twitch fibres (e-g., SOL),
although both groups are known to express CGRP (Piehl et al., 1993; Forsgren et al., 1992).
Since the SOL motoneurons contain relativeiy low basehe levels of CGRe (Piefd et al.,
1993), and the SOL muscle appears quite susceptible to exercise-hduced darnage (Smith et
al., 1997; Ogilvie et al., 1988; Armstrong et al., 1983), perhaps the SOL motoneurons up-
regulate this neuropeptide in response to the induced growth and repair processes within the
muscle. Altemately, if muscle fibre type is the important variable (Friden et al., 1988; Friden
and Lieber, 1992), then the motoneurons associated with the LG or MG may show an
increased expression of CGRP.
4.5 -3. Neuromzîscular Plasriciïv
The sprouting of the motor nerve terminal at the neuromuscular junction has been
charactensed following pharmacological blocks of neuromuscular transmission (Brown,
1984), and is accompanied by increased irnmunoreactivity for CGRP in the pre-synaptic
elements during the sprouting phase (Sala et al., 1995), and an increased CGRP m W A
expression in the ce11 bodies of the motoneurons (Tarabai et al., 1996b). Tarabal and CO-
workers (1996) have s h o w a positive correlation between CGRP content in the
motoneurons and the amount of intrarnuscular nerve sprouting following local paralysis with
botulinum toxin. As well, during post-natal development, the levels of CGRP are high in
muscle pnor to the elhination of polyneuronal innervation, and decrease to adult levels as
the neuromuscuIar junction matures (Matteoli et al., 1990; Andreose et al., 1994). The time-
course of the changes in CGRP mRNA expression in motoneurons following
pharmacological blockade or surgicd interruption of neuromuscular connectivity, which
result in sprouting at the neuromuscular junction (Sala et al., 1995), shows a similar temporal
profile foilowing downhiil exercise to the one demonstrated in the present study. Wernig et
al. (1991a) used a zinc-iodide osmium tetroxide stalliing technique to show sprouting of the
motor nerve terminals in the SOL in mice, 3 days d e r a single 9 hour bout of voluntaxy
wheel-mnning, providing evidence that exercise can produce morpho1ogicaI changes at the
neuromuscular junction; however, they did not investigate any changes in the motoneuron
cell bodies. Therefore, at the present tirne, the weight of evidence suggests a role for CGRP
in the remodelling and plasticity of the adult neuromuscular junction in response to exercise
demands although, apart f?om the present study, this response has not been previously
investigat ed.
4 -5 -4. Muscle D-e and Unaca~storned Activitv
Unaccustomed, strenuous exercise causes both microscopic and macroscopic muscle
damage in animals (Smith et al., 1997; Ogilvie et al., 1988) and humans (Stauber, 1989). In
the present study, a mode1 of downhill running was chosen based on reports that eccentric
activity would induce more disruption in a working muscle than concentric activity (Ogilvie
et al., 1988). In vivo, non-voluntary, electrical stimulation models of repetitive eccentric
contractions in rats (Wong and Booth, 1990) and rnice (Lowe et al., 1995) have shown that
eccentric exercise increases protein synthesis. As well, recent work by Smith and CO-workers
(1997) has provided evidence for increased muscle damage, followed by the expression of
developmental isomyosins and activated satellite cens in the SOL foliowing a single bout of
downhiil running exercise. Therefore, eccentnc running was used in Our study simply as a
mode1 that would be expected to induce muscle remodehg. Our purpose was to determine
if this muscle r emodehg would be associated with an increased neuropeptide expression in
motoneurons innervating the af3ected muscles.
4.6. CONCLUSION
We have documented the tirne-course of the changes in the number o f CGRP-positive
rnotoneurons innervating the TS, but not in the AC muscles following downhill exercise. One
explmation for the increase in C m immunoreactivity in the TS motoneurons, but not in
the AC motoneurons, may be the greater demand for neuromuscular remodehg at the TS
motor nerve temhals. We hypothesise that the elevated levels of CGRP in the TS
motoneurons are in response to increased target muscle demands for an enhanced synaptic
contact a d o r the subsequent stabilisation of these affected neuromuscular junctions. Further
studies are required to identify which of the three muscles, (SOL, MG, LG) is the
predominant responding population in the TS, and whether the morphologically detectable
changes at the neuromuscular junctions accompany the time-course of changes in CGRP
levels in the spinal cord.
4.7. Ceil Size Distribution of CGRP+Ve Motoneurons Following Downhill Exercise:
4.7.1 .. Introduction.
Motoneurons in the mammalian spinal cord are generally classified into two types
based on their cell size: alpha and gamma motoneurons. Alpha motoneurons innervate
extrafusal muscle fibres via alpha motor axons and intrafusal muscle fibres via beta motor
axons. Alpha motoneurons generally have a higher firing threshold than gamma motoneurons
due to their larger ce11 sue (Burke, 1981). kvons of the smailer gamma motoneurons only
innervate muscle spindles (Burke, 1981; Hunt, 1990). Efferent input from gamma or beta
motor axons that innervate the intrafùsal muscle fibres actively regulates the spindle poles
during contraction and evoke a sensory response within the muscle spindle (Hunt, 1990).
Forsgren et al. (1992) have reported CGRP-like immunoreactivity on the surface of
cap-like structures of intrafùsal fibres in the polar regions of the muscle spindle. These cap-
like structures likely constitute the motor endplates of the gamma motoneurons, since their
motor nerve tenninals are present on both the bag and chah fibres in this region of the
muscle spindle (Hunt, 1990). Based on motoneuron celi size, CGRP immunoreactivity is
most often observed in the alpha motoneurons of large rnotor units (Arvidsson et al., 1993;
Piehl et al., 1991, 1993). Small cell bodies, considered to be gamma motoneurons, appear to
have little CGRP immunoreactivity (Arvidsson et al., 1993) or to lack it entirely (Piehl et al.,
1991, 1993).
Since eccentnc lengthening contractions most Iikely require enhanced tonic spindle
activity via increased gamma motoneuron input, we wondered whether an elevation in the
numbers of CGRP +ve gamma rnotoneurons might help to explain Our results. In support of
this idea, we noticed intensely immunopositive profiles in muscle spindles (this data remains
to be quantitated).
4.7.2. Methods:
The materials and methods are the sarne as described in Sections 2.3 -2, 2.3.4., and
2 . 3 5 Motoneuron ceil bodies were divided into two groups based on average diarneter
measurement, where cells with diameters greater than 20pm were counted as alpha
motoneurons. Ce11 bodies with average diameters equal to or less than 19.9pm were counted
as gamma motoneurons. This method was similar to that established by Henneman et al.
(1965) based on electrophysiological studies, and anatomical studies of Campa et al. (1970,
1971; Burke, 198 1, 1982).
4.7.3. ResuIts and Discussion
The cell size distributions of aU CGRP+ve motoneurons in the TS and AC motor
nuclei were determined at ali t h e points following exercise. No signifïcant daerences were
observed in any of the groups, compared to control, in either of the two motoneuron pools
(Fig. 9) (P>0.05). There appeared to be an increase in smaiier diarneter rnotoneurons at 72 h
post-exercise; however, this value was not significantly daerent from either the control or
two week values. The data obtained in this 'pilot' study were based ody on CGRP-
immunoreactive profiles, without adequate knowledge of their parent motoneuron pools. To
examine this isssue more ngorously, ce11 body measurements will need to be made using
FluoroGold to retrogradely-label CGRP+ve motoneurons.
Since CGRP has been observed at intrafùsal endplates in rodent muscle (Forsgren et
al., 1992), we have made the assumption that CGRP may be present in gamma motoneurons
even though there have been conflicting reports (Arvidsson et al., 1993). Furthemore,
although it has been well established in the feline that alpha and gamma motoneurons c m be
distinguished based on ce11 size (Burke, 1981; Henneman et al., 1965), Our acceptance of
19.9pm and smaiier as the average diameter sue of a gamma motoneuron in the rat may have
resulted in the inclusion of srnall alpha motoneurons in the sarnple. There is currently no
evidence in the Literature distinguishg rat alpha and gamma rnotoneurons on the basis of ce11
body size. In consideration of this latter fact, perhaps subsequent experiments will
circumvent the technical Limitations of identimg gamma motoneurons by size, perhaps by
developing a phenotypic marker for this ceii type. Neuroanatomical markers that can
distinguish between alpha and gamma motoneurons are thus required.
g a m m a size motoneumns
O aipha sée rnotoneurons
time following exercise
Figure 9. CeIl size distribution of CGRP +ve rnotoneurons as a hction of t h e following exercise.
A) The response of CGRP+ve small, (presurnptive gamma) and large (presumptive aipha) triceps
surae motoneurons before and after downhill exercise. B) The response of CGRP+ve small and large
anterior crural rnotoneurons before and after ciownhiil exercise.
CHAPTER V.
STUDY 2:
E C C W C EJERCISE PREFERENTLALLY INCREASES CGRP IN
MOTONEURONS INNERVATING FAST GLYCOLYTIC MUSCLE FIBRES
5.1 ABSTRACT (modified fkom J. Appl. Physiol. (1999) submitted)
We have recently reported an increased number of CGRP+Ve motoneurons supplying the
h d h b extensors, but not in the flexon, following downhill exercise. The purpose of the
present study was, first, to idente the responding population with respect to muscle and
motoneuron pool, and second, to investigate whether corresponding changes in CGRP could
be observed at fibre type-identified endplates. Thirty-four, adult, female rats were divided
into three groups: control, 72 h and 2 wk post-exercise. FluoroGold (15 pl) was injected into
the SOL, LG, and both the proximal (mixed fibre type), and distal (fast twitch glycolytic)
regions ofthe rnediai gastrocnemius @MG and MG, respectively). The experimental groups
ran d0wn.h.U for 3 0 minutes. The number of FluoroGold/CGRP+ve motoneurons within the
pMG and dMG motor nuclei increased by 72 h after exercise (P<0.05). No significant
changes were observed in the SOL or LG motor nuclei at any post-exercise interval. The
number of aBuTx/CGRP+ve motor nerve terminais was found to increase exclusively at fast
twitch glycolytic muscle fibres at 72 h and 2 wk post-exercise (P<O.05).
5 -2 INTRODUCTION
Immunocytochernical (Forsgren et al., 1993; Piehl et al., 1993) and in situ
hybridisation (Blanco et al., 1997) studies in control animals have shown that motoneurons
supplying fast-twitch muscles (e-g., extensor digitorum longus) show higher levels of CGRP
staining than motoneurons imervating muscles of slow-twitch fibre type (e-g., soleus). A
sunilar pattern of CGRP expression is observed in the muscle, with CGRP found
predominantly at rnotor endplates on fast-twitch muscle fibres (Forsgren et al., 1993;
Forsgren et al., 1992). However, none of these studies have correlated CGRP expression
patterns in identified (Le., retrogradely labelled) motoneurons with motor endplates identified
according to muscle fibre type.
While the role of CGRP in the normal adult motor system is not entirely clear, the
current fiamework of evidence suggests that it is associated with pre-synaptic sprouting and
post-synaptic structural changes at the neuromuscular junction (Fontaine et al., 1986, 1987;
Osteriund et al., 1989; Changeux, 1991; Sala et al., 1995; Tarabal et al., 1996). Any form of
experimental manipulation that disrupts the connection between the motor nerve and the
neuromuscular junction, either through surgical (Streit et al., 1989; Arvidsson et al., 1990;
Piehi et al., 199 l), or pharmacological (Sala et al., 1995; Tarabal et al., 1996b) intervention,
results in an up-regulation of CGRP peptide andor mRNk CGRP expression also increases
foilowing spinal cord transection (Arvidsson et al., 1989; Piehl et al., 1991) and androgen
depnvation (Popper and Micevych, 1989; Popper et al., 1992). To further investigate the
role of CGRP in the normal, intact, adult, neuromuscular system, Our approach was to
develo p a 'non-interventional' experimental paradigm, which provides a p hysiological
challenge to the motoneuron and its target. We recently demonstrated that CGRP expression
in hindlimb motoneurons increases following an acute bout of downhill running exercise in
sedentary anirnals (Homonko and Thenault, 1997). The results showed that CGRP
expression remained elevated over a 2 week penod, returning to baseline by 4 weeks, in the
motoneurons of the ankle extensors (triceps surae; Le., muscles perfoming eccentric
contractions), but not in the ankle flexors (antenor crural; Le., muscles perforrning concentx-ic
work).
We now idente the respondmg motoneurons, their fibre type association and the
the-course of change in CGRP expression foilowing unaccustomed eccentrk exercise.
Intrarnuscular injections of FluoroGold were used to retrogradely identG motoneurons
supplyhg the soleus, lateral gastrocnemius, and the proximal and distal regions of the medial
gastrocnemius (SOL, LG, pMG and dMG, respectively). Changes between control and
experimental groups were quantified using double-labelling immunofluorescence techniques.
5.3 MATERIALS AND METHûDS
5 -3.1 hiperimental Protocoi and Tissue Processing
In order to idente the responding population(s) of motoneurons, a total of thirty-
four, adult, female, Wistar rats, 250 - 275 g, were used for this study. All animds, with the
exception of the animais used in the glycogen depletion study below, were aven
intramuscular injections of 4% FluoroGold (Fluorochrome Inc., Inglewood CA).
Identification of the topographie locations of the SOL, LG, and MG motor nuclei in the
spinal cord was completed in a series of retrograde labelling experiments that has been
described previously (Sections 2.1.3, 3.3.1). FluoroGold (1 0 pl) was injected into the belly of
the left SOL muscle of I I animals and into the right LG muscle (15 pl; belly portion) of 9
animals. In 14 animals, the proximal-media1 region of the medial gastrocnemius @MG) of the
left leg and the distal-media1 region of the medial gastrocnernius (dMG) of the right, were
injected with 15 pl of FluoroGold (see Fig. 10). Three days following FluoroGold injection,
animals were randomly placed into either a control (non-exercise) or exercise group.
Exercising animals foliowed the standard protocol (see Section 2.1.1) and were then allowed
to recover for a period of 72 h or 2 wk. These t h e points were selected based on the results
nom Study 1 (Chapter IV).
FluoroGold injection
O overlap regions
Figure 10. Schematic repraentation of the rat hindlimbs (lefi and right MG) as viewed fiom the
dorsal plane. Dark shaded regions indicate FluoroGold injection sites in the proximal region of the
left MG, and the distalregion of the right MG. Muscle fibre type is indicated in the clear and hatched
regions (FG,FOG,SO).Overlap regions = area of FluoroGoId injection and muscle fibre type.
Calibration bar = 1.5 cm.
At the tirne of sacrifice, the anirnals were adrninistered an overdose of sodium
pentobarbitol(l.0 ml; 60 mg- ml-'). Pnor to the fixation perfùsion and under anaesthesia, the
MG was quickly excised, with the proximal and distal regions separated, sliced into 1 mm
thick cross-sections, mounted in OCT embedding media on cardboard, and snap fiozen in 2-
methylbutane (Baxter Canlab, Mississauga, ON) imrnersed in liquid nitrogen (-80°C). The
spinal cord was harvested foIlowing transcardial perfùsion with 500 ml of 4%
paraformaldehyde (pH 7.35-7.45; BDH, Oakville, ON). The lumbar region of the spinal cord
(L2 to L5) was removed intact, post-ked in 4% paraformaldehyde for 18 h, and
cryoprotected overnight in 20% sucrose in O.1M phosphate bufEer, pH 7.2 (BDH, Oakville,
ON). Tissues were then fkozen in 2-methylbutane at -80°C for subsequent muscle enryme
histochemistry and irnmunocytochernistry.
5.3 -2. JmmunocytochemishV q f the S~inal Cord
The immunocytochernical methodologies have been reported previously (Homonko
and Theriault, 1997; TheriauIt et al., I993), and are briefly descnbed here. The iumbar
regions (L2-L5) of the fi-ozen spinal cord were serially sectioned at 10 Pm. In an attempt to
reduce inter-expenmental variability, the cross-sections of the spinal cord fkom each of the
experimental groups were placed on the same slide (Le., control, 72 h, 2 wk). The sections
were then washed in 0.1M phosphate buffered saline (PBS), pH 7.1, blocked with 10%
normal goat serum (Gibco, Baxter Canlab, Mississauga, ON), and then incubated overnight
at 4°C with a polyclonai antibody to CGRP [rabbit a-CGRP (Rat, 1-3 7) antibody; Genosys,
TX] at a 1:2000 dilution. The tissues were then processed using the ABC kit with a goat
anti-rabbit secondary antibody (Vector Laboratories, Mississauga, ON), followed by
incubation with avidin-conjugated Texas Red fluorophore (Vector Laboratories,
Mississauga, ON). An immunofluorescent secondary antibody was used instead of DAB
based staining in order to detect CGRP+ve motoneurons that CO-localised the fluorescent
FluoroGoId signal in the sarne tissue sections. The slides were coverslipped with Mowiol
(Theriault and Tator, 1994).
5 -3.3. Acetylcholinesterase Histochemistrv and I m u n o c v t o c h e m ~ of Muscle Tissue
The rnotor endplates were ùiitidy identified by acetylcholinesterase (AChE)
histochernistry in order to determine the innervation pattern and location of the endplate
zones in the two regions of the MG. Subsequent immunocytochemistry experhents directed
at CO-localising the CGRP response at the motor endplate employed fluorescein conjugated
aBuTx to identi@ the neuromuscular junctions in MG muscle sections. Frozen, unfked,
muscle tissue was serially sectioned at 12 p. Three series of samples were collected every
200 pm and placed directly onto slides. The fkst series was processed for AChE
histochemistry, the second series for immunocytochemistry, and the third series for
myofibrillar adenosine triphosphatase (myosin ATPase) determination (see below). Briefly,
for AChE histochemistry (Pestronk and Drachrnan, 1978a), cross-sections on slides were
incubated in 20% sodium sulphate (BDH, Oakville, ON) for 3 minutes, foiiowed by a wash in
deionized water, and then incubated in a reaction solution pH 7.2 (5-bromoindoxyl acetate,
ethanol, &Fe(CN)6, K4Fe(m6-3Hfl, TRIS-HCl, TRIS-Base, CaC12; Sigma, USA) for 15
minutes, washed in deionized water, quickly dipped in eosin (Sigma, USA), then defatted,
and coverslipped with Entellan (BDH, OakvilIe, ON).
For immunocytochernistry, eozen serial sections fiom unfïxed muscle tissue were
coiiected on slides as descnbed above. The slides were washed in 0.1M PBS, pH 7.1, and
then immersed in 4% parafomaldehyde fixative (pH 7.4; BDH, OakvilIe, ON) for 30
minutes. The slides were then washed in 0.1M PBS, pH 7.1, blocked with 10% normal goat
serum (Gibco, Baxter Caniab, Mississauga, ON), and incubated ovemight at OC with CGRP
antisera, at a 1:1000 dilution. The tissue sections were processed using an ABC kit with a
goat anti-rabbit secondary antibody (Vector Laboratories, Mississauga, ON), followed by
incubation with avidin-conjugated Texas Red (TR; Vector Laboratories, Mississauga, ON).
After washing in 0.1M PB, the tissues were incubated overnight in fluorescein conjugated
alpha-bungarotoxin (FITC-aBuTx; 1 : 1500; Sigma, USA). The slides were then coverslipped
with Mowiol as described above.
5.3.4. Mvo fibnnllar A Pase Histochemisty
The muscle fibres were identified and classified as slow twitch oxidative (ST or type
1), fast twitch oxidative glycolytic (FOG or type IU), or fast twitch glycolytic (FG or type
IIB) fkom the ATPase stain (cf, Brooke and Kaiser, 1970). Briefiy, fiozen muscle sections
were mounted on slides and placed in Coplin jars containing acid medium (NaC2H30b KCI,
pH 4.6; BDH, Oakville, ON) for 4 min., washed in basic medium (C2HsNO2, CaCI2, NaCl,
NaOH, pH 9.4; BDH, Oakville, ON) for 30 seconds, placed in incubation medium (basic
medium + ATP, pH 9.4, 30 min., 37OC; Sigma, USA), foiiowed by a series of washes in
CaClz @DY Oakville, ON), rinses in CoCh (Fisher, Mississauga, ON), distilled H20, ending
with a 1 min. incubation in 20%(NH& (Fisher, Mississauga, ON). Slides were then cleared
and coverslipped with Enteiian (BDH, Oakville, ON).
5.3.5. GZvco~en Stuc&: Tissue S m l i n g and Anabses
In order to evaluate whether the exercise protocol activated both the proximal and
the distal muscle regions of the MG and ail muscle fibre types, we exarnined the pattern of
glycogen depletion in both regions of the muscle afler downhill running. Two groups of
animals were divided into control (non-exercise; n=4) and downhill ninners (n=4). The
ninning group completed the exercise protocol and was sacrifced 20 minutes later. The MG
(2 per animal) was quickly dissected out and snap frozen as described above. Frozen control
and exercise pMG and dMG muscle were serially sectioned, placed on the same siide, and
histochemicaliy analy sed for glycogen content using the Perïodic-Acid Schiff stain (PAS;
Drury and WaiIington, 1990a) and on separate slides for myosin ATPase Prooke and
Kaiser, 1970).
The relative fibre populations were quantifïed by counting and categorising 100-120
fibres in pMG and 500-600 fibres in dMG fiom each sample in randomly chosen fascides
distnbuted throughout the entire cross-sectional area of the sample (see Table 2). These
values represent an estirnated 10% of the total number of al1 muscle fibre types present in the
proximal compartment (FG+FOG+SO; 300/2900) and 12.5% of the total number of muscle
fibres present in the distal compartment (500/4000). Using bright-field settings with a Leica
DM/RB microscopey the staining intensities for glycogen content in the fibres were analysed
by the relative optical density (ROD) measurement using the MCID Image Analysis Software
(Irnaging Research Inc., Ste. Catherine's, ON). Baseline grey scale values were standardised
at the be-g of the analysis and used to establish a cnterion illumination throughout the
entire analysis.
5 -3 -6. Quanti fation of CGRP+ ve Motoneurom and Motor EnajAafes
The quantitation of the CGRPive motoneurons and motor endplates was completed
using a Leica D m fluorescence microscope. The counts of the motoneurons staining
positively for both CGRP and FluoroGold were obtained by selecting only those somata with
observable nuclei; any profles not containing a nucleus/nucleolus were not quantitated (cc
Thenault and Diamond, 1988a). The motoneurons of the SOL, LG, pMG and dMG are
topographically located in the medial-ventral region of the spinal cord beginning at the L4
ventral root entry zone and extend rostrally approximately 1800-2000 pm (Homonko and
Therïault, 1997). The FluoroGold-labeiled motoneurons exhibited cell bodies and dendrites
Wed with granules of bright gold fluorescence. In contrast, CGRP-TR immunofluorescence
was characterised as cytoplasmic and punctate, with the fluorescent red granules behg
preferentially located in the soma and in the proximal parts of the major dendrites.
Percentages of CGW+ve motoneurons were cdculated as the number of labelled cells versus
unlabelled cells within each group.
CGRP staining at the motor nerve temiinal was evaluated in twelve animals that were
randomiy divided into three groups: control, 72 h and 2 wk post-exercise. The motor nerve
temiinals were initially identified in each transverse section by acetylcholinesterase (AChE)
staining. Based on the results fYom a separate series of experirnents where we evaluated
AChE-stained longitudinal muscle sections taken from the belly of the MG (see section
5.3.3), we were able to determine that the average length of a MG motor endplate was
approxirnately 200 Pm. This method permitted us to establish a sarnpling distance within the
muscle region that would not result in duplicate counts of motor endplates that were
evaluated for aBuTx. In subsequent adjacent sections, motor endplates were identifïed with
aBuTx fluorescence using a blue filter (525 nm; 10x PL FLUOTAR objective) for
fluorescein detection, foilowed by a green filter (625 nm; 100x/1.25 N PLAN OL) for
detecting TR immunofluorescence, allowing CO-localisation of the CGRP+ve signal. CGRP
immunofluorescence was detected as a punctate staùung pattern visible in regions of the
junctional folds, overlapping the aBuTx l abehg which fiüed the entire junctional area (Fig.
ISC,D). Approximately 140 endplates were quantitated per muscle sample (Table 3). The
slides with CGRP+ve motor nerve terminais were then compared with adjacent serial
sections stained for myosin ATPase to deternine the fibre type of the identifïed
neuromuscular junction.
Importantly, all tissue analyses were done blinded to the experïmental condition
throughout all the procedures described in these studies. Ln order to permit statistical
analyses, the identity of each expenmental group was subsequently decoded foIlowing the
completion of the data collection. Statistical significance was determined by Student's t-test,
ANOVA, and where necessary, the Kniskal Wallis Test for non-parametric distributions
(SigmaStat, v1 .O, Jandel Scientific, USA).
5.4. RESULTS
5 -4.1. CGRP Reqonse in the SOL. L G. oMG and &G Motoneurons
The FluoroGold-IabeUed motoneurons within the motor nuclei of the SOL, LG, pMG
and dMG were readily identified under ultraviolet iight (Fig. 1 l q C ) . The double-Iabelled
TR-CGRP+ve ceIls had a punctate staining pattern, making this staining easïiy discernible
fiom the more homogeneous h e l y granular FluoroGold signal (Fig. 1 ID). The numbers of
- - -
Fimire 1 1. Photomicrographs of double-IabelIed MG rnotoneurons 72 h fzowing exercise. (A)
FluoroGold Wed motoneuron in rat lumbar @A) spinaI &rd retrogradely labeiled from the proximal
region of the MG that lacks an immunofluorescent TR-CGRP+ve signal. Filied arrow indicates the
Iocation of the unlabelleci rnotoneuron in B. (C) FluoroGold-filleci motoneuron retrogradefy labelled
f?om the proximal region of the MG that is imrnunofluorescently detected to be TR-CGRP+ve 0).
Calibration bars = 3 0 p.
Fiare 15, Photomicrographs of the neuromuscular junctions in the proxunal MG double-labeiied
with FITC-aBuTx and TR-CGRP in MG 2 wk foiIowing one bout of downhill exercise. (A) FITC-
d u T x identified motor endplate CO-localised with (E3) immunofluorescent TR-CGRP lacking a
CGRP+ve signai (i.e., CGRP-Ve). (C) FITC-aBuTx identified pMG motor endplate CO-ldsed with
an @) immunofluorescent TR-CGRP+ve signal. Calibration bars = 10 p.
Fiaure 17. CGR.P immunoreactivity is present at motor nerve temiinals on type W muscle fibres in
the MG. (A) Serial fiozen muscle cross-section from the proximal MG identified as a type IIB muscle
fibre by mATPase histochemistry (B) Subsequent serial fiozen cross-section ident-g a FITC-
aBuTx labelled neuromuscular junction that is (C) CGRP+ve as detected by Texas Red
immunofluorescence. Calibration bars = 30 pm (A): 10 pm (B,C).
double-labelled motoneurons in the identified SOL motor nucleus did not change foflowing
d o W exercise over the experimental tirne period (Fig. 12A). A sirnilar observation was
found for the double-labelled motoneurons of the LG motor nucleus (Fig. 12B).
In both regions of the MG, however, the do* exercise resulted in a signincant
increase in the numbers of double-Iabeiied CGRP+ve motoneurons 72 h after exercise when
compared to control @MG: P = 0.003; dMG: P = 0.03; ANOVA, Fig. 12C). While it was
SOL
1 T
time post-exercise
Figure12 Profile of double-labelled FluoroGold and CGRP+ve soleus (SOL) and lateral gastrocnemius (LG) and medial gastrocnemius
(MG) motor nuclei following downhill exercise. (A) The percentage of CGRPtvelFluoroGold labelled SOL motoneurons. (B) The
percentage of CGRP+ve FluoroGold labelled LG motoneurons. (C) The percentage of double-labelled CGRPtve/FluoroGold
motoneurons in MG motor nuclei in lumbar section L4 of the rat spinal cord (* = P<0.05; ANOVA). The double-labelled cells fiom
SOL and LG were not identified in the same cross-sections.
not apparent nom these data whether there was a preferential association of CGRP with
muscle fibre type, a more robust response was observed in the pMG motoneurons (27%
increase versus 14% increase in dMG). A si@cant ciifference was not observed between the
pMG at 72 h and the M G at 72 h post-exercise. Signifïcant dserences in the number of
C G W v e motoneurons were not observed between the control and 2wk post-exercise data
in the pMG or dMG e0.05), indicating that CGRP expression in these motoneurons had
retumed to baseline levels by this tirne.
5 -4.2. Giyco~en Dedetion Ej-erïments
Based on our observation of the increased n iumber and dMG CGRP+ve
motoneurons, it was of interest to ascertain whether both regions of the muscle were actively
recruited by the downhill running protocol. DEerences in the activity patterns of the muscle
fibres would permit us to interpret the physiological state of the two MG regions. Therefore,
the glycogen content of both compartments was detennined by PAS histochernistry (Fig.
13B,D).
Table 2. Number of muscle fibres analyseci per fibre type in the proximai and distal regions of
MG for glycogen depletion studies.
region of MG
proximal
distal
# of fibres
analyse#animal
100
100
1 O0
500
average # of
fibres/cross
section
2900
2900
2900
4000
fibre type
ST
FOG
FG
FG
# of
miimals
8
8
8
8
Relative optical density (ROD) measurements (Fig. 14) showed that all fibre types (type 1,
P=0.00002; type El, W.0002; type IIB, P=0.0005; T-test) in the pMG and dMG (type
IIB, P=0.0006; T-test) were signincantly depleted of glycogen stores following this eccentric
exercise paradigm. Whiie indicating that both proximal and distal regions of the MG were
active, these results did not ailow us to interpret either the relative amounts of glycogen
breakdown in the digerent muscle fibre types or the contribution of dBerent motor units to
the exercise protocol.
5.4.3. CGRP Rewonse at Motor Endplares in the pMG and M G Afier Eccentric fiercise.
In the experiments focused on motor endplate identifkation with aBuTx, bleed-
through nom the green aBuTx labelhg to TR/CGRP+ve staining was obviated by
differentiating the two signals based on the different morp hological staining patterns (Fig.
15). The bright aBuTx signal was easy to detect, and it was also easy to determine whether
there was signal bleed-through, Le., a false positive result. Specifïcaily, CGRP stained
dflerently at motor endplates than aBuTx, with CGRP being more pepperylpunctate in the
folds, s i d a r to the pattern of staining in the motoneurons, and the region of CGRP
immunoreactivity did nqt overlap the entire neuromuscular junction as stained by aBuTx
(Fig. 15D). In contrast, fluorescein-conjugated aBuTx filled the entire neuromuscular
junction region which was equdy visible at the bright field level (Fig. 15 4 C ) .
When viewed in transverse section, the motor endplates in the MG were easily
identified by FITC-aE3uTx staining, as they foliowed a distinct pattern throughout the belly
of the muscle. Interestingly, the CGRP+Ve endplates were most often observed in regions
where clusters of neuromuscular junctions were found, although not al1 of the neuromuscuIar
Figure 13. Photomicrographs of myosin ATPase and PAS stained muscle fiom
the proximal region of MG demonstrating the glycogen depletion patterns
before and after downhill exercise. Frozen serial cro ss-sections stained for
myosin ATPase (A and C) and PAS (B and D) in the control condition (B) and
20 minutes after downhill running exercise @). Calibration bars = 5 0 ~
FOG
1 1 exercise
Figure 14. Glycogen depletion profiles of ST, FOG, and FG muscle fibre types in the proximal and distal MG 20 minutes post-
exercise, as indicated by relative optical density (ROD) measurements. The asterisk (*) denotes significant differences as compared
to controls at the P<0.001 level; T-Test.
Table 3. Total number of motor endplates quantSecl in the proximal and distal regions of
MG in the controi and expehental groups.
region of MG
proximal
junctions within a cluster were CGRP+ve (Fig. 15B), with the majority being CGRP-ve (Fig.
15B). The elevated numbers of imrnunofluorescent CGRP+ve motor endplates were
observed in both the pMG and dMG following downhiii exercise. In the pMG, a region of
mixed fibre type, a significant increase (10%) in the numbers of CGRP-Unmunoreactive
neuromuscular junctions was observed at 72 h post-exercise (P=0.03; ANOVA) and
remained sigruticantly elevated 2 wk later (F'=0.03; ANOVA) (Fig. 16A). In the dMG, which
is comprised entirely of fast twitch glycolytic fibres, a 25% increase in the number of
CGRP+ve motor endplates, compared to control, was observed at 72 h post-exercise
(P=0.002; ANOVA) (Fig. 168). Interestingly, this percentage continued to be signifïcantly
elevated in the dMG compared to control, increasing to 33% by 2 wk after exercise. The
sampling of motor endplates every 200 Pm of 2 mm thick sections in the MG could
potentiaily result in an underestimate of CGRP+ve endplates. Furthermore, on average, 140
1 distai 1 12 1 1700 1 400 ( O ) O 1 4 0 0 1
# of
anÏrnaZs
12
total # of
FE-aBuTx
ena@Iates
1700
total#of
CGRP+ve
motor en@lates
150
fioretypeofCGRPtve
em@Iutes
FG
148
ST
1
FOG
1
endplates were counted per muscle sample, suggesting that a portion of CGRP+ve motor
endplates may have been excluded fiom the database.
time post-exercise
Fi.mre 16. The increase in the percentage of CGW+ve motor endplates in the proximal and distal
regions of the MG after downhill exercise. (A) The increased numbers of FITC-aBuTx and TR-
CGRP+ve motor endplates in the proximal cornpartment of the MG 72 h and 2 wk post-exercise as
compared to control (P=0.03; ANOVA). (B) The increased nwnbers of FITC-aBuTx and TR-
CGRP+ve motor endplates in the distal cornpartment of the MG 72 h and 2 wk post-exercise
compared to control (P=0.002; ANOVA).
5 -4.4. CGRP immunoreactivifv and Muscle Fibre Type
The analyses of the serial cross-sections of the pMG and dMG stained
histochemically for myosin ATPase showed that the CGRPtve motor endplates CO-localized
almost exclusively to the FG (type IIB) muscle fibres (Fig. 17A,B,C). Out of approximately
three thousand endplates exarnined in both control and exercise conditions, only one
CGRP+ve endplate was found at a FOG (type IIA) muscle fibre, and o d y one ST (type I)
fibre was found to CO-localize CGRP (Table 3).
5.5. DISCUSSION:
In a previous report (Homonko and Theriault, 1997), we demonstrated that one 30
minute bout of downhill ninning resulted in increased numbers of CGRP+ve motoneurons in
hindlimb extensor, but not in fiexor motoneurons. Significantly, CGRP remained elevated in
those flexor motoneurons for two weeks post-exercise, returning to baseline values by four
weeks. Our present shidies reveal that the changes in CGRP were exclusively associated with
the MG motoneurons, and that w i t h the muscle, this response was specifically localised to
motor endplates on FG muscle fibres. These results are the &st to demonstrate increases in
CGRP levels as a result of physiological neuromuscular exercise activity, rather than a lack
of activity, Le., as induced by surgical or pharmacological paralysis or by hormone
deprivation.
The increased CGRP levels in MG motoneurons couid be due to a variety of factors.
For example, the exercise regime may result in ftank histopathological damage to the muscle,
thereby initiating repair and regenerative mechanisms within the muscle that would require
new endplates on de novo myofibrils. Aitemately, unaccustomed exercise may induce
growth-related morphological alterations within the individual myofibrils, and subsequently,
at their neuromuscular junctions, leading to morphological changes at the motor endplates. A
more subtle process could be that the particular demands of eccentnc exercise may modulate
the interaction between specifk motoneurons and their targets, thereby leading to an increase
in synaptic efficacy at the affected neuromuscular junctions.
5.5.1. DownhiII Runnin~ Produces C k e s in MG Motoneurons in the Absence qf Muscle
Damugg
In this downhill running model, the ankle nexors are perforrning essentiaiiy concentric
exercise (shortening whiie Ioaded), while the ankle extensors (SOL, LG, MG) are
undergoing eccentric contractions (Iengthening white Ioaded). The eccentric exercise
protocol we have used is acute and not very physiologicdy demanding. In contrast, previous
studies that have used chronic andior extended bouts of eccentric activity report substantid
SOL muscle damage with indices of pathology being clearly observed 3-5 days post-exercise
(Ogilvie et al., 1988; Stauber, 1989; Armstrong et al., 1983, 1991). While the majority of
studies reported eccenticdy-induced damage almost exclusively in the SOL, several
investigations have reported that eccentncally-biased activity preferentially affects fast twitch
muscle fibres (Lieber and Fnden, 1988; Fnden and Lieber, 1992).
Smith et al. (1997) detected the formation of new muscle satellite cells in the rat
SOL, foIiowed by significant increases in developmental rnyosin isoforms, and the
appearance of new myofibrils. However, neither the MG nor the LG showed any significant
trends, thus making the need for de novo endplates in the MG unlikely. Since we, and others,
have shown that there is no significant histopathology or inflammation in the MG, as
compared to the SOL, following either chronic or acute exercise protocols (Smith et al.,
1997; Homonko and Theriauit, 1997), it seems unlikely that the changes in CGRP levels we
reported cm be attributed to myofibrillar damage and repair processes. Therefore, increased
numbers of CGRP+ve motoneurons in the MG motor pool is not related to muscle damage
as induced by eccentric exercise.
The relative lack of baseline CGRP immunoreactivity in the almoa exclusively slow
twitch SOL motoneurons and the absence of change in CGRP levels foilowing exercise
(despite the histopathology data) argues against the idea that CGRP may be related to
extensively used or easily recruited (e.g., SOL) motoneurons (Arvidsson et al., 1993). Our
results however, do support the view that motoneurons innervating fast glycolytic fibres,
which are known to be larger and faster motor units (Kanda and Hashinime, 1992; Bakels
and Kemell, 1993; Gardiner, 1993), have a higher baseline of immunoreactivity for CGRP.
5 -5 -2. CGRP and Sprout;ina ut the NeuromuscrrIar Jzmction
Sprouting of the motor nerve terminal in the adult has been correlated with changes
in CGRP peptide and mRNA expression in motoneurons following either pharmacological
blockade (Tarabal et al., 1996b) or surgical interruptions of neuromuscuIar comectivity (Sala
et al., 1995). Both of these approaches involve either a significant injury to the muscle nerve,
or a substantial interruption of neuromuscular comectivity with the resultant sprouting
response characteristic of that seen following nerve crush (Blake-Bru& et al., 1997) and
axotomy (Streit et al., 1989; A ~ d s s o n et ai., 1990; Piehl et ai., 1991). In Our study,
however, no darnage was incurred by the MG muscle nerve and extensive macroscopic
muscle damage is absent. Furthemore, it is unlikely that the intrarnuscular injection of
FluoroGold produced a nerve injury that initiated a sprouting response and a subsequent up-
regulation in motoneuronal CGRP expression. Data tiom Our first study showed elevated
numbers of CGRP+ve motoneurons following eccentric exercise when muscles were not
injected with FluoroGold (Homonko and Thenault, 1997). In addition, other uivestigators
Popper et al., 1992) were unable to see changes in CGRP immunoreactivity and
aCGRPmRNA in the bulbocavemosus muscle in sham and buffer treated groups when using
a multiple muscle injection protocol.
While it is known that exercise can effect morphological changes at the
neuromuscular junction (Wernig et al., 199 1; Waerhaug et al., 1992; Deschenes et al., 1993),
in motoneurons, and in axons (Edstrom and Grimby, 1986), a 9% hcrease in the number of
axon collaterals (nodal and terminal axon extensions) at mouse motor endplates could only
be observed after an exercise protocol that was more demanding and longer in duration than
ours (Le., 3 x 3 h with 30 min. rest penods; 14 m-mine1, 6" ; (Wernig et al., 1991a). In
contrast to the latter study, the differences we reported in the MG were relatively subtle, as
the greatest changes in CGRP levels could only be observed in 23.5% (400A700) of the FG
motor endplates sampled in the M G . While the literature and Our pilot studies of the extent
of histopathological darnage in the SOL and TA would suggest that Our 30 minute bout of
exercise would not induce significant muscle damage, fbrther studies need to be cornpleted.
In order to conclusively determine whether a single 30 minute bout of exercise elicits
sprouting at motor endplates, morphoIogica1 measurements would need to be made on
teased, single muscle fibre populations, and muscle examined over a longer tirne-course (e.g.,
up to 4 weeks).
5 -5.3. A neuromodulato~ role for CGRP at the motor endplate @ f e r exercise
CGRP7s rapid neurotransrnitter actions on the nicotinic acetylcholine receptor
(AChR) are weii documented dong with its trophic functions at the neuromuscular junction
(reviewed in Arvidsson et al., 1993; and see section 6.4). In W o , CGRP has been shown to
directly affèct AChR metabolism in chick muscle membrane preparations (New and Mudge,
1986; Fontaine et al., 1986; Jinnai et al., 1989; Changeux et al., 1992). Adaptation in
synaptic activity may also involve changes in acetylchohesterase (AChE) enzymes. Recent
studies (Femandez and Hodges-Savola, 1 996) have suggested a correlat io n between CGRP
and AChE G4 which is an isoform of the enzyme located at the neuromuscular junction
(Femandez et al., 1996), and known to be up-regulated by enhanced activity in fast-twitch
muscles (Fernandez and Donoso, 1988; Jasmin and Gisiger, 1990; Gisiger et al., 1994).
Compeliïng evidence from in vitro studies on mouse myotubes (Boudreau-Lariviere and
Jasmin, L997), where CGRP more than doubled AChE and AChR a-subunit mRNA
expression, fùrther suggests a trophic role for CGRP in regulating gene expression of these
neuromuscular junctional proteins, at least in the developing neuromuscular system.
Although the possibility that CGRP regulates ACIS G4 and AChR subunit expression at the
adult neuromuscular junction following exercise appears prornising, fùrther investigations in
vitro and in vivo need to be completed.
5 -5.4. Motonewonal and Motor E e l a t e CGRP l3p-ession us a Funcfion of Motor Unit
Recnrifment
Both the LG and MG rnotoneurons have equivdent baseline levels of CGRP, with
60% of the motoneurons being CGRP+ve in the sedentary animal. The fact that there are no
changes in CGRP expression in the LG motoneurons following downhill running, suggests a
subtle and peculiar effect of this exercise paradigm on the MG that is not elicited in the LG.
The rat MG is a highly compartmentalised muscle with distinct fibre type distributions
(Vanden Noven et al., 1994; DeRuiter et al., 1995, 1996). MorphoIogicalIy, the proximal
region ofthe muscle, innervated by the proximal and lateral branches of the MG nerve, is a
mixture of fast twitch (FOG and FG) and slow twitch fibres, while the distal region,
innervated by the distal branch of the MG nerve, is exclusively FG (Vanden Noven et al.,
1994; DeRuiter et al., 1995). Presently, there is some evidence for regional specialisation of
muscle fibre type influencing motor unit recruitment patterns in the performance of dif5erent
motor tasks (English et al., 1985; Hutchison et al., 1989; Roy et al., 1991; Vanden Noven et
al., 1994; Sebum and Gardiner, 1995; DeRuiter et al., 1995, 1996). During uphill mnning in
the rat, higher intensity workloads evoke the preferential recruitment of the fast twitch region
of the MG as measured by EMG activity (Hutchison et ai., 1989b; Roy et al., 1991b), while
Iower intensity workloads preferentiaüy recruit muscle fibres in the proximal compartment
(DeRuiter et ai., 1996b). In addition, PAS staining has shown a differential increase in
glycogenolysis in FG fibres of the proximal compartment versus those in the distal
compartment following uphill running @eRuiter et al., 1996b).
Based on these anatomical and physiological observations, DeRuiter et al. (1996b)
have proposed a partitioned organisation of segmental rnotor control that results in the
differential activation of proximal or distal compartments of the MG, according to the
locomotor task. Our findings of a more robust response in CGRP+ve neuromuscular
junctions in the dMG may support the idea that there is a dEerential activation of this
cornpartment durhg the downhill running protocol.
Motor control strategies for downhiil ninnùig are suggested to dEer fkom those of
uphilI running (Nardone et al., 1989; Enoka, 1995, 1996). To date, cornpartment-related
recruitment strategies and the influence of muscle fibre regionaikation as pertainhg to
downhdi mnning activity have not been addressed. Based on our findings, it is tempting to
speculate that changes in CGRP expression at FG motor endplates rnay reflect motor unit
recruitment strategies that rnay be inherently difFerent for downhill running activity.
Histological glycogen depietion anaiysis of muscle cross-sections is a crude indicator
of motor unit recmitment (Armstrong and Taylor, 1993), although the technique does
provide insight into the usage of different compartments within a muscle (e-g., Sullivan and
Armstrong, 1978; Theriault and Diamond, 1988; DeRuiter et al., 1996). Although not
quantitative with respect to the degree or rate of glycogen depletion, Our PAS results showed
that both regions and ali three muscle fibre types in the MG were active during downhiil
mnning. We did not match CGRP+ve endplates with the relative levels of glycogen depletion
in individual muscle fibres. Nor did we electrophysiologically measure the activity of the
dinerent MG compartments. Therefore, in order to determine if an enhanced CGRP
expression at the FG motor endplates is associated with a selective recmitment of a certain
fast motor unit type [fast fatiguable (F'F) or fast fatigue resistant (FR)] (Seburn and Gardiner,
1995; DeRuiter et al., 1996), such expenments would need to be done.
5.6. CONCLUSIONS
We have shown that following downhill running, CGRP expression is elevated
exclusively in MG motoneurons and at MG motor endplates on FG muscle fibres. In
addition, this CGRP response is not associated with muscle darnage or repair processes. One
possibility is that the response may be due to an enhanced activity in FG motor units elicited
by eccentric work. The eEects of exercise-induced increases in CGRP expression rnay
therefore, play a role in the remodelling of the post-synaptic membrane of the adult
neuromuscular junction.
CHAPTER VI.
Experirnents Desimed To Investieate the Role of CGRP at the Neuromuscular Junction:
Two studies were completed that attempted to clarify the mechanism behind the
increased number of CGRP+ve motoneurons and motor nerve terminais in the MG following
exercise. The first experirnent (Section 6.1) was designed to detect changes in the
acetylcholinesterase subunit G4 in the MG, and to correlate those obsenrations with the
CGRP response just described. The second experiment (Section 6.2.) was designed to test
whether there was a correlation between CGRP and a potent target-derived neurotrophic
factor known to be correlated with the onset of motor nerve sprouting.
6.1. Acetylcholinesterase (Ga Activity in the Medial Gastrocnemius Muscle Following
Downhill Exercise.
6.1.1. Introduction:
A fiinctional association between CGRP and AChE G4 has been suggested (Hodges-
Savola and Femandez, 1995; Femandez and Hodges-Savola, 1996). Intrapentoneal injection
of CGRe (0.1 rnl-kg-') down-regulated G4 isozyrne expression following denervation of the
fast twitch gracilis muscle, an effect that was reversible in the presence of an ktraperitoneal
injection of CGRP antagonist (Hodges-Savola and Femandez, 1995). As well, an
intrarnuscular injection of exogenous CGRP, following treadmill walking in rats, was
reported to reverse the exercise-induced increase in G4 isozyme in the rat gracilis muscle
(Femandez and Hodges-Savola, 1996). Clearly, there are problems with the interpretation of
these studies, in that the relative sensory, motor, and systernic effects of exogenous CGW
are not known. In addition, the relative contribution of the endogenous release of CGRP that
occurs during exercise is also not understood (Schifier et al., 1995).
In vitro, mouse myotubes treated with CGRP exhibited a large increase in AChE and
AChR a-subunit mRNA (Boudreau-Lariviere and Jasasmin, 1997). These studies, in particular,
support a role for CGRP as a trophic factor at the developing neuromuscular junction.
Therefore, we wondered ifthe changes we observed in CGRP in MG motoneurons following
acute eccentric exercise would be correlated with an increase in AChE G4 activity in the adult
muscle.
6- 1.2. Materials and Methods:
6.1.2.1. merimental Design and Tissue Handing:
Twelve, female, Wistar rats were randomly divided into three groups: control, 72 h
and 2 wk post-exercise. Rumers foUowed the sarne standard exercise protocol as described
in section 2.1.1. In all groups, both the right and left MG muscles (n=24) were included for
analysis.
At the tirne of sacrifice, the left and right MG were excised, placed in a g l a s petrie
dish containing ice-cold saline buffer (0.9% NaCI; 4°C) on ice and each muscle quickly
dissected into two pieces. Two sections, the proximal-medial and distal-lateral portions (see
Fig. 6) were isolated and quickly frozen in liquid nitrogen. These regions contained the
anatornicd injection sites for FluoroGold in Study 1 (Chapter IV) and Study 2 (Chapter V)
retrograde labelling protocol. Muscle pieces were subsequently stored at -80°C until AChE
analysis.
6.1 -2.2. Sedimen fation AnaiysiS of AChEMolecular Foms:
AU AChE analyses were completed in the laboratory of Dr. Victor Gisiger, Dept. of
Anatomy, Université de Montréal, during a 4 week period in May-June, 1996. The method
for measurement of AChE activity has been described previously (Gisiger et al., 1994).
Briefly, muscle pieces were homogenised on ice (2 x 15s) with a Polytron in a high salt
b a e r (TESB; 1% Triton-X-100, 0.20 M Tris-HCI, pH 7.0-7.2, 1M NaCI, 0.10M EDTA, 1
mg-mi-' Bacitracin (Sigma, USA), + aprotinin (Boehringer Mannheim, Montreal, PQ.). The
homogenate was then centrifuged at 12,000 g for 12 minutes. The supernatant was retained
and immediately used or stored at-80°C.
Aliquots (50 pl) of muscle extract were [oaded on a 5-20% sucrose gradient (TESB;
1% Triton-X-100, 0.10 M Tris-HCI, pH 7.0-7.2, 1M NaCI, 50 nM MgCl2 , 1 mgml-'
Bacitracin (Sigma, USA), then ultracentrifbgated for 21 h at 4"C, 40,000 rpm in a Beckman
SW 41 rotor. Fractions of the gradient were collected and cornbined with reaction medium
(0.1M PO4 , pH 7.0, DTNB 5x104 M, AcThCh 7Sx1O4 M +IO5 M cholinesterase inhibitor
iso-OMPA; Sigma, USA) for measurement of AChE activity by a spectrophotometric
method (Jasmin and Gisiger, 1990; Gisiger and Stephens, 1988; E h a n et al., 1961). The
protein concentration of the muscle extracts were determined according to the method of
Smith et al. (1985).
For this data set, a Student's t-test and ANOVA were used to deterrnine the
statisticd signïficance between groups, and where applicable, the Mann Whitney Rank Sum
Test was employed. As before, an alpha level of0.05 was accepted as significant.
6.1.3. Results and Discussion:
We obtained the sedimentation profle of all five molecular forms. Graphical
representation of AChE activity in the control, 72 h and 2 wk post-exercise groups are
presented in Figs. 18-20, respectively, with values corrected for specinc activity and protein
content. Table 4 sumrnarises the spec5c activity of the combined left and right pMG and
dMG muscle sarnples analysed. No signincant dBerences were observed in G4 activiw at any
- - - -
Proximal Remi of the MG Disral Reghm of the MG
Cori trol
3.068 k 0.3 13
0.551 f 0.078
4.475 +_ 0,403
4.182 f 0,244
1.600 f 0,148
Table 4. Specific activity measurements of acetylcholinesterase subunits in the proximal and distal regions of the medial gastrocnemius
in the control, 72 hours and 2 weeks post-exercise groups. Data represent left and right leg measurements, n = 8 muscles/condition, x
+ sem.
72 h
2.2537 & O. 1877
0.3737 f O.0261
5.136 f 0.442
4,1713 f 0,3933
1.79 i 0.2087
tirne-point foilowing exercise, or between the proximal and distal regions following exercise,
although a trend towards an uicrease in the Gd isozyme was observed at 72 h post-exercise.
As weU, trends towards a decrease in AIZ and As isozymes were aiso observed.
6.1-4. Cortclusions:
We could not detect a statistically significant elevation in Gq activity level in the
proximal or distal regions of the MG muscle foiiowing a mild, acute bout of eccentnc
exercise using the descrïbed methodologies. However, one limitation of the methodologies
employed was to cut the muscle in sections so as to separate the proximal distai regions of
the muscle. This may, in fact, have reduced our capabiiïties of acquiring the entire G4 pool in
Our sample, as weil as the other 4 pools (Gisiger and Stephens, 1988). In fact, significant
changes were observed in the activity of the other 4 isozymes that are considered artifactual
and attributed to errors of technical rneasurement. Subsequent investigations should use the
entire intact muscle to maximise acquisition of the entire pool of acetylcholinesterase
subunits. Ln addition, the SOL and LG should also be investigated.
6.2. GDNF Expression at MG Motor Endplates Following Downhill Exercise.
6.2.1 Introduction:
The most potent s u ~ v a l factor for motoneurons identified to date is glial-derived
neurotrophic factor - GDNF (Henderson, 1996). In 1993, it was initially charactensed as a
neurotrophin for midbrain (substantia nigra) dopaminergic neurons in culture (Lin et al.,
1993, I994), and was later found to be neuroprotective for these cells (reviewed in Birling
and Price, 1995). A member of the TGF-P farnily, it shares a common receptor, the
serinehhreonine kinase (Henderson, 1996). In vitro, cultures with minimal concentrations of
GDNF are able to sustain viable embryonic rat motoneurons (Henderson et al., 1994). In
vivo, GDNF attenuates prograrnmed cell death in chick motoneurons (Oppenheim, 1996),
and reverses axotomy-induced ceil death and atrophy in rat motoneurons (Henderson, 1994,
1996; Li et al., 1995; Yan et al., 1995; Oppenheim et al., 1995). GDNF mRNA is synthesised
in the peripheral nerve, at the base of the iimb bud prior to and during progammed ceii death
(Henderson et al., 1994), most likely by Schwann ceU precursors (Henderson, 1996), since
the mRNA is present in Schwann cells during developmental stages (Henderson et al., 1994).
It is retrogradely transported by motoneurons in the neonatal rat (Yan et al., 1995) in
response to naturai celi death, and following sciatic nerve transection in the young rat, where
increases in GDNF mRNA in peripheral nerve are also observed (Trupp et al., 1995).
In models of axotomy, where motoneurons are maintained or rescued by other
agents, GDNF is a very effective neurotrophic factor in reducing atrophy and loss of choline
acetyltransferase (CHAT) imrnunoreactivity (Henderson, 1996). GDNF can enhance the
chohergic development of motoneurons in culture (Zum et al., 1994). GDNF is up-
regulated in denervated rat skeletal muscle for 1-2 weeks following transection of the MG
nerve (Springer et al., 1995). Aithough the major evidence for GDNF supports a role for a
suMval factor during prograrnmed ce11 death, its physiological role in the mature
neuromuscular system remains a topic of debate and investigation.
Pnor reports have described the up-regulation of motoneuronal CGRP (peptide and
mRNA) following nerve injury or muscle paralysis (Piehl et al., 1991; Sala et al., 1995;
Tarabal et al., 1996). Furthermore, an increase in CGRP in rat muscle is associated with
sprouting at the rnotor endplates (Tarabal et ai., LW6a). Irnportantly, pensynaptic Schwann
cells have been shown to participate actively in the induction and guidance of sprouting
motor nerves at the motor endplate d u ~ g development and following BoTx-induced
pardysis in the adult rat (Son and Thompson, 1995a). As well, CGRP has been shown to
increase Schwann ce11 proMeration in culture (Cheng et al., 1995).
These observations have been taken to suggest a direct correlation between CGRP
and motor nerve terminal sprouting or remodehg. In Our model, the changes in CGRP
immunoreactivity have been well documented, and may be correlated with exercise-induced
remodehg at type III3 neuromuscular junctions. Since we observed changes in CGRP at
type IIB motor endplates foiiowing eccentric activity, we proposed to examine whether
changes in CGRP expression were preceded by an hcreased expression of GDNF at these
'crem~deUing" neuromu~cular junctions. Therefore, we anaiysed portions of the MG muscle
for changes in GDNF expression at the CGRP+Ve neuromuscular junctions using double-
l a b e h g irnrnunohistochemical procedures. Our hypothesis was that, following
unaccustomed exercise, changes in CGRP would be preceded by changes in GDNF
expression at type IIB motor endplates.
6.2.2. Methods:
6.2.2.1. Procehre:
Sixteen, female, Wistar rats, 250-275g were randomly divided into 4 groups: control;
18h, 24h, and 72h post-exercise. The animals followed a standard exercise protocol (see
Section 2.1.1) and the muscle tissue was harvested as descnbed in Section 2.1.2.
6 -2.2 -2. Muscle Immunocytochemishy:
Frozen, muscle tissue (12 pm) was seriaiiy sectioned through the belly of the MG
approximately 12 mm posterior to the ongin of the muscle. Serial sections were collected in
duplicate every 200 pm and placed directly ont0 slides. The first series was used for GDNF
(R&D Systems Inc., Minneapolis, MN) immunocytochemistry, and the second series for
CGRP (Genosys, TX) irnrnuno~ytoche~stry. Prior to ~nocy tochern i s t ry , the slides were
placed into fixative: 4% paraftormaldehyde for 30 min. for CGRP series and ice cold 1:l
acetonehethanol fixative for 30 min. for GDNF senes. Three 10 min. washes in O. 1M PBS
pH 7.1 were completed before incubation in blocking serum. Standard irnmunocytochemical
procedures were followed as described in section 2.1.4. Both series were incubated for a
second overnight in FITCzrBuTx (1 : 1500; Sigma, USA) for motor endplate identification.
The slides were then coversiipped with Mowiol as described previously.
6.2.2 -3. Quantitalion of CGRP + ve NeuromzmscuZar Junc f ions:
The quantitation of CGRP+ve and GDNF+ve motor endplates was completed in a
similar manner to that described in Section 5.3.6. Approximately 100 endplates were
quantitated per muscle sample. The slides immunostained for CGRP and GDNF motor
endplates were evaluated separately, then compared with the adjacent section once a
positive-staining (aBuTx) endplate was identified. As established in previous experiments, al1
tissue analyses were done blinded to the experimentd condition throughout all the
procedures described in the study. The identity of the experimental groups was subsequently
decoded following completion of the data collection in order to permit statistical analysis.
Percentages of CGRP+ve, GDNF+ve, and CGRP+ve/GDNF+ve motoneurons were
calculated as the number of labelled endplates versus unlabelled endplates within each group.
Statistical significance was detemiined by ANOVA (SigmaStat, v1.0, Jandel Scientific,
USA).
6.2.3. Results and Discussion:
6.2.3.1. GDAF Nnmunoreactivity at MG newomziscuIar junctions:
GDNF immunoreactivity at the neuromuscular junction 0 was unchanged within
the first 72 hours following exercise. No significant dserences were observed in the
GDNF+ve immunofluorescence profles of MG NMTs at any tirne-point versus control
(W.984; ANOVA) (Fig. 21A). In general, in both sedentary and exercised anirnals, few
endplates (15%) presented with a positive imrnunofluorescent GDNF signal. As weU, there
did not appear to be a pattern or specific anatomicai location within the muscle where the
GDNF signal was most often obsenied or recognised (Fig. 22).
6.2.3 -2.CGR.P immunoreactivity at MG neuromziscuZar junclions:
Within the f is t 72 h post-exercise, CGRP expression at the MG NMJ increased in a
directly proportional manner (Fig. 21B). Although a 10% increase was observed in the
membranes of the immunofluorescently labelled endplates, compared to control; the increase
was not sigmfïcant (P=0.07; ANOVA). Under high powered (100~-oii immersion)
immunofluorescence microscopy, the CGRP+ve NMls were visibly scattered throughout the
muscle cross-section in a nondescript pattern. At tirnes, the CGRP+ve NMJs were found to
occur in small clusters within the muscle.
6.2.3 -3. GDAF und CGRP ïmmunoreactivity at MG neuromtiscylarjunc fions:
The percentage of CGRP+ve, GDNF+ve (Le., double-labelled) MWs increased over
the experimental time period (P=0.053; ANOVA) (Fig. 21C) in a sirnilar pattern to that
observed with the CGRP expression (Fig.2 1 A). However, very few endplates (4.3 5%) CO-
Iocabed the two peptides at 72 h post-exercise.
6.2.4. Concfusion
The changes in GDNF expression did not precede changes in the CGRP expression at the
MG motor endplates of the type IIB muscle fibres.
A B CGRP GDNF
ctrl 18 24 72 ctrl 18 24 72
time following exercise (hours)
ctrl 18 24 72
time following exercise (hours)
Figure 21. The profile of CGRP+ve and GDNF+ve irnmunofluorescence at aBuTx identified
W J s in the MG following downhill exercise. (A) The CGRP response at MG NMJs within
the first 72 h following exercise and (B) the response of GDNF in the sarne muscles. (C) Co-
localized CGRP and GDNF immunofluorescent stainùig at MG NMJs within the fïrst 72 h
following exercise.
Figure 2 2 . Photornicrographs o f GDNF immunof luorescent staining at neuromuscular junctions (NMJ) of the MG muscle. Setial cross-sections of MG muscle 72 h post-exercise with FITC- aBuTx identified NMJ (A) double- labelled with TR-GDNF, indicating a GDNF-ve NMJ in (B). FITC-aBuTx identified NMJ in the MG muscle 72 h post-exercise (C) double-labelled with TR- GDNF indicating a GDNF+ve NMJ in @). Calibration bars = 1 0 Pm.
C W T E R VII.
GENERAL DISCUSSION:
The objective of this work was to investigate the role of CGRP in the adult rnotor
system. Needless to Say, over the course of these studies, we obtained results that have raised
more questions and interesthg possibilities for future experimental work than initidy
imagined. The multidisciptinary approach to the development of this model was unique
(combining exercise physiology and neuroscience), as was the ap proac h taken t O explaining
the role of CGRP in the motor system. It is comrnon for researchers to remain within the
constraints of one discipline to explore research issues and to explain their experimental
findings. The work in tbis thesis has atternpted to Iink two dEerent disciplines or areas of
research to resolve basic questions. We have developed a novel, versatile and physiologically
relevant experimental protocol in which questions pertaining to the motor and sensory
system may be posed and then tested. Specifically, one can derive experirnents in which to
test the role of CGRP and many other factors, at the pre- and post-synaptic membrane, of the
neuromuscular junction, within the motor system in the normal adult animal. This "rnodel" or
constmct is a signifïcant contribution to the scient& literature. Experimental designs and
intact systems are required which can test observations that are made in minimalkt systems
(i. e. in vitro and in silu systems) .
Another impo~ant result of this work is that we have shown a change in a
neuropeptide in response to physiologically reasonable physical exercise that produced little
to no damage within the muscle, in contrast to previous reports which used surgical or
pharmacological rnethods to induce macroscopic changes in neuromuscular connectivity.
These are the first experiments that begin to elucidate the role of CGRP in the normal adult
mammalian neuromuscular system. Our exercise protocol can be used to study
neuromuscular adaptations in mammals performing natural activity patterns.
The first part of this chapter will consist of a discussion of the hypothesised
mechanisms that may eticit altered CGRP expression in motoneurons and at their NMJ,
followed by suggestions for future experiments. The hypotheses developed in Section 1.2
will be addresed in a discussion of the general significance and novelty of this work. The last C
part of this chapter wili discuss the major limitations encountered and consider their Ulfluence
on the outcome of the results in the respective studies (Chapters II-V).
7.1. PART 1. DISCUSSION OF THE FUNCTIONAL ROLE OF CGRP AT THE
NEUROMUSCULAR JZTNCTfON
The results of the studies reported in this thesis have produced rnany interesting
questions regarding the functional role of CGRP at the adult neuromuscular junction and in
spinal cord motoneurons. This part of the discussion will address the three main hypotheses
that were presented in Chapter I, and then further expand on the possible role of CGRP in
the aduft mammalian motor system.
7.1.1. A Re-assessrnent of the Hvuotheses:
Hypothesis 1: DownhiZI nmning exercise causes an increase in CGRP in fJ7e
motonezcro~ts of eccentricaZly confrocting muscles. The objective of the first series of
experiments in Chapter N was to determine if changes in CGRP expression were obsenred
in motoneurons innenrating two contrasting muscle groups following downhill exercise in the
rat. The results of these studies have shown clearly that downhiii running exercise increases
the number of CGRP+ve motoneurons projecting to muscles that are reported to be engaged
in eccentric contractions. The concentncally contracthg muscle group, the AC, showed no
significant differences in numbers of CGRP+Ve motoneurons. In addition, the t h e course of
these changes post-exercise occurred within the first 48 hours following activity, and
retumed to baseline by 4 weeks post-exercise. As mentioned earlier in this chapter (see
Section 6.1.1 .), we did not perform electro physiological experiments on the activity profile of
the TS muscle group that underwent changes in CGRP to determine if, in fact, they were
eccentrically contracting. However, the histopathologic work of other investigators (Ogilvie
et al., 1988) who have used indices of muscle darnage to assess eccentric muscle activity
suggests that this was the case for the exercise regirne we selected. The fact that we did not
see a similar CGRP response in motoneurons of the concentncaliy contracting AC muscle
group suggests that the particular type of neuromuscular activity in the TS muscle group was
a factor in the changes in CGRP that were observed. Our report was the first in the literature
to descnbe changes in motoneuronal CGRP as a result of "naturaï' activity in a
physiologicaily intact neuromuscular system. Thus, the hdings reported in his t hesis add
new and important information on the effect of physiological exercise on CGRP expression in
the intact, adult, mammalian motor system.
The second hypothesis in Chapter 1B stated: CGRP preferentially increases at the fast
g&coIyfiyfic (FG) type IIB endplales and in lheir motonewons following eccenîric exercise.
This hypothesis was supported by the observations fiom the experiments completed and was
descnied in Chapter V. The responding population of motoneurons was found to be within the
medial gastrocnemius of the ankle extensor (TS) group. At the motor endplate in the MG, CGRP
expression was associated exclusively with the fast glycolytic (FG) type IKB muscle fibres. This
discovexy of elevated numbers of CGRP+Ve motornerve terrninals on one type of muscle fibre in
the MG is the first in the literahire to directly comect changes in CGRP expression following
experimental intervention with motor endplates of an identioed muscle fibre type. Previous work
by other investigators (Piehl et al., 1993; Forsgren et ai., 1993; Blanco et al., 1997) had only
shown an association between CGRP and muscles predominantly composed of fast-twitch fibres.
Although we have identifieci the predominant muscle fibre type, the physiological
characteristics of the fbt twitch motor units that are affecteci by changes in CGEW expression as a
fùnction of activity rernain undetermined. It is not known if the motoneurons that stained
positively for CGRP are fast fitiguable (FF) or fàst fatigue resistant (FR) motor units. As well, it
is as yet undetennineci if the motor endplates are part of FF or FR motor units. Recent studies
have shown that significant overlap exists between the FF and FR motor units in ternis of their
electrophysiological properties that does not always correlate directly with the histochernically
identifïed muscle fibre type (Kanda and HaShinime, 1992; Gardiner, 1993; DeRuiter et al., 1996).
The elegant studies of DeRuiter et al. (1995b7 1996b) were able to clearly demonstrate this
overlap within the different compartments of the rat MG. Knowledge of the motor unit type in
Our activity protocol would provide fiirther information as to the fùnctionai role of CGRP in the
motor system. Currently, a major constn.int is that there are no biochemical or neuroanatomical
characteristics that distinguish the motoneurons innervating FF or FR rnotor units.
The third hypothesis presented in Chapter 1B States: Changes in CGRP eqvression are
preceded by chunges in GDNF expression at fast glycolylic (FG) type IIB rnotor endplales
following eccentric exercise. In the last part of this work, an attempt was made to determine
whether the changes we documented in motoneuronal NMJ CGRP levels could be due to the
iduence of target-denved neurotrophic factors integral to sprouting at the neuromuscular
junction.
The studies of Son and Thompson (1995qb) demonstrated that Schwann ceiis play
an early role in stimulating and guiding nerve sprouting (Son and Thompson, 1995a). GDNF
is a known s u ~ v a l factor for motoneurons in vitro and in vivo, and is present in penpheral
nerve, muscle and Schwann cells (Henderson et al., 1994; Yan et al., 1995). Recently, the
over-expression of GDNF in neonatal transgenic mouse muscle has been shown to
substantially increase junctional sprouting (Nguyen et al., 1 998).
The third hypothesis was constructed to evduate whether the initial events of
junctional sprouting were induced by an increased production of GDNF at the neuromuscular
junction. Our results did not support this hypothesis. The data fkom the small senes of
experiments completed indicated that GDNF expression did not change within the 6rst 72
hours following exercise, i. e., it did not precede the changes in CGRP expression. Therefore,
GDNF is unlikely to be a target-derived factor that directly initiates changes in CGRP
expression at FG motor endplates within the first three days post-exercise. However, we
cannot conclude from these data that sprouting is absent at the rat MG neuromuscular
junction, or that GDNF plays no role in these events. Nor can we conclude that CGRP is not
involved in sprouting events. If sprouting was present in our protocol, then it is possible that
GDNF expression may have changed at a later time-point Le., afler 72 hours post-exercise. It
is possible that GDNF may be associated with other aspects of sprouting that may or rnay not
involve CGRP. Perhaps GDNF expression at the neuromuscular junction is linked to the
magnitude of sprouting that foiiows a certain the-course; alternately the exercise protocol in
this study was insufficient to elicit sprouting. The completion of experiments that address the
time-course of GDNF expression following acute and chronic exercise is warranted.
It has been previously documented that exercise can effed rnorphological changes at
the adult, mammalian neuromuscular junction resulting in morphological (sprouting) changes
at the NUT &er an exercise protocol (Wemig et al., 1991a), which was more demanding and
much longer (Le-, 3 x 3 h with 30 min. rest periods; 14 m-&-', 64 than used in the present
study. In Our study, the greatest changes in CGRP levels in the MG were found in one third
of the FG motor endplates sampled in the dMG (e.g., 24 % of the total number of muscle
endplates sampled). It is diicult to comment on the possible incidence of sprouting in Our
rnodel. We did not test nor complete methodologies that would aiiow us to determine in
fact, morphological changes (sprouting) were underway at any experimental tirne point
foilowing exercise. In order to demonstrate sprouting, morphological measurements of
endplates on single muscle fibre populations or whole muscle slice preparations would be
required.
It is possible that a continual enlargement and strengthening of neuromuscular
junctions on the FG fibres in the MG is occumng, as suggested by the work of Balice-
Gordon and Lichtman (1990, 199 1). These investigators found that in young mice, as muscle
fibres hypertrophied, the neuromuscular junctions grew in concert. However, this type of
remodelling was not observed to a great extent in the sedentary adult mouse (Balice-Gordon
and Lichtman, 1990). However, when the adult junctions underwent change, they grew
substantially by 'intercalary expansion' (Balice-Gordon and Lichtman, 199 1). If this type of a
dynamic process was operative in our model, then the role of CGRP may help in the
maintenance of signalling efficiency at the post-synaptic membrane (see below).
In summary, the experiments in this thesis describe an increase in CGW expression in
the motoneurons innervating the MG muscle foliowing downhill exercise, with the increased
expression of CGRP being observed only at the type IIB motor endplates. The greatest
changes in motoneuronal CGRP levels were observed at 72 h post-exercise, and returned to
baseline by 2 wk; CGRP levels at the NMJ, however, remained elevated at 2 wk post-
exercise. Within the first 72 h post-exercise, the increase in CGRP was not preceded by an
increase in GDNF expression at the motor endplate in the MG, suggesting that the altered
CGRP expression in this protocol may not be due to motoneurai sprouting.
7.2. CGRP ar an effector peptide in tvpe IIB motor uni&
Apart fiom its 'fast' neurotransmitter-like actions on the Ncotinic acetylcholine
receptor (AChR), CGRP also exerts longer tasting trophic fùnctions at the neuromuscular
junction, presumably mediated by specifïc CGRP receptors localised to the post-synaptic
membrane at the neuromuscular junction (reviewed in Arvidsson et al., 1993) (Figure 23). In
cultured chick myotubes, CGRP has been shown to up-regulate the appearance, number, and
insertion of nicotinic AChRs into the muscle membrane (New and Mudge, 1986; Fontaine et
aI., 1986; Jimai et al., 1989; Changeux et al., 1992). Fontaine et al. (1986) have shown that
CGEW increases the mRNA codhg for the stabilising a-subunit in the AChR cornplex.
Although it is not known whether CGRP up-regulates aAChR subunit expression in our
exercise paradigm, ifit does, one potential outcome may be to strengthen the neuromuscular
junction for the demands of a subsequent eccentnc bout of activity.
Acetylcholinesterase (AChE) is predominately located at the endplates of developing
and adult muscle fibres, where it hydrolyses unbound acetylcholine at the synaptic defi
following synaptic transmission (Massoulie, 199 1; Massoulie et al., 1993) (Figure 23,24).
There are three main forms of AChE: the asymmetnc, junction-associated Aiz; the tetrarneric
Figure 23. Schematic diagram of the known short-term effects of CGRP at the neuromuscular j unction.
Ga and GI; and two minor forms, Ag and G2 (Bon et al., 1979). The catalytic subunits of the
A form are linked to a coliagen tail, which anchor them to the basal lamina (Haii and Sanes,
1993). AChE mRNA is highly concentrated within the post-synaptic membrane at the
junctional folds (Jasmin et al., 1993)( Fig. 24). Recent work on mouse myotube cultures by
Jasmin et al. (1995), suggests that the subneural Golgi apparatus may be involved in the
intraceIlular routing of AChR and AChE to the motor endplate in both the adult and neonate.
The synthesis of the A form of AChE is infiuenced by muscle activity (a calcium
mediated process) and neural factors (Massoulie, 1991). FoUowing denervation, synaptic
AChE vanishes rapidly, but may be induced to accumulate at the rnotor endpiates when the
denervated muscle membrane is directly stimulated (Hall and Sanes, 1993). The varying
foms of AChE are known to respond dserently to neurornuscular activity (Massoulie et al.,
1993). The tetrarneric G4 form is significantly and exclusively increased in fast twitch muscles
following a variety of chronic exercise activities (Femandez and Donoso, 1988; Jasmin and
Gisiger, 1990; Gisiger et al., 199 1; Femandez and Hodges-Savola, 1992; Gisiger et al., 1994;
Femandez et al., 1996), whereas the junctional Alz fom is unchanged in fast twitch muscle,
and is slightly down-regulated in slow twitch muscle in these exercise paradigms (Gisiger et
al., 1994). Since G4 found at the perïjunctional sites of the neuromuscular junction (Gisiger
and Stephens, 1988), is involved in the fast clearance of ACh at the junctional receptors
(Massoulie et al., 1993; Femandez et al., 1996), a functional role for G4 in the regdation of
motor unit activity during penods of enhanced muscle performance has been proposed
(Gisiger et al., 1994). Furthemore, the intramuscular injection of exogenous CGRP has been
reported to reverse the treadrnill exercise-induced increase in G4 in the rat gracilis muscle
synapse
Figure 24. Schematic representation of a neurornuscular j unction. (Adapted from Hall, 1993)
(Femandez and Hodges-Savola, 1996). Recent studies have reported that mouse myotube
cultures, treated with CGRP, displayed a 2.5 fold increase in both AChE and AChR a-
subunit mRNq strongly implicating a role for CGRP as a trophic factor regulating the gene
expression of integral post-synaptic molecules at the developing neuromuscular junction
(Boudreau-Lariviere and Jasmin, 1997). Similar work by Choi et al. (1998) demonstrated
that CGRP infiuenced A c h . synthesis in chick myotubes by a CAMP mediated process.
In the studies presented in this thesis (Chapter V., section 5.7.1), we did not observe
signifcant increases in AChE G4 activity at any of the tirne-points foilowing downhill exercise
However, a trend towards an increase in Gq was noticed at 72 h post-exercise. This
observation suggests that, in fact, there may have been an interaction of CGRP with AChE
G4 in the MG after eccentnc exercise that was masked by technical difficulties. It is possible
that a longer and more intense exercise protocol may have significantly increased AChE G4 in
the MG.
7.2.1 . Szrmesfrsfrms for Future Erperiments:
Many questions remain to be answered conceming the role of CGRP at the
neuromuscular junction. These questions c m be addressed by completing experïments that
involve the utilisation of molecular techniques to detemine interaction between CGRP and
AChRs and AChEs at identified motor endplates. For example, which AChE and/or AChR
subunit is undergohg adaptation? How is CGRP involved in the time-course of the adaptive
response? What is the AChE subunit activity within whole muscle preparations following
chronic downhiil exercise?
7.3. Influence of Motor Unit Recruitment on the remZation of CGRP activiq in FG motor
mifs
The idea that muscles are divided into functionally separate compartments is not
without precedent and remains a veq hotly debated topic. As discussed in Chapter V
(section 5.5.4), many studies have demonstrated motor unit recruitment of one region of a
muscle over another for the successfùl performance of a rnotor task. Many questions rernain
about compartmentalised recnutment in concentric based muscle activity (Le., uphill running,
walking, etc.), and it is stiil not known ifcompartmentalised recruitment is a requirement for
more muscle power, increased fatigue resistance, or a combination of both. However, based
on the results of the present study, it may be interesting to speculate whether a preferentid
recruitment of motor units in the MG occurs, and if the changes in CGRP expression are a
result of motor unit recruitment strategies that may be inherently dserent for downhill,
versus uphill or level mnning activity.
The present theory of the neural control of eccentric work suggests a unique motor
control strategy for eccentnc versus concentnc work (Nardone et al., 1989; Enoka, 1995,
1996). Studies completed in humans (reviewed in Enoka, 1996) have proposed that the 'size
principal', which governs concentnc work (Hennernan, 1957), may in fact, not be
appropriate for expiaining motor unit recruitment patterns in muscles performing eccentnc
work. The "size principle" States that small, slow motor units are recmited before larger, fast
motor units. It is important to note that this hypothesis is based on studies of isometrically
contracting muscle. Investigations of a variety of neuromuscular parameters during eccentric
work in humans, (maximal voluntary contractions, motor unit behaviour, motor evoked
potentials, muscle fatigue) have provided evidence for the selective recruitment of high
threshold motor units over slow twitch motor units (Enoka, 1996). Therefore, in the present
study, the performance of eccentric work may result in a preferentid activation of a subset of
FG motoneurons in the MG to complete the motor task, thereby eliciting the up-regulation of
CGRP levels in a certain population of FG motor units.
7.3.1. &gesfisfions for Fuhrre &ve&enb
One of the cnticisms of the theory of neural control for eccentric activity has been
that al1 the evidence has been obtained tiom human subjects in experimental models. The
mode1 developed in this thesis is an excellent one in which to test this hypothesis in anïmals.
Data fiom experiments which would incorporate etectrophysiological techniques would
provide exciting and valuable information in an already complex system.
Many questions arise from this study including: (i) whether the changes in CGRP
expression are simply activity driven, a result of enhanced activity in the neuromuscular
circuits, andor (ii) are they an example of a unique motor control strategy? In order to
address the former, future experiments need to be designed to look at different workloads
and types of activity to address the hypothesis that CGRP expression is regulated by the type
or magnitude of muscle activity (e-g., workload). It is suggested that a cornparison of a
variety of motor activities be used (i-e., training studies, overload, sprinting, endurance, etc.)
to determine if changes in CGRP are predominately intluenced by neuromuscular adaptations
at the junction, or within particular muscle fibres.
7.3 -2. beriiments with Neurotrophic Factors
Neurotrophic factors can be broadly defined as polypeptides that are necessary for
neuronal survival during development. They also play a role (as yet poorly-defined) in the
maintenance of phenotype in the adult. Presently, a large number of trophic agents from a
varïety of gene families that interad with particular receptors are known to innuence s u ~ v a l
of motoneurons in many developing systems (reviewed in Thoenen et al., 1993 and Lindsay
et al., 1994) (Figure 25). Some of the candidates that most highly warrant consideration as
motoneuronal trophic factors are target-derïved and include: N T 4 (Funakoshi et al., 1995),
IGF-1 (Caroni and Grandes, 1990), GDNF menderson et al., 1994), BDNF (Koliatsos et al.,
1993), while others are not target-derived, such as CNTF (Sendtner et al., 1992). Presently,
there is still a substantid amount of information Iacking as to the basic actions and
înteractions of these factors (Figure 25). For exarnpIe, the fibroblast growth factor, FGF-5, is
postulated to be a target-derived neurotrophin for motoneurons Vughes et al., 1993). Piehl
et al. (1995) tested this hypothesis by performing sciatic newe transection on adult rats and
then injecting bFGF (1 pg) subcutaneously to determine changes in a- and PCGRP in the
spinal cord. Treatment with bFGF abolished aCGRP up-regdation in the spinal cord as
revealed by in silu hybridisation. In contrast, BDNF (15pg) had no effect on aCGRP or
PCGRP in motoneuron cultures or in vivo (Son et al., 1996). In a subsequent study by the
sarne authors, intrarnuscular injection of IGF-1, IGF-2 and CNTF did not increase aCGRP
or GAP43 M A in rat motoneurons following sciatic nerve transection (Piehl et al.,
1998a). These results are in contrast to others that have shown an increase in CGRP
expression at the motor endplate following axotomy and CNTF treatment (Tarabal et al.,
1996a), as weli as in the motoneuron (Kidokoro and Saito, 1988; Yan and Miller, 1993;
Kwon and Gurney, 1994). Therefore, the conflicting data in the literature demonstrate that
the role of various neurotrophic factors in this, and other, models has yet to be clearly
understood.
Most investigations of neurotrophic factors have been completed in vitro. and often
the in vivo results do not correlate. The sarne c m be said for the use of dEerent species and
the age of the animal (developmental versus adutt anirnds). Furthermore, many "trophic"
factors have been manipulated in models that have produced interesting results that are not
yet fùiiy understood. Our understandmg of the role of neurotrophins will o d y increase when
we can clarify the contribution of the supporting effectors in biologicaliy relevant models and
physiological constructs.
7.3 -2.1. ,!&gestions for Future Ejieriments: Erreriments with nezrrohophins and CGRP:
To date, the muscle derived target factors most affecting motoneurons are GDNF,
NT-4, and BDNF. IGF is excluded because it is not retrogradely transported, although
experiments to detennine its interaction with neural components are still warranted. NT-4
(Funakoshi et al., 1995), and most recently GDNF (Nguyen et al., 1998), are the best
candidates for motoneuronal trophic support in the adult. Experiments in a natural physical
activiiy mode1 should be used to hrther describe the actions of NT-4 and GDNF on
motoneuron properties in vivo.
Future studies in this area of research are needed to address whether the level of
motoneuronal CGRP is differentially regulated by a target/muscle-derived factor(s) that is
elicited by unaccustomed exercise, specifically eccentnc exercise, as well as by other forms of
activity (Le., concentnc exercise). Although numerous experimental options present
themselves, investigations using knockout mice, with specific deletions of CGRP or
neurotrophins (CNTF, BDNF, NT-4, GDNF, etc.), rnay have the best potential for producing
interesting results.
Figure 25. Schematic diagram depicting the sites of origin of target-derived growth factors that may influence motoneuronal CGW expression. (a) onginating from the post-synaptic membrane, @) muscle-derived (Le., muscle spi ndle) ( c ) extracellular matrix in origin, (d) sensory (Le., DRG spinal, supraspinal-origin) (e) autocrinelparacrine ( f ) of humoral or systemic origin. (Adapted from GrinneIl, 1995).
7.3 -2.2. Erperiments with muscle growtMkirnover pro feins and CGRP
One of the questions that arises fkom this study is the effect of changes in the muscle
itself as a remit of the eccentric exercise paradigrn. As discussed previously, chronic
eccentric activity is known to produce muscle fibre damage (Ogilvie et al., 1988; Armstrong
et al., 1983). Even though we do not attribute the changes in motoneurond CGRP in Our
study to an effect of myofibriiIar damage, adaptations that are a part of regular muscle
growth and turnover are known to occur foliowîng the prescribed activity (Wong and Booth,
1990; Booth and Kirby, 1992; Lowe et al., 1995). This adaptation may be in the form of
minor repair or enhancement of myofibrillar structure (Smith et al., 1997), changes in muscle
metabolism and fatigue resistance (Batnave and Thompson, 1993; Schwane and Armstrong,
1983), and may involve changes in growth hormone (Roy et al., 1996), IGF (Roy et al.,
1996; Yan et ai., 1993), satellite ce11 activity @am and Schultz, 1987) and their precursors,
developmental as well as adult myosins (Smith et al., 1996), skeletai muscle structural
proteins, such as troponin-l (Sorichter et al., 1997), or co~ec t ive tissue components
(desmin, fibronectin) (Friden and Lieber, 1998; Lieber et al., 1994, 1996). Initial work shouid
describe the changes that result fiom eccentric exercise, and then determine if there is a
correlation with motoneuronal and motor endplate CGRP response.
7.4. PART 2. LIMITATIONS OF EXPERMENTS LN CHAPTERS III, IN, and V.
Inherent in all the experimentd investigations reported are limitations of the
methodologies employed. These limitations generaiiy result fiom technicai difficulties or
procedural complications that contribute variability to the study. Many of these problems
have been discussed in the Methods and Materials sections of Chapters II, III, IV, and V, as
weli as in the Results sections. Below is a consideration of other factors.
7.4.1 .Limitations of Experiments in Cha~ter IV: The Exercise Model.
In using the downhill running exercise model, we made the assumption that the TS
muscles are performing eccentric contractions, while the AC muscles are performing
concentric contractions, as reported by Ogilvie et al. (1988) and Armstrong and Ogilvie
(1983). However, neither they nor we have completed electrophysiological measurements
documenthg the involvement of these muscle groups or their recmitment patterns while
perfomiing the d o d running activity. Most data on eccentric contractile activity have
been generated fiom human studies. The applicability of the human model to that obsewed in
the animal, remains to be determined. Biomechanical differences in gait between bipeds and
quadrupeds is an issue that also needs to be addressed in conjunction with the clear
demonstration (EMG) of the contribution of dBerent muscle groups to the successful
completion of the downhill running motor program.
The series of experiments reported in this thesis were based on an acute exercise
protocol. The effects of chronic concentric, or repeated bouts of ninning exercise with
respect to muscle are weil known in both humans and rodents (Edstrom and Grirnby, 1986;
Booth and Thomason, 2991). I f a chronic, more intense, period of exercise was utilised in
our experiments, it may have produced different results in the MG, other muscles, and their
motoneurons. For example, chrmic andior long penods of eccentnc activity, have been
shown to result in substantial SOL muscle darnage, with various indices of pathology being
clearly observed 3-5 days post-exercise (Armstrong et al., 1983, 1991; Ogilvie et al., 1988;
Stauber, 2989). Therefore, fiirther studies of the effects of chronic eccentric exercise on the
MG and the extent of tissue darnage associated with such exercise are required.
Concomitantly, changes in motoneuronal and motor endplate CGRP leveIs need to be
correlated with indices of myofibriiiar damage and repair processes. Experiments designed to
address this issue wïii fùrther our knowledge of neuron target-derived interactions.
7.4.2. Limitations of Experiments in Chapter V.
In addition to the factors already discussed in Chapter V (section 5.3), these
experiments presented other Limitations relating to the injection of tracer into two dEerent
muscle areas, and the potential muscle damage associated with the intramuscular injection.
The giycogen depletion studies will also be discussed in this section in terms of the technical
restrictions and the interpretative limitations of the data.
7.4.2.1. Iqection of tracer Ïnto the two r e m s of MG.
The decision to inject neuroanatornical tracers into two different areas of the MG
was based on work completed by DeRuiter et al. (1995a). These investigators elegantly
demonstrated anatomical and physiological dBerences in the rat MG @eRuiter et al., 1995a,
1996b). However, the terminology used by these authors regarding identification of a muscle
"compartment" differs &om that used in the present study, which needs to be noted.
The MG motor nerve divides into many intramuscular nerve branches in a proxirno-
distal sequence after it enters the MG muscle. DeRuiter et al. (1995a) refer to the proximal
"compartment" as being the part of the MG that is innervated by the proximal brunch of the
MG nerve and the dis& "compartment" as being innervated by the disfa[ nerve (DeRuiter et
al., 1995a). In Chapter V, experiments utilising the intramuscular injections of FluoroGold
were restricted to two areas of the MG, what we have cded the proximal and the distal
regonsS These regions incorporated the respective proximal and distal nerve branch
territones, but also included a larger volume of muscle tissue (see Figure 26). Intra-muscular
injections of FluoroGold into the proximal muscle region med the area innervated by the
Figure 26. Schematic representation of the Ieft rat MG describing the innervation o f the and distal cornpartrnents as described by DeRuiter et al., (1995). White box = FG muscle fibres; light- filled box = FG; FOCi; SO fibres and red filled box = FCI; FOG, SO fibres of the "proximal" compartrnent innervated by the proximal branch of the MG nerve. Cdibration bar = 2 cm.
proximal, intermediate, and proximal-lateral branches of the MG nerve. The distal region of
the muscle was injected in an area innervated by the primary and secondary distal branches of
the MG nerve (Figure 26). These injection areas incorporate dzerent '%ompartrnents' than
those descnbed by DeRuiter et al. (1995a,b, 1996qb). It is important to distinguish between
the temiinology used by ourselves and DeRuiter et al. (1995a) in order to correctly
extrapolate &om their conclusions. See Figure 10 for a diagram of our injection protocol into
the "distd and proximal" MG regions.
7.4.2.2. Mzïscle &mage as a resuli of the infrm11scz1Iar injection technique.
As discussed briefly in Chapter V (sec 5.5.2), it is unWcely that the intrarnuscular
injections of FluoroGold resulted in a detectable nerve injury that evoked nerve sprouting
and an elevation in motoneuronal CGRP expression. In the fkst study, Chapter IV,
motoneurons were deliberutely not retrogradely labelled due to the concem that the
intramuscular injection might cause sufficient damage to initiate a sprouting response. The
data fiom that study clearly indicated that an increase in the numbers of CGRP+ve
motoneurons fol lohg downhill exercise occurred when the muscles were not injected with
FluoroGold (Homonko and Theriault, 1997). In addition, the injection of FluoroGold into
the SOL and LG did not a e c t the expression of CGRP at either the endplates or
motoneurons supplying these muscles. Other investigators (Popper et al., 1992), who usecl a
multiple injection protocol to demonstrate the retrograde effects of a ccmuscle factor" on the
bulbocavernosus muscle, were unable to detect changes in CGRP immunoreactivity and a-
CGRP rnRNA in either the sham or buffer treated groups. Therefore, based on Our initial
study and the findings of other investigators, it appears unlikely that the intrarnuscular
injection technique used in these studies could be responsible for the si&cant and
proionged changes in CGRP expression in MG motoneurons and at their NMJs.
7-4.2.3. Limitations of the alycogen - dep letion studies
Quantification of muscle glycogen depletion c m be employed to estimate the
metabolic or physiological activity of a given muscle or muscle fibre population under the
appropnate experimental paradigm. Hence, utilisation of the PAS stain for the hiaological
anaiysis of glycogen content in muscle cross-sections provides a rough indication of the
spatial recruitment of motor units within a given muscle (Armstrong and Taylor, 1993). It
provides some insight into the proportionate rates of activity of a particular muscle or fibre
population within the muscle and the usage of different cornpartments or regions within a
muscle (e.g., Sullivan and Armstrong, 1978; Theriault and Diarnond, 1988; DeRuiter et al.,
1996). However, glycogen depletion data contributes no uiformation about power output or
tension generated by that muscle.
Another factor to consider is the sarnpling method used to assess muscle fibre
glycogen content. Due to the globular distribution of glycogen within a muscle fibre, random
cross sections may not provide a completely accurate profile of the muscle's glycogen status
(Sullivan and Armstrong, 1978). The MG muscle has a pennate architecture. If the muscle is
not oriented correctly when sectioned into transverse sections, certain muscle fibres could
appear to be "missing" or only partially represented. Therefore, a lack of glycogen content,
or an uneven distribution of glycogen could be reported for those muscle fibres.
In ternis of using glycogen depletion data to determine recruitment of muscle fibres
during task performance, Sullivan and Armstrong (1978) have noted that, during running
activity, the recruitment of motor units may "cycle" either during various stages of a stride or
during sequential step cycles. Therefore, a reduced glycogen content in al1 fibres would not
necessarily imply that sirnultaneuus activity in all motor units had been achieved at any given
tirne. These data suggest that di motor units were active at one tirne-point, but not
necessarily equdy active; in addition, no information is garnered for the the-course of the
activity or the proportion that were active.
The Limitations of this technique apply directly to attempts at deriving more
"quantitative" estimates of muscle fibre activity. The present study used muscle glycogen
depletion data only as a gross measure to evahate whether one region of the MG was active,
and the PAS results hdicated that ali muscle fibre types in both regions were active during
downhill runnuig. In order to determine whether our exercise paradigm preferentiaiiy
recruited one region or a specific fibre type, expenments need to be completed which match
CGRP+ve endplates with the relative levels of glycogen depletion in individual muscle fibres.
In addition, eIectrophysiological measurements of the activity of the different MG
compartments matched with PAS staining would be in valuable. These experhents would
need to be done to determine if CGRP expression at FG motor endplates is associated with
selective recmitment of either fast fatiguable (FF) or fast fatigue resistant (FR) motor units.
CHAPTER VIII:
SUMMARY AND CONCLUSION
Cellular, organ, and systemic modifications that favour the survival of an animal or a
human to an environmental change are said to be adaptive. Physical exercise, like
environmental change, is disruptive to the intemal or homeostatic condition (Booth and
Thornason, 1991). Alterations in tells, tissues, organs or systems that remain after, or as a
consequence of, physicaf training are considered exercise adaptations. One fùnction of
exercise adaptation is to minimise dismption of the homeostasis during a subsequent exercise
bout. This enhanced maintenance of the intemal condition via exercise adaptation favours the
fûnctional effectiveness of the non-sedentary animal. Less disruption in homeostasis allows
the individual (animal or human) to perforrn physical activity for extended periods of time at
the same absolute power before expenencing fatigue.
We have shown that folIowing downhill running, CGRP expression was elevated in
MG motoneurons, and more specifically, at their motor endplates on FG muscle fibres
exclusively. The long-term effects of exercise-induced increases in CGRP expression may
play a role in the remodelling of the post-synaptic membrane of the neuromuscular junction.
As well, this mode1 provides a suitable system which lends itself to firther investigations of
the relationships between neuropeptide expression, muscle compartmentaiisation, and motor
unit recniitment strategies for unaccustomed tasks.
Andersen, S.L.V., and Clausen, T. (1993). Calcitonin gene-related peptide stimulates
active Na+-K+ transport in rat soleus muscle. Amer. J PhysoI. Z64:C4 lg-C429.
Andreose, J. S ., Sala, C., and Fumagalli, G. (1 994). ImmunolocalUation of chromogranin
B, secretogranin II, calcitonin gene-related peptide and Substance P at developing and
adult neuromuscular synapses. Neurosci. Le#. 174: 177- 180.
Appeli, H.J. (1984). ProMeration of motor endplates induced by increased muscular
activity. Inter. J Sports Med. 5 : 125- 129.
AppeD, H. J., Soares, J.M.C., and Duarte, J. AR. (1 992). Exercise, muscle darnage, and
fatigue. Sport. Med. 13:108-115.
Armstrong, RB., Ogilvie, R.W., and Schwane, I.A. (1983). Eccentric exercise-induced
injury to rat skeletal muscle. J. Appl. Physol. 54:80-93.
Armstrong, R.B . (1 990). Initial events in exercise-induced muscular injury. Med Sci.
Sport. ber. 22:429-43 5.
Armstrong, RB., Warren, G.L., and Warren, J.A (2991). Mechanisms of exercise-
induced muscle fibre injury. Sport. MM. 1 2: 1 84-207.
Armstrong, R.B., and Phelps, R.O. (1984). Muscle fiber type composition of the rat
huidhb. Amer. J Anat. 17 1 :259-272.
Armstrong, R.B., and Taylor, C.R. (1993). Glycogen loss in rat muscles during
locomotion on difFerent inclines. J. Exp. Biol. 1 76: 1 3 5- 144.
Arvidsson, U., Cdheim, S., Ulfhake, B., Holdèlt, T., and Terenius, L. (1989). Altered
levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity of cat lumbar
motoneurons after chronic spinal cord transection. Brain Res. 48W 87-39 1.
AMdsson, U., Johnson, R, Piehl, F., Cullheim, S., HolGelt, T., Risling, M., Terenius, L.,
and Ulfhake, B. (1990). Peripheral nerve section induces increased levels of calcitonin
gene-related peptide (CG=)-Wce immunoreactivity in axotomized motoneurons. Exp.
Brain Res. 79212-216.
Arvidsson, U., Piehl, F., Johnson, H., Ulfhake, B., Cullheim, S., and HolGelt, T. (1993).
The peptidergic motoneurone. NewoReport 4: 849-856.
Atwood, H.L., Nguyen, P.V., and Mercier, A J. (1 99 1). Acfivty-dependent adaptation in
neuromuscular systems: comparative observations. In A Wernig (Ed.), Plarricitv of
Motoneurd Connections. @p. 10 1-1 14). Amsterdam: Elsevier Scientific Publications
BV.
Atwood, H.L., and Lnenicka, G.A. (1987). Role of activity in determining properties of
the neuromuscular system in crustaceans. Amer. 2001. 27:977-989.
Bakels, R-, and Kernell, D. (1993). Matching between motoneurone and muscle unit
properties in rat medial gastrocnemius. J. Physiol. 463 :3 07-3 24.
Balice-Gordon, RJ., and Lichtman, J.W. (1990). In vivo visualization of the growth of
pre- and postsynaptic elements of neuromuscular junctions in the mouse. J. Neurosci.
10:894-908.
Balice-Gordon, R.J., and Lichtman, J.W. (1991). The plasticity and stabiiity of
neuromuscular synapses in living mice. In A. Wernig (Ed.), PZastici~ of Motoneuronal
Connections. @p. 7 1-84). Amsterdam: Elsevier Scientific Publications BV.
Balnave, CD., and Men, D.G. (1995). 1ntracelIula.r calcium and force in single mouse
muscle fibres following repeated contractions with stretch. J. PhysioI. 488- l:25-36.
Balnave, CD., and Thompson, M.W. (1993). Effect of training on eccentric exercise-
induced muscle damage. J. Appl. PPhysoZ. 75: 1545-1 55 1.
Barker, D., and Ip, M.C. (1966). Sprouting and degeneration of marnrnaiian motor axons
in normal and de-afEerentated skeletal muscle. Proc. Ray. Soc. Ser. B. 163538-554-
Birling, M., and Price, J. (1995). Influence of growth factors on neuronal differentiation.
Curr. Op. Neurobiol. 7:878-884.
Blake-BnizPni, KM., Borke, R.C., Anders, J.J., and Potts, J.D. (1997). Calcitonin gene-
related peptide and a-CGRP mRNA expression in cranid motoneurons f i e r hypoglossal
nerve injury during postnatal development. J. Narrocyfol. 26: 163- 179.
Blanco, C.E., Popper, P., and Micevych, P.E. (1992). a-CGRP mRNA expression in
motoneurons is related to myosin heavy-chah composition of specific rat hindlimb
muscles. Soc. Neurosci. Abs. l8:85 8
Blanco, C.E., Popper, P., and Micevych, P.E. (1997). a-CGRP mRNA levels in
rno toneurons bervathg specinc rat muscles. Mol. Brain. Res. 44: 253 -26 1.
Bon, S., Vigny, M., and Massoulie, J. (1979). Assymetric and globular forms of
acetylcholinesterase in marnmals and birds. Proc. Nat. Acud Sci. USA. 762546-2550.
Booth, F.W., and Kirby, C R (1992). Changes in skeletal muscle gene expression
consequent to altered weight bearing. Amer. J Physol. 262:R3 29-R3 3 2.
Booth, F.W., and Thornason, DS. (199 1). Molecular and cellular adaptation of muscle in
response to exercise:perspectives of various models. Phys. Rev. 7 1 5 4 1-585.
Soudreau-Lariviere, C., and Jasmin, B.J. (1997). Trophic regdation of
acetylcholinesterase expression in cultured mouse myotubes. Soc. Narrosci. Abs. 22: 1453
(abstract)
Brooke, M.H., and Kaiser, K.K. (1970). Muscle fiber types: how many and what kind?
Arch, Neural, 23 :369-379.
Brown, M C , Holland, ILL., and Hopkins, W.G. (1981). Motor nerve sprouting. A m .
Rev. Neurosci. 4 : 1 742.
Brown, M.C. (1984). Sprouting of motor nerves in adult muscles: a recapitulation of
ontogeny. IïNS. 7: 10- 14.
Burke, R.E. (198 1). Motor units: anatomy, physiology, and finctional organization. In
V.B. Brooks (Ed.), Hand6ook of PhyJioZogy. Section I r ïhe Nervous System. @p. 345-
423). Bethesda, MD: American Physiologicai Society.
Burke, R.E., Dum, R.P., Fleshman, J. W., Glenn, L.L., Lev-Tov, A, OtDonovan, M. J., and
Pinter, M.I. (1 982). An HRP study of the relation between cell size and motor unit type in
cat ankle extensor motoneurons. J. Comp. Nml. 209: 17-28.
Byrd, S.K. (1992). Alterations in the sarcoplasmic reticulum: a possible link to exercise-
induced muscle damage. Med Sci. Sport. fier. 2453 2-536.
Bymes, W.C., Clarkson, P.M., White, J.S., Hsieh, S.S., Frykman, P.N., and Maughan,
RJ. (1985). Delayed onset muscle soreness following repeated bouts of downhiII running.
J. Appl. Physol. 59:710-715.
Cannon, J.G., Fielding, RA, Fiatarone, M.A., Orencole, S .F., Dinarello, C.A., and Evans,
W.J. (1989). Increased interleukin lb in human skeletal muscle d e r exercise. Amer. J.
Physid. 257:R4S 1 -R455.
Cannon, J.G., Meydani, S.N., Fielding, RA, Fiatarone, M.A., Meydani, M.,
Farhangmehr, M., Orencoie, SE., Blumberg, J.B., and Evans, W.J. (199 1). Acute phase
response in exercise. II. Associations between vitamin E, cytokines, and muscle
proteolysis. Amer. J. Physiol. 260-XI23 5-R1240.
Cardasis, CA, and Padykula, B A . (1981). Ultrastructural evidence indicating
reorganization at the neuromuscular junction in the normal rat soleus muscle. Anat. Rec.
200:41-59.
Caroni, P., and Grandes, P. (1990). Nerve sprouting in innervated adult skeletal muscles
hduced by exposure to elevated levels of insulin-like growth factors. J. Cell Bioi.
1 lO:l307-13 17.
Changeux, J.P. (1991). Cornpartmentaiized transcription of acetylcholine receptor genes
d u ~ g motor endplate epigenesis. New Bol. 3 :4 13-429.
Changeux, J.P., Duclert, A, and Sekine, S. (1992). Calcitonin gene-related peptides and
neuromuscular interactions. Ann. NY. Acad Sci. 3 6 1-3 78.
Cheng, L., Khan, M., and Mudge, A.W. (1995). Calcitonin gene-related peptide promotes
Schwann ce11 proliferation. d Cell Bid. 129:789-796.
Choi, R.C.Y., Yung L.Y., Dong, T.T.X., Wan, D.C.C., Wong, Y.H., Tsim, K.W.K., and
Rieger, F. (1998). The calcitonin gene-related peptide-induced acetylchohesterase
synthesis in cultured chick myotubes is mediated by cyclic AMP. J. Neurochem. 71 : 152-
160.
Clarkson, PM., Nosaka, K., and Braun, B. (1992). Muscle fünction after exercise-induced
muscle damage and rapid adaptation. Med Sci. S W - Exer. 245 12-520.
Cooper, R.L., Marin, L., and Atwood, H-L. (1995). Synaptic dïerentiation of a single
motor neuron: conjoint definition of transmitter release, presynaptic calcium signals, and
~Itrastructure. J. Nmosci- 15:4209-4222.
Darr, K.C., and Schultz, E. (1987). Exercise-induced satellite cell activation in growing
and mature skeletal muscle. J; Appl. Physioi. 63 : 18 16- 182 1.
DeRuiter, C.J., DeHaan, A., and Sargeant, A.I. (1995a). Repeated force production and
metaboiites in two media1 gastrocnernius muscle compartments of the rat. J. AppZ.
Phusid. 79: 1855-1861.
DeRuiter, C. J., DeHaan, A., and Sargeant, A. J. (1 995 b). Physiological characteristics of
two extreme muscle compartments in gastrocnemius medialis of the anaesthetized rat.
Acta Physiol. Scm. 153:3 13-324.
DeRuiter, C.J., DeHaan, A., and Sargeant, A.J. (1996a). Fast-twitch muscle unit
pro perties in difFerent rat medial gastrocnemius muscle compartment S. J. Neurophysiol.
75:2243 -2254.
DeRuiter, C.J., Habets, P.E.M.H., DeHaan, A.., and Sargeant, A.J. (1996b). Ln vivo IIX
and IIB fiber recruitment in gastrocnernius muscle of the rat is compartment related. J.
AppZ. Phpiol. 8 1 : 93 3 -942.
Deschenes, MJL, Maresh, CM., CnveUo, J-F-, Armstrong, L.E., Kraemer, W. J., and
Covault, J. (1993). The effects of exercise training of different intensities on
neuromuscular junction morphology. J. Neurocytol. 22:603-6 15.
Deschenes, M.R, Covault, J., Kraemer, W.J., and Maresh, C.M. (1994). The
neuromuscular junction: muscle fibre type dierences, plasticity and adaptability to
increased and decreased activity. Spors Med. 17(6):358-372.
Dorlochter, M., Irintchev, A, BMikers, M., and Wernig, A (1991). Effects of enhanced
activity on synaptic transmission in mouse extensor digitorum longus muscle. J PhysoZ.
4361283-292.
Dmry, R.A.B., and Wallington, E.A. (1990). Carleton's H .o lo~cuZ Technime. Odord,
N.Y., Toronto: Oxfiord University Press.
Duan, C., Delp, M.D., Hayes, D.A., Delp, P.D., and Armstrong, R.B. (1990). Rat skeletal
muscle mitochondrial [Ca"] and injury from downhill waiking. J. AppL Physiol- 68: 1241-
1251.
Dubner, R., and Ruda, M.A (1992). Activity-dependent neuronal plasticity following
tissue injury and idammation. 7INS. 1 5 : 96- 1 03.
Edstrom, L., and Grirnby, L. (1986). Effect of exercise on the motor unit. Muscle Neme
9: 104-126.
Ehan, G.L., Courtney, K.D., Andres Jr., V., and Featherstone, R.M. (1961). A new and
rapid colorimetric detemination of acetylcholinesterase activity. Biochem. P h . 7:88-
95.
English, AW., Donahue, S.P., and Weeks, 0.1- (1985). Compartmentalization of rat
lateral gastrocnemius muscle. Amer. Zool. 25: 107A(abstract)
Enoka, ILM. (1995). Morphological features and activation patterns of motor units. J.
Clin Neurop?ysioZ. 12: 5 3 8-5 59.
Enoka, RM. (1996). Eccentnc contractions requue unique activation strategies by the
nenrous system. J. AppL Physiol. 8 1 :2339-2346.
Evans, W.J., and Cannon, J.G. (199 1). The metabolic effects of exercise-induced muscle
damage. fier. Sport. Sci. Rev. 99-125.
Fernandez, H.L., Moreno, R.D., and Inestrosa, N.C. (1996). Tetrameric G4
acetylcholinesterase:structure, locaiïzation and p hysiological regulation. J. Neurochem .
66: 1335-1346.
Femandez, H.L., and Donoso, J.A (1988). Exercise selectively increases G4 AChe activity
in fast-twitch muscle. J. AppL Physiol. 65:2245-2252.
Femandez, H.L., and Hodges-Savola, C.A. (1992). Trophic regulation of
acetylcholinestrase isoenzymes in adult mammalian skeletal muscles. Neurochem. Res.
17:115-124.
Femandez, H.L., and Hodges-Savola, C.A. (1996). Physiological regulation of G4 AChe
in fast-twitch musc1e:effects of exercise and CGRP. J. Appl. Physd. 80:357-362.
Fielding, RA, Manfkedi, T.J., Ding, W., Fiatarone, M.A-, Evans, W.J., and Cannon, J.G.
(1993). Acute phase response in exercise III. Neutrophil and IL-lb accumulation in
skeletal muscle. Amer. J. Physiol. 26S:Rl66-R172.
Fontaine, B., Klarsfeld, A, HoMelt, T., and Changeux, 19. (1986). Calcitonin gene-
related peptide, a peptide present in spinal cord motoneurons increases the number of
acetylcholine receptors in primary cultures of chick embryo myotubes. Neurosci. Lett-
71 59-65.
Fontaine, B., Klarsfeld, A, and Changew J.P. (1987). Calcitonin gene-related peptide
and muscle activity regulate acetylcholine receptor a-subunit mRNA levels by distinct
intracellular pathways. J Cell Biol. 105: 13 3 7- 1342.
Forsgren, S., Bergh, A, Carlsson, E., and Thornell, T.E. (1992). Studies on the
distribution of calcitonin gene-related peptide-like and substance P-like immunoreactivities
in rat hind-limb muscles. Histochem. J. 24:345-353,
Forsgren, S., Bergh, A., Carlsson, E., and Thomell, L.E. (1993). Calcitonin gene-related
peptide expression at endplates of difFerent fibre types in muscles in rat hïnd limb muscles.
Cell Tiss- Res. 274:43 9-446.
Friden, J., Seger, J., and Ekblom, B. (1988). Sublethal muscle fibre injuries d e r high
tension anaerobic exercise. Eur. J. AppL PhysioZ- 57:360-368.
Fnden, J., and Lieber, R.L. (1992). Structural and mechanical basis of exercise-induced
muscle injury. Med Sci. Sport. fier. 24521-530-
FrÏden, J., and Lieber, EL. (1998). Segmentai muscle fiber lesions after repetitive
eccentric contractions. Ce22 Tiss. Res. 293 : 165- 17 1.
Funakoshi, H., Belluardo, N., Arenas, E., Yamamoto, Y., Casabono, A., Persson, H., and
Ibanez, C.F. (1 995). Muscle-derived neurotroph-4 as an activity-dependent trophic
signal for adult motor neurons. Nature 268: 1495-1499.
Gardiner, P.F. (1993). Physiological properties of motoneurons innervating different
muscle unit types in rat gastrocnemius. J. NeurophysioIl. 69: 1 160- 1 170.
Gibala, M.J., MacDougaiI, JD., Tamopolsb, M A , Stauber, W.T., and Elomaga, A
(1995). Changes in human skeletd muscle ultrastructure and force production &er acute
resistance exencse. J. Appl. PhysioZ. 78:702-708.
Gibson, S.J., Polak, J.M., Bloom, S R , Sabate, LM., Muldeny, P.M., Ghatei, M A ,
McGregor, G.P., Momson., J-FB., Kelly, J.S., Evans, RM., and Rosenféid, M.G. (1984).
Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other
species. J. Neurosci. 4 3 10 1-3 1 11.
Gisiger, V., Belisle, M., and Gardiner, P.F. (1991). Swimming training increases the G4
acetylchoiinesterase content of both fast ankle extensors and flexors. FEBS. 278:27 1-273.
Gisiger, V., Belisle, M., and Gardiner, P.F. (1994). Acetylcholinesterase adaptation to
voluntary wheel running is proportional to the volume of activity in fast, but not slow, rat
h i n d h b muscles. Eur. J. Neurosci. 6:673-680.
Gisiger, V., and Stephens, H.R. (1988). Localization of the pool of G4
acetylcholinesterase characterizing fast muscles and its alteration in murine muscular
dystrophy. J. Neurosci. Res. 19:62-78.
Grinnell, AD. (1995). Dynamics of nerve-muscle interaction in developing and mature
neuromuscular junctions. Physiol. Rev. 75:789-834.
Grinnell, AD., and Herrera, A A (1981). Specificity and plasticity of neurornuscular
connections: long-term regulation of motoneuron fûnction. Prog. Neurobiol. 17203-282.
Hd, Z., and Sanes, J.R (1993). Synaptic structure and development: the neuromuscular
junction. Cell72:99- 12 1.
Henderson, CE., Philips, H-S., Pofloclg RA, Davies, AM., Lemeulie, C., Armanini, M.,
Simpson, L.C., Moffet, B., Vandlen, RA, Koliatsos, V., and Rosenthal, A (1994).
GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.
Science 266: 1062-1064,
Henderson, C.E. (1996). RoIe of neurotrophic factors in neuronal development. C m . Op.
In Neuro biol, 6: 64-70.
Hememan, E. (1957). Relation between size of neurons and their susceptibility to
discharge. Science 126: 1345-1346.
Henneman, E., Somjen, G., and Carpenter, D.O. (1965). Functional signifïcance of celi
size in spinal motoneurons. J. Neurophysiol. 28560-580.
Hememan, E., and Mendell, L.M. (1981). Functional organization of motoneuron pool
and its inputs. In V.B. Brooks (Ed.), Hmdbook of Phvsiolo~. Section 1: n e Nervous
Svstem. @p. 423-507). Bethesda, MD: Arnerican Physiological Society.
H e ~ e m a n , E., and Olson, C.B. (1965). Relations between structure and fiinction in the
design of skeletal muscles. J. Physiol. 2858 1-598.
Hodges-Savola, C.A., and Femandeq H.L. (1995). A role for calcitonin gene-related
peptide in the regdation of rat skeletal muscle G4 acetylcholinesterase. Nmrosci. Lea.
190:117-120.
Homonko, D.A., and Theriault, E. (1997). Calcitonin gene-related peptide is increased in
h indhb motoneurons after exercise. Int. J. Sports. Med. 18: 1-7.
Hughes, R-A, Sendtner, M., Goldfarb, M., Lindholm, D., and Thoenen, H. (1993).
Evidence that fibroblast growth factor 5 is a major muscle-derived survival factor for
cultured spinai motoneurons. Neuron 1 O:3 69-3 77.
Hunt, C. (1990). Mammalian muscle spind1e:periphera.l mechanisms. PhysoLRev. 70:643-
663.
Hutchison, D.L., Roy, RR, Bodine-Fowler, S., Hodgson, J.A., and Edgerton, V.R.
(1989a). Electromyographic @MG) amplitude patterns in the proximal and distal
compartments of the cat semitendinosus during various motor tasks. Brain Res. 47956-
64.
Hutchison, D.L., Roy, R.R-, Hodgson, J.A, and Edgerton, V.R (1989b). EMG amplitude
relationships between the rat soleus and medial gastrocnemius during various motor tasks.
Bruin Res. SO2:23 3 -244.
Ingds, C.P., Warren, G.L., and Armstrong, RB. (1998a). Dissociation of force
production fiom MHC and actin contents in muscles injured by eccentric contractions. J.
Musc. Res. Cell Motil* 1 9: 2 1 5 -224.
Ingalls, C.P., Warren, G.L., Williams, J.H., Ward, C.W., and Armstrong, R.B. (1998b). E-
C coupling fdure in mouse EDL muscle after in vivo eccentnc contractions. L Appl.
Physiol. 8558-67.
Jasmin, B.J., Lee, RK., and Rotundo, R.L. (1993). Compartmentalization of
acetlcholinesterase mRNA and enzyme at the vertebrate neuromuscular junction. Neuron
1 1 :467-477,
Jasmin, B. J., Antony, C., Changeux, JP., and Cartoud, J. (1 995). Nerve-dependent
plasticity of the golgi complex in skeletal muscle fibers: compartmentalization withui the
subneural sarcoplasm. Eur. J. Nmrosci. 7:470-479.
Jasmin, B.J., and Gisiger, V. (1990). Regulation by exercise of the pool of G4
acetylcholinesterase characterizhg fast muscles: opposite eEect of running training in
antagonist muscles. J. Neurosci. 10: 14441454.
Jinnai, K., Chihara, K., Kanda, F., Tada, K., and Fujita, T. (1 989). Calcitonin gene-related
peptide enhances spontaneous acety lcholine release nom the rat motor nerve terminal.
Neurosci. Lett. 1 O3 :64-68.
Jones, D.A., Newham, D.J., and Torgan, C. (1989). Mechanical iduences on long-lasting
human muscle fatigue and delayed-onset pain. J. Physiol. 4 lZ:4 1 5-427.
Kanda, K., and Hashizume, K. (1992). Factors causing difEerence in force output arnong
motor units in the rat medial gastrocnemius muscle. J. Physiol. 448:677-695.
Kashihara, Y., Sakaguchi, M., and Kuno, M. (1989). Axonai transport and distribution of
endogenous calcitonin gene-related peptide in rat peipheral nerve. J. Nezrrosci. 9:3796-
3802.
Kawabuchi, M., and Kosaka, T. (1993). Variations o f colateral branching in the motor
nerve fibres in the skef etal muscle of adult rats. Acta Histochem. 94: 173 - 1 84.
Kidokoro, Y., and Saito, M. (1988). Early cross-striation in twitching Xenopus myocytes
in culture. Proc. Nat. Acad Sci. USA. 85: 1978-1982.
Koliatsos, V.E., Clatterbuck, RE., Winslow, J., Cayouette, M.H., and Price, D.L. (1993).
Evidence that brain-derived neurotrophic factor is a trophic factor for rnotor neurons in
vivo. N i o n 10:359-367,
Koyanagi, I., Tator, C H , and Theriault, E. (1993). Silicone rubber microangiography of
acute spinal cord injury in the rat. Neuromrg. 32:260-268.
Kuipers, H- (1 994). Exercise-induced muscle damage. Int. J.Spors Med 1 5: 132- 13 5.
Kwon, Y.W., and Gumey, M.E. (1994). Systernic injections of ciliary neurotrophic factor
induce sprouting by adult motor neurons. NeuroReport 5:789-792.
Laufer, R., and Changeux, J.P. (1987). Calcitonin gene-related peptide elevates cyclic
AMP in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal
messenger. M O . 6:90 1-906.
Li, J., and DahIstrom, A (1992). Development of calcitonin gene-related peptide,
chromagranin A, and synaptic vesicle markers in rat rnotor endplates, studied using
imrnunofluorescence and confocal laser scanning. Miscie Nerve 1 5 :984-992.
Li, L., Wu, W., Lin, L.H., Lei, M., Oppenheim, R.W., and Houenou, L.J. (1995). Rescue
of adult mouse motoneurons fiom injury-induced cell death by glial ceil Iine-denved
neurotrophic factor. Proc. Nat. Acad Sei. USA. 92:977 1-9775.
Lieber, R.L., Woodburn, T.M., and Fiiden, J. (1991). Muscle damage induced by
eccentric contractions of 25% strain. J. AppL Physioi. 70:2498-2507.
Lieber, R.L., Schmiîz, M C , Mishra, D.K., and Fnden, J. (1994). Contractile and cellular
remodelling in rabbit skeletal muscle after cyclic ccentric exercise. J. Appl. Physiol.
77: 1926-1934.
Lieber, E U . , Thornell, LE., and Friden, J. (1996). Muscle cytoskeletal disruption occurs
within the tint 15 min of cyclic eccentnc contraction. J. Appl PhysioL 80:278-284.
Lieber, U., and Friden, 1. (1988). Selective darnage of fast glycolytic muscle fibres with
eccentric contraction of the rabbit tibialis anterior. Acta Physiol. Scan. 133 587-588.
Lin, L.F., Doherty, D.H., Lile, J.D., Bektesh., S., and Cohs , F. (1993). GDNF: a glid ceil
line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130-
1132.
Lin, L.F., Zhang, T.J., Coilins, F., and Armes, L.G. (1994). Purification and initial
characterization of rat B49 glial cell line-derived neurotrophic factor. J. Neurochem.
6362:758-768.
Lindsay, R.M., Wiegand, S.J., Altar, C.A., and Distefano, P.S. (1994). Neurotrophic
factors:fiom molecule to man. TINS. 17: 182- 190.
Liou, J., and Fu, W. (1995). Additive effect of ADP and CGRP in modulation of the
acetylcholine receptor channel in Xenopus embryonic myocytes. Br. J. P h m . 115563-
568.
Lnenicka, G.A, Atwood, H.L., and Marin, L. (1986). Morphological transformation of
synaptic tenninals of a phasic motoneuron by long-term tonic stimulation. J. Neurosci.
Lnenicka, G A , and Atwood, H.L. (1985). Age-dependent long-term adaptation of
crayfïsh neuromuscuIar phasic motor axon synapses to dtered activity. J; Neurosci. 5:459-
467.
Lowe, D-A, Warren, G.L., Ingds, C.P., Boorstein, D.B., and Armstrong, RB. (1995).
Muscle fiinction and protein metabolism after initiation of eccentnc contraction-induced
injury. J. Appl. Physiol. 79: 1260- 1270.
Lu, B., Fu, W., Greengard, P., and Poo, M. (1993). Calcitonin gene-related peptide
potentiates synaptic responses at developing neuromuscular jundion. Nature 3 63 : 76-79.
Lu, B., and Fu, W. (1995). Regulation of postsynaptic responses by calcitonin gene-
related peptide and ATP at develo ping neuromuscular junctions. Cm. J. Physiol.
Pharmacol. 73: 1050-1056.
Lynn, R., Talbot, J.A, and Morgan, DL. (1998). DifFerences in rat skeletal muscles after
inciine and decluie running. J. Appl. Physol. 85 :98- 104.
Massoulie, J., Bacou, F., Barnard, E., Chatomet, A, Doctor B.P., and Quinn, D.M.
Cholinesterases. Structure. Function. Mechanism. Genetic, and Cell Bioloav.
Washington, DC : Arnerican C hernical Society.
Massoulie, J., Peuementi, L., Bon, S., Krejci, E., and Vailette, F.-M. (1993). Molecular
and cellular biology of choluiesterases. Prog. Neurobiol. 4 1 :3 1-9 1.
Matteoli, M., Hairnann, C., Tom-TareLi, F., Polak, J.M., Ceccaretli, B., and DeCamilli, P.
(1988). DEerentiaI effect of a-latrotoxin on exocytosis fiorn srnall synaptic vesicles and
f'rom large dense-core vesicles containhg cdcitonin gene-related peptide at the fi-og
neurornuscular junction. Proc. Ncrt. Acad Sci. USA. 85:7366-7370.
Matteoli, M., Balbi, S., Sala, C., Chini, B., Cimino, M., Vitadello, M., and Fumagalli, G.
(1990). Developmentally regulated expression of calcitonin gene-related peptide at
mammaiian neuromuscular junction. J. Mol. Neumci. 2: 175- 184.
McCully, K.K., and Faulkner, J-A (1985). Injury to skeletal muscle fibers of mice
following lengthenuig contractions. J. Appl. PPhyso[. 59: 1 19-126.
McCully, KX., and Faulkner, J.A. (1986). Characteristics of lengthening contractions
associated with injury to skeletal muscle fibers. J. Appl. Physiol- 6 l:293-299.
Miles, K., Greengard, P., and Huganir, R-L. (1989). Calcitonin gene-related peptide
regulates phosphorylation of the nicotinic acetylcholine receptor in rat myotubes. Neuron
2: 15 17-1 524.
Mulle, C., Benoit, P., Pinset, C., Roa, M., and Changeuq JP. (1988). Calcitonin gene-
related peptide enhances the rate of desensitization of the nicotinic acetylchohe receptor
in cultured mouse muscle cells. Proc. N d . Acad ScL USA. 8557284732.
Nardone, A., Romano, C., and Schieppati, M. (L989). Selective recruitment of high-
threshold human rnotor units during voluntary isotonic lengthening of active muscles. J.
Physioi. 409:45 1-471.
New, H.V., and Mudge, A W. (1 986). Calcitonin gene-related peptide regulates muscle
acetylcholine receptor synthesis. Nature 323 :809-8 1 1.
Newham, D.J., McPhail, G., Mills, KR., and Edwards, R.H-T. (1983). Ultrastructural
changes after concentnc and eccentric contractions of human muscle. J. Neurol. Sci.
61: 109-122.
Newham, D.J. (1988). The consequences of eccentric contractions and their relationship
to delayed onset muscle pain. Eur. J. Appl. Physioi. 57:353-359.
Nguyen, Q.T., Parsadanian, AS., Snider, W.D., and Lichtman, J.W. (1998).
Hyperinnewation of neuromuscular junctions caused by GDNF overexpression in muscle.
Science 279: 1 725- 1 729.
Noguchi, K., Senba, E., Monta, Y., Sato, M., and Tohyarna, M. (1990). a-CGRP and b-
CGRP mRNAs are differentidy regulated in the rat spinal cord and dorsal root ganglion.
MOI. Brain Res. 7:299-3 04.
O@e, R. W., Armstrong, R B , Baird, K.E., and Bottoms, CL. (1988). Lesions in the rat
soleus muscle following eccentncdy biased exercise. Amer. J. Ana?. 182:3 3 5-346.
Ohlen, A, Lindbom, L., Staines, W., HoMeIt, T., Cuello, A C , Fischer, KA., and
Hedqvist,P. (1987). Substance P and calcitonin gene-related peptide:immunohistochemicd
localization and rnicrovascular effects in rabbit skeletd muscle. Arch. Pharmacol. 3 3 6:8%
93.
Oppenheim, R.W., Houenou, L.J., Johnson, J.E., Lin, L.H., Li, L., Lo, AC., Newsome,
AL., Prevette, D., and Wang, S. (1995). Deveioping motor neurons rescued fiom
prograrnmed and axotomy-induced ce11 death by GDNF. Nature 373:344-346.
Oppenheim, R.W. (1996). Neurotrophic suMval molecules for motoneurons: an
embarassrnent of riches. Neuron 17: 1 95- 197.
OsterIund, M., Fontaine, B., Devilliers-Thiery, A., Geoffroy, B., and Changeux, J.P.
(1989). Acetylcholine receptor expression in primary cultures of embryonic chick
myotubes-1. Discoordinate regulation of a-,gamma-d- subunit gene expression by
calcitonin gene-related peptide and by muscle electncal activity. Neurosci. 32:279-287.
Padykula, BA, and Gauthier, G.F. (1970). The ultrastructure of the neuromuscular
junctions of mammaiian red, white and intermediate skeletal muscle fibers. J. Cell Biol.
46W-4 1.
Peeze Binkhorst, FM., Kuipers, H., Heymans, J., FrederiIg P.M., S l a D.W., Tangelder,
G. J., and Reneman, R S. (1 989). Exercise-induced focal skeletal muscle fiber degeneration
and capilIary morphology. J. Appl. PhysioZ. 66:2857-2865.
Peeze Binkhorst, F.M., Slaaf, D.W., Kuipers, H., Tangelder, G.J., and Renernan, R.S.
(1 990). Exercise-induced sweliing of rat soleus musc1e:its relationship with intrarnuscular
pressure. J. Appl. Physiol. 69:67-73.
Pestronk, A., and Drachman, D.B. (1978a). Motor nerve sprouting and acetylchoiine
receptors. Science 199: 1223-1225.
Pestronk, A., and Drachman, D.B. (1978b). A new stain for quantitative measurement of
sprouting at neuromuscular junctions. Muscle Nerve 1 :70-74.
Piehl, F., Arvidsson, U., Johnson, H., Cullheim, S., Villar, M., Dagerlind, A., Terenius, L.,
Hokfelt, T., and Ulfhake, B. (1991). Calcitonin gene-related peptide (CGRP)-like
irnmunoreactivity and CGRP mRNA in rat spinal cord motoneurons after diBerent types
of lesions. Eur. J. Neurosci. 3:737-757.
Piehl, F., Arvidsson, U., Hokfelt, T., and Cullheim, S. (1993). Calcitonin gene-related
peptide-like imrnunoreactivity in motoneuron pools innervating d ie ren t hind-limb muscles
in the rat. E i p Braï'n Res. 96:29 1-303.
Piehl, F., Ru-Rong, J., CuIlheim, S., Hokfelt, T., Lindholm, D., and Hughes, R. (1995).
Fibroblast growth factors regulate calcitonin gene-related peptide mRNA expression in rat
motoneurons after lesion and in culture. Eur. J. Neztrosci. 7: 1 73 9- 1750.
Piehl, F., Hammarberg, H., Hokfelt, T., and Cuiheim, S. (1998a). Regulatsry effects of
trophic factors on expression and distribution of CGRP and GAP-43 in rat motoneurons.
J. Neurosci. Res. 5 1 : 1 - 14.
Piehl, F., Hammarberg, 8, Tabar, G., Hokfelt, T., and Cuiiheim, S. (1998b). Changes in
the mRNA expression pattern, with special reference to calcitonin gene-related peptide,
after axond injuries in rat rnotoneurons depends on age and type of injury. Exp. Brain
Res. 119:191-204.
Plyley, M. J., Sutherland, G.I., and Groom, C. (1 976). Geometry of the capillary network
in skeletal muscle. Micro. Res. 1 1 : 16 1 - 173.
Plyley, M.J., and Groom, C. (1975). Geometncal distribution of capillaries in mammalian
stristed muscle. Amer. J, Physiol. 228: 13 76- 13 83.
Popper, P., Ulibam, C., and Micevych, P.E. (1992). The role of target muscles in the
expression of calcitonin gene-related peptide mRNA in the spinal nucleus of the
bulbocavemosus. Mol. Brain Res. 13 :43-5 1.
Popper, P., and Micevych, P.E. (1989). The effectof castration on calcitonin gene-related
peptide in spinal motor neurons. Neuroendocrinol. 50:338-343.
Roa, M., and Changeux, J.P. (1991). Characterization and developmental evolution of a
high affinity binding site for calcitonin gene-related peptide on chick skeletai muscle
membrane. Neurosci. 41563-570.
Rosenfeld, M.G., Merrnod, I.J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier,
J., Vaie, W.W., and Evans R.M. (1983). Production of a novel neuropeptide encoded by
the calcitonin gene via tissue-specific RNA processing. Nature 3 04: 129
Roy, RR., Hutchison, DL., Pierotti, D.J., Hodgson, J.A., and Edgerton, V.R. (199 1).
EMG patterns of rat ankle extensors and flexors during treadrnill locomotion and
swimmllig. J. Appl. P@si0ZI 70~2522-2529.
Roy, R.R-, Tri, C., Grossman, E.I., Talmadge, RJ-, Grindeland, R.E., Mukku, V.R., and
Edgerton, V.R. (1996). IGF-1, growth hormone, andor exercise effects on non-weight-
bearing soleus of hypophysectomized rats. J. Appl. Physiol. 8 1 :302-3 1 1 .
Sakaguchi, M., Inaishi, Y., Kashihara, Y., and Kuno, M. (1991). Release of calcitonin
gene-related peptide nom nerve terminais in rat skeletal muscle. J. PhysioI. 434:257-270.
Sala, C., Andreose, J.S., Fumagalli, G., and Lomo, T. (1995). Calcitonin gene-related
peptide:possible role in formation and maintenance of neuromuscular junctions. J.
Neurosci. 15520-528.
Schifter, S., Breum, L., Niclasen, B., Vollmer-Larsen, A., Rasmussen, H-S., and GraE-
Larsen, 0. (1995). Calcitonin gene-related peptide during exercise and training. Hor. Met.
Res. 27:473-475.
Schwane, I.A., and Armstrong, R.B. (1983). Effect of training on skeletal muscle injury
fiom downhill ninning in rats. J. Appl. Physiol. 55:969-975.
Seburn, K.L., and Gardiner, P.F. (1995). Adaptations of rat laterd gastrocnemius motor
units in response to voluntary running. J: AM. P PhysioZ- 78: 1673- 1678.
Sendtner, M., Stock., KA, and Thoenen, K. (1992). Synthesis and location of ciliary
neurotrophic factor in the rat sciatic nerve of the adult rat &ter Iesion and during
regeneration. J. CeZl BioZ. 1 1 8: 139- 148.
Smith., H.K., Plyley, M-J., Rogers, C.D., McKee, N-Et, and Theriault, E. (1996).
Expression of developmental isomyosins and morphologicd changes in rat skeletal muscle
fibers foilowing downhill exercise. A C M (abstract)
Smith, H.K. (1997). Skeletal muscle damage and repair in the adult rat hindlimb foliowing
downhill treadrnill exercise. PhD Thesis, Dept. of Community Heaith, University of
Toronto,
Smith, H.K., Plyley, M.J., Rogers, C.D., and McKee, NH- (1997). Skeletal muscle
damage in the rat hindhb foiiowing single or repeated daily bouts of downhiit exercise.
Int. J. Sports Med. 18 :94- 100.
Smith, L.L. (1991). Acute intlammmation: the underlying mechanism in delayed onset
muscle soreness? Med Sci. Sport. Ber . 23 542-55 1.
Smith, P.K., Krohn, RI., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano,
M.D., Fujimoto, E.K., Goeke, N.M., Olson, BI, and Klenk, D.C. (1985). Measurement
of protein using bicinchoninic acid. Anal. Biochem. l50:76-85.
Son, Y.J., Trachtenburg, J.T., and Thompson, W.J. (1996). Schwann cells induce and
guide sprouting and reinnervation of neuromuscular junctions. TïïVS. 19:280-285.
Son, Y.-J., and Thompson, W.J. (1995a). Nerve sprouting in muscle is induced and guided
by processes extended by s c h w a ~ ceiis. Neuron 14: 133-141-
Son, Y.-J., and Thompson, W.J. (1995b). Schwann ceii processes guide regeneration of
peripheral axons. Newon 14: 125-132.
Sonchter, S., Mair, J., Koller, A, Gebert, W., Rama, D., Calzolari, C., Artner-Dworzak,
E., and Puschendort; B. (1997). Skeletal troponh 1 as a marker of exercise-induced
muscle damage. J. Appl. Physiol. 83 : 1076-1 082.
Springer, JE., Seeburger, JL., He, J., Gabrea, A, Blankenhom, E X , and Bergman, L.E.
(1995). cDNA sequence and diffierential mRNA regulation of two foms of glial ceil line-
derived neurotrophic factor in schwann celis and rat skeletal muscle. Erp. Neural. 13 1:47-
S tauber, W.T. (1 989). Eccentric action of muscles:physiology,injury, and adaptation.
Eker. Sport. Sci. Rev. 7: 157- 185.
Stauber, W.T., Clarkson, P.M., Fritz, V.K., and Evans, W.J- (1990). Extracellular m a t h
disruption and pain d e r eccentnc muscle action. J. Appl. Physiof. 692368-874.
Stebbins, CL., Schultz, E., Smith, R.T., and Smith, E.L. (1985). Effects of chronic
exercise during aging on muscle and end-plate morphology in rats. J. Appl. PhysioI.
58:45-5 1.
Streit, W., Dumoulin, F.D., Raivich, G., and Kreutzberg, G.W. (1989). Calcitonin gene-
related peptide increases in rat facial rnotoneurons after peripherd nerve transection.
Neurosci. Lerr. 101: 143-148.
Sullivan, T.E., and Armstrong, R.B. (1978). Rat locomotory muscle fiber activity dunng
trotting and galloping. J AppI. PPhysol. 44:3 58-3 63.
Swett, J.E., Wikholm, R.P., Blanks, RH.I., Swett, A.L., and Conley, L.C. (1986).
Motoneurons of the rat sciatic nerve. merf NeuroZ. 93 :227-252.
Takami, K., Hashimoto, K., Uchida, S., Tohyama, M., and Yoshida, H. (1 98Sa). Effect of
calcitonin gene-related peptide on contraction of stnated muscle in the mouse. Neurosci.
Lett 60:227-230,
Takami, K., Kawai, Y., Shiosaka, S., Lee, Y., Girgis, S., Wyard, C.J., MacIntyre, I.,
Emson, P.C., and Tohyama, M. (1 98Sb). Immunohistochemicai evidence for the
coexistence of calcitonin gene-related peptide and choline-acetyltransferase-like
immunoreactivity in neurons of the rat hypoglossal, facial, and ambiguus nuciei. Brmn
Res. 328:386-3 89.
Takami, K., Hashimoto, K., Uchida, S., Tohyama, M., and Yoshida, H. (2986). Effect of
calcitonin gene-related peptide on the cyclic AMP level of isolated mouse diaphragrn. Jq.
J, Pharmacol. 42:345-350.
Tarabal, O., Caldero, J., and Esquerda, LE. (1996a). Intramuscular nerve sprouting
induced by CNTF is associated with increases in CGRP content in mouse motor nerve
terminais. Neurosci. Lett. 2 19:60-64,
Tarabal, O., Caldero, J., Ribera, J., Sorribas, A, Lopez, EL, Molgo, J., and Esquerda, J.E.
(1996b). Regdation of motoneuronal calcitonin gene-related peptide (CGRP) during
axonal growth and neuromuscular synaptic plasticity induced by botufinim toxin in rats.
Eur. J. Neurosci. 8329-836.
Theriault, E., Marshail, K.W., and Homonko, D.A. (1993). Maintained peptidergic
innervation of the knee joint in an animal mode1 of antigen-induced arthritis. Regul. Pep.
46:204-207.
Thenault, E., and Diamond, 1. (1988a). Nociceptive stimuli evoke localized contractions
in a skeletal muscle. J. Neurophysiol. 60:446-462.
Theriault, E., and Diamond, J. (1988b). Intrinsic organization of the rat cutaneous trunci
motor nucleus. J. Neuruphysiol. 60:463-477.
Theriault, E., and Tator, C.H. (1994). Persistence of mbrosphd projections foliowing
spinal cord injury in the rat. J. Comp. Neural. 342:249-258.
Thoenen, Et, Hughes, R.A, and Sendtner, M. (1993). Trophic support of
motoneuronç:physiologicai, pathophysiological, and therapeutic implications. Exp. Narrol.
124147-55.
Tidball, J.G. (1995). Idammatory ce11 response to acute muscle injury. Med Sci Sport.
fier. 27: 1022- 1 O3 2.
Trupp, M., Ryden, M., Jomvali, H., Funakoshi, K-, Timmusk, T., Arenas, E., and Ibanez,
C.F. (1995). Peripheral expression and biological activities of GDNF, a new neurotrophic
factor for avian and mamrnalian peripheral neurons. J. CeU Biol. 13 0: 137- 148.
Tsujimoto, T., and Kuno, M. (1988). Calcitonin gene-related peptide prevents disuse
induced sprouting of rat motor nerve terminais. J. Neurosci. 8:395 1-3 957.
Tuffery, A-R (1971). Growth and regeneration of motor endplates in normal cat hindlimb
muscles. J. And . 1 10:22 1-247.
Uchida, S., Yamamoto, H., Ido, S., Matsumoto, N., Wang, X., Yonehara, N., Imai, Y.,
Inoki, R., and Yoshida, H. (1990). Release of calcitonin gene-related peptide-like
immunoreactive substance from neuromuscular junction by nerve excitation and its action
on stnated muscle. J Neurochem. 54: 1000- 1003.
Van der KIoot, W., Benjamin, W.B., and Balezina, O.P. (1998). Calcitonin gene-related
peptide acts presynapticdy to increase quantai size and output at frog neuromuscular
junctions. J. Physiol. 507:689-695.
Vanden Noven, S., Gardiner, P-F., and Sebum, K.L. (1994). Motoneurons innervating
two regions of rat medial gastrocnemius muscle with differing contractile and
histochemicd properties- Acta Anut. 150:282-293.
Waerhaug, O., Dahl, BA, and Kardel, K. (1992). DifEerent effects of physical training on
the rnorphology of motor nerve terminds in the rat extensor digitorum longus and soleus
muscles. Anai. Embryol. 186125-128.
Warren III, G.L., Williams, J.H., Ward, C.W., Matoba, H., Ingalls, C.P., Hermann, KM.,
and Armstrong, R.B. (1996). Decreased contraction economy in mouse EDL muscle
injured by eccentric contractions. J. Appl. Physiol. 8 1:2555-2564.
Warren, G.L., Lowe, D.A., Hayes, D.A., Karwoski, C.J., Prior, B.M., and Armstrong,
R.B. (1993). Excitation failure in eccentnc contraction-induced injury of mouse soleus
muscle. J. Physiol. 468:487-499.
Warren, G.L., Hayes, DL., Lowe, D.A., Williams, J.H., and Armstrong, R.B. (1994).
Eccentric contraction-induced injury in normal and hind-lirnb suspended mouse soleus and
EDL muscles. J. Appl. Physol. 77: 142 1 - 143 0.
Wemig, A., Irintchev, A., and Weisshaupt, P. (1990). Muscle injury, cross-sectional area
and fibre type distribution in mouse soleus after intermittent whell-running. J. Physiol.
428 :639-652.
Wernig, A, Salvini, T.F., and Irintchev, k (1991a). Axonal sprouting and changes in fibre
types &er running-induced muscle darnage. J Neurocyfol. 20: 903 -9 13.
Wernig, A., Saivini, TI., Langenfeld-Oster, B., Irintchev, A., and Dorlochter, M.
(1991 b). Endplate and motor unit remodehg in vertebrate muscles. In A Wernig Ed.),
Plmciitv cf MotuneuromI Connections. @p. 85- 100). Amsterdam: Elsevier Scientific
Publications BV.
Wernig, A., and Herrera, Ak (1986). Sprouting and remodehg at the nerve-muscle
junction. Prog. Neurobiol. 27:25 1-29 1.
Wojtowicz, JM., Marin, L., and Atwood, H.L. (1994). Activity-induced changes in
synaptic release sites at the crayfkh neuromuscular junction. J. Neurosci. 14:3688-3703.
Wong, T.S., and Booth, F.W. (1990). Protein rnetabolism in rat tibialis anterior muscle
after stirnulated chronic eccentric exercise. J. Appl. Physiol. 69: 17 18- 1724.
Yamada, M., Ishikawa, T., Fujimon, A, and Goto, K. (1997). Local neurogenic
regdation of rat hindlimb circulation: role of calcitonin gene-reIated peptide in
vasodiiation after skeletal muscle contraction. Br. J- P h m . 122:703-709.
Yan, Q., Matheson, C., and Lopez, O.T. (1995). In vivo neurotrophic effects of GDNF on
neonatal and adult facial motor neurons. Nature 373 :34 1-347.
Yan, Q., and Miller, J.A. (1993). The use of trophic factors in degenerative motoneuron
diseases. m e r . Neurol. 124:60-63.
Yan, Z., Biggs, R.B., and Booth, F.W. (1993). hsulin-Like growth factor
ïmmunoreactivity increases in muscle after acute eccentric contractions. J. Appl. Physiol.
74:410-414.
Zenker, W., and Scheidegger, EP. (1991). Quantitative aspects of muscle innervation. In
A Wemig (Ed.), PlaSfiaSficiq of Motoneuronal Connections- @p. 3 7-47). Amsterdam:
Elsevier Scientific Publications. BV.
Zum, AD., Baetge, E.E., Hammang, J.P., Tan, S A , and Aebischer, P. (1994). Glial celi
he-derived neurotrophic factor (GDNF), a new neurotrophic factor for motoneurones.
NeuroReport 6:113-118.
A~pendix 1.
Experhentd Desim of Animal Assiments in Cha~ters EL IV. V. and VI.
i. Chapter III:
. - 1.1,
- .- LU-
3-D Reconsbuction of Muscle
1 rat
SOL + TA SOL +- TA G) @)
Silicon Rubber Microangiography
3 rats
SOL + TA SOL + TA 6) 0 9
ii. Cha~ter N:
Ki. Retrograde Tracing Experiments with Fluorochromes
12 rats
3 EDL
I rats (L + R)
6 rats TA (R) + SOL (L)
ii .ii Immunocytochemistry for CGRP+ve Motoneurons in Control vs. Exercise Anirnals
callId (m 48hk(rP4) 7 2 w € (,k4) 2d~Ex(rP4) 4 ~ k I x ( . ) =+TA =+TA =+TA. SOL+TA SOL +TA @+W + &+W (L+R (L+R
iii. Cha~ter V.
iiii. Co-localisation of FluoroGold and CGRP+ve TS Motoneurons
I l rats SOL
9 rats LG CR)
control 72 h Ex 2 wkEx control 72 h Ex 2 wk Ex (n=3) (n=4) (w (n=3) (n=3) ( ~ 3 )
14 rats
control 72 h Ex 2 wk Ex control 72 h Ex 2 wk Ex (n=9 (n=4) (n=4) (n4) (n=5) (n=5)
ici. Giycogen Depletion Experiments
8 rats
G.G. aBuTx/CGRP and mATPase Staining at the MG Motor Endplate
12 rats
iv. Chapter VI
iv-i. AChE (G4) Isozyme Quantincation
12 rats
control (n=4) 72 h (n-4) 2 wk (n=4)
iv-ii. GDNF and CGRP Immunoreactivity at MG Motor Endplates
16 rats MG(L+R)