1 Department of Cellular and Molecular Medicine and 2 Centre for Neuromuscular Disease, University of Ottawa, Ontario K1H-8M5, Canada
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ABSTRACT |
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Neonatal sciatic nerve crush results in a sustained reduction of the mass of both extensor digitorum longus (EDL) and soleus (SOL) muscles in the rat. Type IIB fibers are selectively lost from EDL. We have investigated the effects of ciliary neurotrophic factor (CNTF) combined with neurotrophin (NT)-3 or NT-4 on muscle mass, as well as the number, cross-sectional area, and distribution of muscle fiber types and the number of motor neurons innervating EDL and SOL 3 mo after transient axotomy 5 days after birth. Both NT treatments prevented the axotomy-induced loss of muscle mass in both EDL and SOL and of total number of muscle fibers in EDL but not in SOL. Although IIB fiber loss was not prevented, both NT treatments resulted in altered fiber type distribution. Both NT combinations also reduced the loss of EDL motor neurons. These data suggest that a differential distribution of NT receptors on either motor neurons or muscle fibers may lead to different levels of susceptibility to neonatal axotomy.
motor neurons; myosin heavy chain; fiber size
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INTRODUCTION |
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NEONATAL NERVE INJURY in the rat results in a long-term loss of motor neurons, an 80% decrease in the number of motor neurons innervating the jaw muscles (5, 31, 38, 63), and a 50-60% decrease in the number of motor neurons innervating the hindlimb muscles (45, 60, 77). In contrast, transient nerve injury in an adult rat results in the loss of <10% of the motor neuron population (9). The relative extent of reduction in the number of lumbar motor neurons after neonatal axotomy corresponds well with the extent of muscle atrophy and reduction of force generation (45, 47). In the hindlimb, muscle weight and maximum tetanic tension of tibialis anterior (TA) and extensor digitorum longus (EDL) are both reduced by ~50% 60 days after axotomy at postnatal day 5 (47). Soleus (SOL), on the other hand, showed only a 20% decrease in muscle weight and a 30% decrease in maximum tetanic tension after 2 mo of recovery (47). The extent of gastrocnemius muscle mass loss is similar to that of SOL (35), suggesting that posterior crural muscles (or the motor neurons innervating them) are less susceptible to neonatal axotomy than the anterior muscles of the hindlimb. Histochemical studies of EDL and TA have shown the disappearance of fast glycolytic fibers (presumably type IIB) after transient neonatal axotomy (44).
Developing motor neurons are susceptible to target muscle ablation (24) and can be rescued with administration of muscle-derived neurotrophins (NTs) (40). Consequently, one possible explanation for susceptibility of a specific population of motor neurons and muscle fibers after neonatal axotomy is the transient block in retrograde transport of muscle-derived NTs to motor neurons. Of these factors, NT-3 (7), NT-4 (16), and brain-derived neurotrophic factor (BDNF) (23, 29) are expressed in skeletal muscle. Previous observations have shown prolonged survival of cultured motor neurons in response to the above-mentioned NTs (3, 29, 31). These NTs have also been reported to enhance the survival of motor neurons after neonatal nerve injury in rat (38, 40, 71, 77). Ciliary neurotrophic factor (CNTF) appears to have both myotrophic and neurotrophic activity and is expressed in Schwann cells (1, 14, 28, 49, 54, 62, 64, 65). However, histological analyses on mice null for NT-3, NT-4, and CNTF have revealed only a modest loss of facial motor neurons (6, 42, 43, 61).
The effect of individual NTs on the survival of specific populations of
motor neurons (those innervating fast vs. slow muscle fibers) after
neonatal nerve injury has not been investigated. However, Sterne et al.
(68) have shown that treatment with NT-3 decreased the
loss of type IIB fibers in denervated gastrocnemius of adult rats. This
could result from enhanced survival of the motor neurons innervating
type IIB fibers or, alternatively, modification of the properties of
the remaining motor neurons. NT-3 and NT-4 exhibit distinct temporal
and spatial patterns of expression in the developing hindlimb
(16, 23, 29). Prenatal expression of NT-3 is observed
throughout the developing muscle, whereas NT-4 expression is restricted
to the epidermis at this stage (23). In the adult, NT-3 is
expressed in muscle spindles (7), whereas NT-4 has been
reported to be restricted to the slow type I fibers of SOL
(16). NT receptors TrkB, TrkC, p75, as well as CNTFR, the cognate CNTF receptor, are all expressed in motor neurons (8,
13, 23, 25, 27, 48). TrkB is the putative high-affinity receptor
for NT-4 (36), and TrkC is the receptor for NT-3
(39), whereas p75 can bind to both NT-3 and NT-4
(2). Studies on mice null for TrkB, TrkC, and CNTFR
have shown varying degrees of motor neuron loss (10, 37,
66).
Whereas TrkC expression seems to be greatest in smaller, presumably gamma, motor neurons (8), Simon et al. (67) have recently reported that TrkC transcript levels are higher in motor neurons innervating EDL than those of SOL. The differential expression of NTs and their receptors in various muscle fiber types and motor neurons may suggest differences in the interactions between these components of motor units, which could account for their susceptibility to neonatal axotomy. Here we decided to investigate whether there is preferential survival of muscle fiber types and motor neurons with administration of different NTs after neonatal nerve injury.
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MATERIALS AND METHODS |
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Neonatal Sciatic Nerve Crush
Thirteen-day-gestation Sprague-Dawley rats were obtained from Charles River Laboratories (Quebec, Canada). Animals were housed in the University of Ottawa animal care facility with 12:12-h dark-light cycle and unlimited food and water. All surgical procedures were approved by University of Ottawa Animal Care and Use Committee. Day of birth of pups was taken as postnatal day 0 (P0). Five-day old pups were anesthetized with isofluorane gas, and the right sciatic nerve was exposed at midthigh level by carefully parting surface muscles (biceps femoris and gluteus maximus). With fine forceps, the sciatic nerve was crushed for 5 s. The wound was sutured with thin prolene suture, and the pups were returned to their mother. The animals were housed in the Animal Care facility for a period between 3 and 5 mo, after which they were subjected to motor unit counting and muscle analysis.NT Application
NTs were provided by Regeneron (Tarrytown, NY). A piece of gelfoam soaked in sterile phosphate-buffered saline (PBS) containing the various NTs was placed on the site of the crush. The following doses were used: in CNTF + NT-3-treated animals, 5 µg of CNTF plus 6.25 µg of NT-3; in CNTF + NT-4-treated animals, 5 µg of CNTF plus 1.45 µg of NT-4. NTs were then injected daily under the skin of the back (28) in a volume of 0.005 ml of sterile PBS per gram of body weight for a period of 10 days. Seven pups were administered a daily injection of 0.3 µg of CNTF and 1.5 µg of NT-3 per gram of body weight. In the CNTF + NT-4 group, three pups were administered 0.3 µg of CNTF and 1.5 µg of NT-4 per gram of body weight. These doses were selected based on those reported in the literature. For example, Helgren et al. (28) used doses of CNTF in the range of 0.3-1 µg/g body wt. Funakoshi et al. (16) implanted genetically modified 3T3 cells, which secreted NT-4 at a rate of 150 ng/day, into the gastrocnemius muscle; given that the mass of this muscle is ~0.6% of total body mass, a dose of 30 µg of NT-4 into rat pups with a mean body weight of 20 g, as in our experiments, would yield values of NT-4 at the level of the muscle comparable to those obtained by Funakoshi et al. (16). Furthermore, doses higher than these have been shown, in preliminary experiments, to induce cachexia. Indeed, because of the death of a second group of three pups injected with this dose of CNTF + NT-4, the amounts of CNTF and NT-4 given an additional two pups in this group were reduced to 0.08 and 0.38 µg/body wt, respectively.Electrophysiological Counting of Motor Units in EDL and SOL
After the survival period, the animals were weighed and then anesthetized with halothane gas. A catheter was placed in the animal's tail vein for continuous anesthesia with injection of pentobarbital sodium (Somnotol; MTC Pharmaceuticals) at a dose of 0.06 mg · kg body wtAfter the motor unit experiment, TA, EDL, gastrocnemius, and SOL were
excised, weighed, and frozen in isopentane chilled in liquid nitrogen.
The muscle blocks were kept at 80°C and subsequently cryosectioned
at 10-µm thickness and placed on positively charged slides
(FisherBrand Suprefrost Plus).
Myosin Heavy Chain Immunohistochemistry
Sectioned muscles were blocked with 5% skim milk solution in PBS for 30-60 min. Primary antibodies for myosin heavy chain (MHC; see below) were placed on the slides in 1:5 dilution for duration of 4 h to overnight. Slides were washed three times for 15 min in PBS followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:100 dilution for 1-2 h). After washes in PBS, slides were placed in 1% diaminobenzidine (DAB) and 0.01% (vol/vol) 30% hydrogen peroxide (H2O2) in PBS in the dark for 5-10 min for color development and were then mounted with Permount and coverslips. MHC monoclonal primary antibodies were A4-840 for type I (Developmental Studies Hybridoma Bank at the University of Iowa), SC-71 for type IIA, and BF-F3 for type IIB (two latter types from German Collection of Microorganisms and Cell Cultures). These antibodies have previously been described (32, 58).Fiber Counting in EDL and SOL
Muscle fiber measurements were performed by using a Zeiss microscope (Axiophot) and a computer equipped with Northern Eclipse (Empix Imaging). Clearly identifiable degenerated fibers (<5 µm in diameter) in operated EDL were individually counted. All other fibers were counted in five different sample areas within the cross sections of muscles at ×200 magnification. By measuring whole muscle surface area and the mean surface area of the sample field, the estimated total fiber number was calculated as
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The cross-sectional area of 100 fibers per type per section was measured. The average cross-sectional area for each fiber type was compared between the operated and contralateral muscles and also between the untreated and NT-treated groups.
Motor Neuron Labeling
Retrograde labeling of motor neurons innervating TA with HRP was done as previously described (70). Briefly, after 2-3 wk of unilateral P5 sciatic nerve crush, TA muscles in both legs were exposed and injected with 20 µl of 20% HRP (Sigma; type VI) solution in sterile PBS. To avoid labeling motor neuron pools of the other muscles, care was taken to clean any leakage of HRP solution. After 24-36 h, the animal was perfused with 50 ml of sterile PBS, followed by 50 ml of 4% paraformaldehyde in sterile PBS. The spinal cord was then excised and incubated in 1:1 solution of OCT (Tissue Tek) and 30% sucrose for 1-2 h, cryosectioned at 40-µm thickness, and placed on gelatin-coated slides. The protocol of Mesulam (50) was followed for visualizing the motor neurons. To avoid multiple counting of labeled neurons, only those with visible nucleoli were counted.Statistical Analyses
All values were normalized against contralateral control side (except for motor unit values) and compared by using one-way ANOVA with least square difference (LSD). ![]() |
RESULTS |
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Effect of Transient Axotomy at P5
Muscle mass and force generation.
Transient denervation of the hindlimb at P5 results in long-lasting
muscle atrophy. The anterior crural muscles of the hindlimb (EDL and
TA) are affected to a greater extent than the posterior crural muscles
(gastrocnemius and SOL). At 3 mo after sciatic nerve crush at P5, the
mass of TA and EDL were each reduced by ~50%, whereas gastrocnemius
and SOL decreased in weight by 30 and 20%, respectively (Fig.
1). At this time, maximum tetanic force
decreased by 64% in EDL and 23% in SOL. When the maximum tetanic
force was normalized per gram of muscle mass (specific force), EDL
specific force was reduced by 30% relative to normal control muscle.
In contrast, SOL specific force was significantly increased (Table
1).
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Muscle fiber number, size, and type distribution. TA and EDL muscles, which had been transiently denervated at P5, exhibited a marked reduction in the total numbers of muscle fibers as detected with MHC immunohistochemisty. Fiber counts were made in EDL muscles of both the operated hindlimb and the contralateral unoperated hindlimb. The total fiber number was significantly lower in the transiently axotomized EDL (2,570 ± 159) than in the contralateral EDL (3,116 ± 206), with a mean loss of 18%. The transiently denervated EDL muscles contained a mean of 153 degenerating fibers (6 ± 1% of the total fiber number). Because these atrophic fibers did not express embryonic MHC (data not shown), we assumed that they were not regenerating fibers. Furthermore, relatively few degenerating fibers (mean = 24 ± 8) were detected in transiently denervated EDL muscles in 5 of 7 rats that were examined 8 mo after sciatic nerve crush at P5. In SOL, P5 transient denervation resulted in a 25% decrease in the total number of fibers from 2,520 ± 126 in contralateral control side to 1,884 ± 61 in the operated side. Degenerating fibers were rarely seen in the transiently denervated SOL.
In the axotomized group, unoperated EDL muscle contained a mean of 1,494 ± 88 IIB fibers, whereas this fiber type was almost completely absent from the EDL on the operated side (Fig. 2). Type I fibers did not change significantly in number (204 ± 76 in operated and 137 ± 26 in contralateral control side). There was a significant increase in the number of type IIA fibers from a control value of 714 ± 68 to 1,406 ± 217 in the transiently axotomized EDL, and because there was no difference in the number of type IIX fibers, this suggested that approximately one half of the fibers destined to express type IIB MHC were lost and, in the remaining fibers, MHC expression was changed. Transient axotomy at P5 resulted in hypertrophy of type I and type IIA fibers in EDL (from 1,069 ± 96 to 2,448 ± 694 µm2 and 1,101 ± 124 to 2,139 ± 221 µm2, respectively), presumably as a compensatory response to the loss of type IIB fibers.
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Effects of NTs Following Transient Axotomy at P5
Muscle mass and force generation. When NTs were administered individually, no reduction was observed in the extent of loss of muscle mass relative to that occurring after axotomy alone. When expressed as a percentage of unoperated contralateral muscle, the mass of axotomized EDL and SOL, respectively, were 55 ± 4%, 82 ± 5% with CNTF treatment; 46 ± 1%, 87 ± 4% with NT-3; and 55 ± 3%, 73 ± 1% with NT-4. In no case were these values significantly different from axotomized muscles without NT treatment.
Because the combined application of NTs has been shown to have a synergistic effect in culture (75, 78), we decided to administer combinations of CNTF + NT-3 or CNTF + NT-4 to determine the effect of muscle-derived NTs (NT-3 and NT-4) on muscle and motor neuron survival after neonatal axotomy. CNTF + NT-3 reduced the loss of EDL mass to 62 ± 9% of contralateral muscle mass, and CNTF + NT-4 had an even greater effect; the transiently axotomized EDL mass was 72 ± 7% of the contralateral control (Fig. 3). These values are both significantly different from the EDL muscles after P5 sciatic nerve section without administration of NTs (50 ± 11% contralateral muscle mass). In the case of SOL, the axotomy-induced loss of muscle mass (82 ± 4% of contralateral control) was completely prevented by treatment with CNTF + NT-3 (99 ± 4%) and CNTF + NT-4 (102 ± 7%). The combined administration of NTs at the doses given did not affect the mass of the contralateral control muscles, which remained ~0.05% of the body weight as in the normal rat.
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Fiber number, size, and type distribution. Because it has been demonstrated that various NTs can save motor neurons from axotomy-induced death, we decided to see whether these NTs would be able to prevent the loss of muscle fibers. In addition, the loss of type IIB fibers from TA and EDL raised the possibility that the transient axotomy at P5 resulted in the death of a specific population of motor neurons which normally innervated type IIB fibers. In the CNTF + NT-3- and CNTF + NT-4-treated groups, the total number of fibers in operated EDL was 3,258 ± 261 and 2,984 ± 250. These muscles had 20 and 11% more fibers, respectively, than the contralateral unoperated muscles. The transiently denervated EDL in the CNTF + NT-3 treated rats contained a mean of 296 degenerating fibers (9 ± 3% of the total number), whereas degenerating fibers were only found in 3 of 5 of the transiently denervated EDL muscles of CNTF + NT-4-treated rats (mean number = 13 ± 10).
In the CNTF + NT-3 group, the proportion of type I in EDL was the same as in the untreated group (twofold higher than the contralateral control side), whereas in the CNTF + NT-4 group the proportion of type I fibers was not different from contralateral control EDL (Fig. 4). In both NT-treated groups, there was an increase in proportion of type IIA compared with the untreated group (Fig. 4). Type IIB fibers were not present in EDL of either NT-treated groups (Fig. 2), and type IIX remained the same in proportion relative to the contralateral control EDLs (Fig. 4). Treatment with either of the NT combinations reduced the hypertrophic response of the type I fibers in EDL (1,524 ± 100 µm2 for CNTF + NT-3 and 1,264 ± 353 µm2 for CNTF + NT-4 ), although type IIA fibers remained enlarged (2,168 ± 151 µm2 for CNTF + NT-3 and 2,366 ± 114 µm2 for CNTF + NT-4).
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DISCUSSION |
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As previously reported (44), neonatal sciatic nerve
crush in the rat results in extensive skeletal muscle atrophy,
especially of the anterior crural muscles (EDL and TA) of the hindlimb.
The posterior crural muscles (gastrocnemius and SOL) are affected to a
significantly lesser extent. The greater loss of muscle mass in EDL and
TA is associated with the virtually complete loss of type IIB fibers,
which form the largest proportion of EDL fibers in control rats but
which are absent from SOL. In the present experiments, the total number
of fibers 3 mo after nerve crush was 18 and 25% lower in EDL and SOL,
respectively, compared with the contralateral control side. The loss of
fibers in EDL was prevented by the administration of CNTF with NT-3 or
NT-4 after neonatal axotomy. In SOL, the restoration of muscle mass by
NTs was due largely to hypertrophy of the surviving fibers (Fig.
6). These data suggest the possibility of
selective effects of NTs on specific subpopulations of developing
skeletal muscle fibers.
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Although it is conceivable that the reduced fiber number after neonatal sciatic nerve crush could reflect a failure of new fiber generation, there is no evidence of any such increase in fiber number in either EDL (20) or SOL (20, 53) after 5 days of age. In fact, the loss of muscle fibers in transiently denervated EDL (and also TA) is paralleled by the loss of a significant proportion of motor neurons normally innervating these muscles. The SOL motor neuron pool, by contrast, was not affected by the transient axotomy, and this is reflected in the smaller reduction of mass in this muscle. There have been several previous reports of loss of a significant proportion of motor neurons following neonatal axotomy (5, 31, 38, 45, 60, 63, 77). We suggest that the relatively specific susceptibility of type IIB fibers, which we have seen after neonatal nerve injury, may be correlated with the loss of a specific population of motor neurons innervating these fibers in EDL and TA. Transient axotomy after 21 days of age does not result in the extensive death of motor neurons or the disappearance of type IIB fibers (9, 46, 55). We have also noted that sciatic nerve crush in 14-day-old rats results in only a 25% loss of TA and EDL muscle mass (data not shown) and that these transiently axotomized muscles contain a nearly normal complement of type IIB fibers that do not appear to be atrophic. Therefore, there is a period, before 14 days of age, during which these motor neurons are vulnerable to transient target removal.
Neonatal motor neurons die within 6 days after neonatal nerve crush, i.e., before reinnervation (46), and can be rescued by the administration of NTs (30, 40), demonstrating their target-derived trophic dependency. Combinations of CNTF with either NT-3 or BDNF have been reported to enhance survival of motor neurons in mice with genetic mutations that result in motor neuron death (26, 51). We have shown a similar NT-dependency in the population of rat EDL motor neurons that are susceptible to transient neonatal axotomy (Table 1). It is worth emphasizing, however, that the different effect of neonatal axotomy on EDL and SOL is not due to a differential sensitivity of fast vs. slow muscle, because the gastrocnemius muscle also was relatively unaffected by the transient axotomy. One possible explanation for this could be a slower rate of maturation of the anterior vs. posterior crural muscles or the motor neurons innervating them. However, there does not appear to be any evidence to this effect, and, indeed, it has been reported that motor neurons are born and develop to maturity in a rostral (EDL and TA) to caudal (gastrocnemius and SOL) fashion during embryonic development (19). Similarly, using dopamine (L-DOPA) to induce overstimulation and apoptosis in the first 12 days after birth, Sanusi et al. (57) have shown that motor neurons innervating SOL are more susceptible to excitotoxicity than those motor neurons innervating EDL, indicative of SOL motor neurons being less mature than those of EDL.
Our data indicate that NT-3 and NT-4, together with CNTF, may induce
some new fiber generation, because the total number of fibers of normal
size in these muscles was 6-10% greater than in unoperated
control muscles. However, the fiber-type distribution of these muscles
was altered. Rescued fibers did not express type IIB MHC, at least up
to 5 mo after the transient axotomy. The number of type IIA fibers
increased, largely offsetting the loss of IIB fibers and suggesting
that fiber-type conversion had occurred. Although this may simply
reflect a response to the overload placed on the IIA fibers, it is
known that changes in the electrophysiological properties of motor
neurons, resulting in an altered firing pattern, may in turn modify
MyHC expression (52, 69). Gonzales and Collins
(17) have reported that application of BDNF (a TrkB ligand) to intact gastrocnemius muscles of the rat produced a shift of
the properties of the motor neurons, which are normally "fast",
toward those typical of SOL ("slow") motor neurons. Our findings
are consistent with a role for NT-4 (and possibly BDNF) not only in
aiding the survival of motor neurons after transient axotomy but also
in modifying the electrophysiological characteristics of EDL motor
neurons such that the fibers they innervate switch their MHC expression
toward the slow end of the continuum of MHC expression (i.e.,
IIB IIX
IIA
I). It has been reported that NT-4
expression in muscle is activity dependent (16, 73), indicative of higher expression in more active muscle fibers. Indeed,
Funakoshi et al. (16) observed higher expression of NT-4
in slow fibers (type I) of SOL, which would tend to support this
hypothesis (see Ref. 56).
We suggest that these target-derived NTs are already expressed at P5 and not only ensure survival of those motor neurons innervating these fibers but also specify them to a slow type (cf. Ref. 17). We further hypothesize that at P5, the secondary myofibers (i.e., those that will eventually express type IIB MHC) are relatively immature and do not express NTs, with the result that the motor neurons innervating these fibers are in some way more susceptible to the axotomy and, furthermore, have not been specified to either a fast or slow type. As a consequence, this population of motor neurons dies, and a proportion of the fibers that would have been innervated by them also degenerate, although others within this cohort are reinnervated by branches of the surviving slow motor neurons. Application of NT-4 at the time of axotomy results in the entire motor neuronal population receiving the NT, which in turn enhances their chances of survival and also specifies them to the slow type. As a result of this, virtually all of the muscle fibers can be reinnervated but will only express type I or IIA MHC.
During development in rodents, expression of both BDNF and NT-3 in muscle is highest during the late fetal and early postnatal stages and then progressively falls to very low levels by 3 wk of age (16, 23). By contrast, expression of NT-4 is low at birth and progressively rises to reach the adult level at about 5 wk (16, 23). Thus the effect of NT-4 which we have shown may normally be carried out in vivo by BDNF, also acting via TrkB receptors. In our experiments, the contralateral unoperated SOL in the NT-treated rats also appeared to undergo a slight (though not statistically significant) change in fiber type distribution. The unoperated SOL of the NT-4-treated group, compared with that of the untreated rats, exhibited a reduction in the ratio of type IIA to I fibers, indicative of a switch towards type I MHC expression in these muscles.
The present study suggests that the EDL muscle fibers and/or motor
neurons that die after transient nerve crush and are rescued by NTs may
be expressing TrkB and/or TrkC together with CNTFR- during the
neonatal period. Because TrkB and TrkC, along with their putative
ligands (NT-4 and NT-3, respectively), are expressed in skeletal muscle
and in motor neurons (8, 12, 18, 29, 33, 56), NT-3 and
NT-4 may have autocrine as well as paracrine effects. For example, TrkB
is localized to both the postsynaptic (18, 74) and the
presynaptic region of the neuromuscular junction (11, 77),
and postsynaptic TrkB activation stabilizes sternomastoid muscle ACh
receptors in mouse (18). In addition, ACh receptors are
disrupted in NT-4-null mice (4). Similarly, NT-3, perhaps through TrkC, increases ACh aggregates on myotubes in nerve-muscle cocultures (15), and it also increases the ACh release
from presynaptic terminals (72). Given that ACh receptor
blockade in neonates leads to motor neuron death (21, 22),
these observations raise the importance of NTs in maintaining the
integrity of neuromuscular properties that are essential for the
survival of motor neurons and muscle fibers. Furthermore, TrkB null
mice display extensive motor neuron loss in the lumbar region of the
spinal cord (37), although it is not clear whether this
loss was restricted to any specific population of motor neurons.
Heterogeneity within the motor neuronal population is suggested by the
following observations: 1) in mice lacking the receptors for
either CNTF or leukemia inhibitory factor, only a proportion of motor
neurons are lost (10, 41); 2) c-Met, the
receptor for hepatocyte growth factor/scatter factor, which also has
neurotrophic activity, is only expressed by a fraction of cultured rat
motor neurons (76); and 3) NT-3 and BDNF
transcripts are localized to different, temporally and spatially
separated subpopulations of motor neurons in the spinal cord of the
developing chick (34). Although Copray and Kernell
(8) reported that TrkB and TrkC are expressed in the motor
neurons of both EDL and SOL motor neuron pools, Simon et al.
(67) recently reported higher transcript level of TrkC in
the adult motor neuron pool of EDL than those innervating SOL. Whether
full-length TrkB, TrkC, and CNTFR-
proteins are more abundant in EDL
than in SOL motor neurons and muscle fibers remains to be determined.
In summary, neonatal axotomy in rat within the first week of life resulted in extensive lumbar motor neuron loss and muscle atrophy. Muscle fibers were lost in both EDL and SOL, but only the motor neurons innervating EDL were selectively lost. Synergistic effects of CNTF and NT-3 or NT-4 selectively rescued the motor neurons and muscle fibers that are destined to die after neonatal axotomy in EDL. The failure of neonatal axotomy to induce loss of SOL motor neurons suggests that these neurons may have already been rendered stable by exposure to their target-derived NT (possibly NT-4) by P5. The loss of muscle fibers from SOL is more difficult to explain, as is the fact that this loss is not reversed by NT treatment.
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ACKNOWLEDGEMENTS |
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NT-3, NT-4, and CNTF (axokine-1) were generously provided by Regeneron Pharmaceuticals.
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FOOTNOTES |
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This study was supported by a research grant from the Medical Research Council of Canada (now known as Canadian Institute of Health Research) to D. J. Parry.
Address for reprint requests and other correspondence: D. J. Parry, Univ. of Ottawa, Faculty of Medicine, Dept. of Cellular and Molecular Medicine, Rm. 3149, Health Sciences Bldg., Ottawa, Ontario K1H-8M5, Canada (E-mail: dparry{at}uottawa.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 22, 2002;10.1152/ajpcell.00081.2002
Received 19 February 2002; accepted in final form 14 May 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arakawa, Y,
Sendtner M,
and
Thoenen H.
Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: comparison with other neurotrophic factors and cytokines.
J Neurosci
10:
3507-3515,
1990[Abstract].
2.
Barbacid, M.
Structural and functional properties of the TRK family of neurotrophin receptors.
Ann NY Acad Sci
766:
442-458,
1995[Abstract].
3.
Becker, E,
Soler RM,
Yuste VJ,
Gine E,
Sanz-Rodriguez C,
Egea J,
Martin-Zanca D,
and
Comella JX.
Development of survival responsiveness to brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4/5, but not to nerve growth factor, in cultured motoneurons from chick embryo spinal cord.
J Neurosci
18:
7903-7911,
1998
4.
Belluardo, N,
Westerblad H,
Mudo G,
Casabona A,
Bruton J,
Caniglia G,
Pastoris O,
Grassi F,
and
Ibanez CF.
Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.
Mol Cell Neurosci
18:
56-67,
2001[ISI][Medline].
5.
Clatterbuck, RE,
Price DL,
and
Koliatsos VE.
Further characterization of the effects of brain-derived neurotrophic factor and ciliary neurotrophic factor on axotomized neonatal and adult mammalian motor neurons.
J Comp Neurol
342:
45-56,
1994[ISI][Medline].
6.
Conover, JC,
Erickson JT,
Katz DM,
Bianchi LM,
Poueymirou WT,
McClain J,
Pan L,
Helgren M,
Ip NY,
and
Boland P.
Neuronal deficits, not involving motor neurons, in mice lacking BDNF and/or NT-4.
Nature
375:
235-238,
1995[ISI][Medline].
7.
Copray, JC,
and
Brouwer N.
Selective expression of neurotrophin-3 messenger RNA in muscle spindles of the rat.
Neuroscience
63:
1125-1135,
1994[ISI][Medline].
8.
Copray, S,
and
Kernell D.
Neurotrophins and trk-receptors in adult rat spinal motoneurons: differences related to cell size but not to "slow/fast" specialization.
Neurosci Lett
289:
217-220,
2000[ISI][Medline].
9.
Crews, LL,
and
Wigston DJ.
The dependence of motoneurons on their target muscle during postnatal development of the mouse.
J Neurosci
10:
1643-1653,
1990[Abstract].
10.
DeChiara, TM,
Vejsada R,
Poueymirou WT,
Acheson A,
Suri C,
Conover JC,
Friedman B,
McClain J,
Pan L,
Stahl N,
Ip NY,
Kato A,
and
Yancopoulos GD.
Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth.
Cell
83:
313-322,
1995[ISI][Medline].
11.
DiStefano, PS,
Friedman B,
Radziejewski C,
Alexander C,
Boland P,
Schick CM,
Lindsay RM,
and
Wiegand SJ.
The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.
Neuron
8:
983-993,
1992[ISI][Medline].
12.
Escandon, E,
Soppet D,
Rosenthal A,
Mendoza-Ramirez JL,
Szonyi E,
Burton LE,
Henderson CE,
Parada LF,
and
Nikolics K.
Regulation of neurotrophin receptor expression during embryonic and postnatal development.
J Neurosci
14:
2054-2068,
1994[Abstract].
13.
Fernandes, KJ,
Kobayashi NR,
Jasmin BJ,
and
Tetzlaff W.
Acetylcholinesterase gene expression in axotomized rat facial motoneurons is differentially regulated by neurotrophins: correlation with trkB and trkC mRNA levels and isoforms.
J Neurosci
18:
9936-9947,
1998
14.
Forger, NG,
Roberts SL,
Wong V,
and
Breedlove SM.
Ciliary neurotrophic factor maintains motoneurons and their target muscles in developing rats.
J Neurosci
13:
4720-4726,
1993[Abstract].
15.
Fu, AK,
Ip FC,
Lai KO,
Tsim KW,
and
Ip NY.
Muscle-derived neurotrophin-3 increases the aggregation of acetylcholine receptors in neuron-muscle co-cultures.
Neuroreport
8:
3895-3900,
1997[ISI][Medline].
16.
Funakoshi, H,
Belluardo N,
Arenas E,
Yamamoto Y,
Casabona A,
Persson H,
and
Ibanez CF.
Muscle-derived neurotrophin-4 as an activity-dependent trophic signal for adult motor neurons.
Science
268:
1495-1499,
1995[ISI][Medline].
17.
Gonzalez, M,
and
Collins WF, III.
Modulation of motoneuron excitability by brain-derived neurotrophic factor.
J Neurophysiol
77:
502-506,
1997
18.
Gonzalez, M,
Ruggiero FP,
Chang Q,
Shi YJ,
Rich MM,
Kraner S,
and
Balice-Gordon RJ.
Disruption of Trkb-mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions.
Neuron
24:
567-583,
1999[ISI][Medline].
19.
Gould, TW,
Burek MJ,
Sosnowski JM,
Prevette D,
and
Oppenheim RW.
The spatial-temporal gradient of naturally occurring motoneuron death reflects the time of prior exit from the cell cycle and position within the lateral motor column.
Dev Biol
216:
611-621,
1999[ISI][Medline].
20.
Gramsbergen, A,
and
Ijkema-Paassen J.
Early cerebellar hemispherectomy in the rat. Effects on the maturation of two hindlimb muscles and on lumbar motoneurons.
Behav Brain Res
56:
85-91,
1993[ISI][Medline].
21.
Greensmith, L,
Ng P,
Mohaghegh P,
and
Vrbova G.
Reducing transmitter release from nerve terminals influences motoneuron survival in developing rats.
Neuroscience
97:
357-362,
2000[ISI][Medline].
22.
Greensmith, L,
and
Vrbova G.
Alterations of nerve-muscle interaction during postnatal development influence motoneuron survival in rats.
Brain Res Dev Brain Res
69:
125-131,
1992[ISI][Medline].
23.
Griesbeck, O,
Parsadanian AS,
Sendtner M,
and
Thoenen H.
Expression of neurotrophins in skeletal muscle: quantitative comparison and significance for motoneuron survival and maintenance of function.
J Neurosci Res
42:
21-33,
1995[ISI][Medline].
24.
Grieshammer, U,
Lewandoski M,
Prevette D,
Oppenheim RW,
and
Martin GR.
Muscle-specific cell ablation conditional upon Cre-mediated DNA recombination in transgenic mice leads to massive spinal and cranial motoneuron loss.
Dev Biol
197:
234-247,
1998[ISI][Medline].
25.
Haas, CA,
Hofmann HD,
and
Kirsch M.
Expression of CNTF/LIF-receptor components and activation of STAT3 signaling in axotomized facial motoneurons: evidence for a sequential postlesional function of the cytokines.
J Neurobiol
41:
559-571,
1999[ISI][Medline].
26.
Haase, G,
Kennel P,
Pettmann B,
Vigne E,
Akli S,
Revah F,
Schmalbruch H,
and
Kahn A.
Gene therapy of murine motor neuron disease using adenoviral vectors for neurotrophic factors.
Nat Med
3:
429-436,
1997[ISI][Medline].
27.
Hammarberg, H,
Piehl F,
Risling M,
and
Cullheim S.
Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat.
J Comp Neurol
426:
587-601,
2000[ISI][Medline].
28.
Helgren, ME,
Squinto SP,
Davis HL,
Parry DJ,
Boulton TG,
Heck CS,
Zhu Y,
Yancopoulos GD,
Lindsay RM,
and
DiStefano PS.
Trophic effect of ciliary neurotrophic factor on denervated skeletal muscle.
Cell
76:
493-504,
1994[ISI][Medline].
29.
Henderson, CE,
Camu W,
Mettling C,
Gouin A,
Poulsen K,
Karihaloo M,
Rullamas J,
Evans T,
McMahon SB,
Armanini MP,
Berkemeir L,
Phillips HS,
and
Rosenthal A.
Neurotrophins promote motor neuron survival and are present in embryonic limb bud.
Nature
363:
266-270,
1993[ISI][Medline].
30.
Houenou, LJ,
Li L,
Lo AC,
Yan Q,
and
Oppenheim RW.
Naturally occurring and axotomy-induced motoneuron death and its prevention by neurotrophic agents: a comparison between chick and mouse.
Prog Brain Res
102:
217-226,
1994[ISI][Medline].
31.
Hughes, RA,
Sendtner M,
and
Thoenen H.
Members of several gene families influence survival of rat motoneurons in vitro and in vivo.
J Neurosci Res
36:
663-671,
1993[ISI][Medline].
32.
Hughes, SM,
and
Blau HM.
Muscle fiber pattern is independent of cell lineage in postnatal rodent development.
Cell
68:
659-671,
1992[ISI][Medline].
33.
Ip, FC,
Cheung J,
and
Ip NY.
The expression profiles of neurotrophins and their receptors in rat and chicken tissues during development.
Neurosci Lett
301:
107-110,
2001[ISI][Medline].
34.
Kahane, N,
Shelton DL,
and
Kalcheim C.
Expression and regulation of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in distinct avian motoneuron subsets.
J Neurobiol
29:
277-292,
1996[ISI][Medline].
35.
Kashihara, Y,
Kuno M,
and
Miyata Y.
Cell death of axotomized motoneurons in neonatal rats, and its prevention by peripheral reinnervation.
J Physiol
386:
135-148,
1987[Abstract].
36.
Klein, R,
Lamballe F,
Bryant S,
and
Barbacid M.
The trkB tyrosine protein kinase is a receptor for neurotrophin-4.
Neuron
8:
947-956,
1992[ISI][Medline].
37.
Klein, R,
Smeyne RJ,
Wurst W,
Long LK,
Auerbach BA,
Joyner AL,
and
Barbacid M.
Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death.
Cell
75:
113-122,
1993[ISI][Medline].
38.
Koliatsos, VE,
Cayouette MH,
Berkemeier LR,
Clatterbuck RE,
Price DL,
and
Rosenthal A.
Neurotrophin 4/5 is a trophic factor for mammalian facial motor neurons.
Proc Natl Acad Sci USA
91:
3304-3308,
1994[Abstract].
39.
Lamballe, F,
Klein R,
and
Barbacid M.
trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3.
Cell
66:
967-979,
1991[ISI][Medline].
40.
Li, L,
Oppenheim RW,
Lei M,
and
Houenou LJ.
Neurotrophic agents prevent motoneuron death following sciatic nerve section in the neonatal mouse.
J Neurobiol
25:
759-766,
1994[ISI][Medline].
41.
Li, M,
Sendtner M,
and
Smith A.
Essential function of LIF receptor in motor neurons.
Nature
378:
724-727,
1995[ISI][Medline].
42.
Liu, X,
Ernfors P,
Wu H,
and
Jaenisch R.
Sensory but not motor neuron deficits in mice lacking NT-4 and BDNF.
Nature
375:
238-241,
1995[ISI][Medline].
43.
Liu, X,
and
Jaenisch R.
Severe peripheral sensory neuron loss and modest motor neuron reduction in mice with combined deficiency of brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4/5.
Dev Dyn
218:
94-101,
2000[ISI][Medline].
44.
Lowrie, MB,
Krishnan S,
and
Vrbova G.
Recovery of slow and fast muscles following nerve injury during early postnatal development in the rat.
J Physiol
331:
51-66,
1982[Abstract].
45.
Lowrie, MB,
Krishnan S,
and
Vrbova G.
Permanent changes in muscle and motoneurons induced by nerve injury during a critical period of development of the rat.
Brain Res
428:
91-101,
1987[Medline].
46.
Lowrie, MB,
Lavalette D,
and
Davies CE.
Time course of motoneuron death after neonatal sciatic nerve crush in the rat.
Dev Neurosci
16:
279-284,
1994[ISI][Medline].
47.
Lowrie, MB,
and
Vrbova G.
Different pattern of recovery of fast and slow muscles following nerve injury in the rat.
J Physiol
349:
397-410,
1984[Abstract].
48.
MacLennan, AJ,
Vinson EN,
Marks L,
McLaurin DL,
Pfeifer M,
and
Lee N.
Immunohistochemical localization of ciliary neurotrophic factor receptor alpha expression in the rat nervous system.
J Neurosci
16:
621-630,
1996[Abstract].
49.
Magal, E,
Burnham P,
and
Varon S.
Effects of ciliary neuronotrophic factor on rat spinal cord neurons in vitro: survival and expression of choline acetyltransferase and low-affinity nerve growth factor receptors.
Brain Res Dev Brain Res
63:
141-150,
1991[ISI][Medline].
50.
Mesulam, MM.
Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents.
J Histochem Cytochem
26:
106-117,
1987[Abstract].
51.
Mitsumoto, H,
Ikeda K,
Klinkosz B,
Cedarbaum JM,
Wong V,
and
Lindsay RM.
Arrest of motor neuron disease in wobbler mice cotreated with CNTF and BDNF.
Science
265:
1107-1110,
1994[ISI][Medline].
52.
Munson, JB,
Foehring RC,
Mendell LM,
and
Gordon T.
Fast-to-slow conversion following chronic low-frequency activation of medial gastrocnemius muscle in cats. II Motoneuron properties.
J Neurophysiol
77:
2605-2615,
1997
53.
Narusawa, M,
Fitzsimons RB,
Izumo S,
Nadal-Ginard B,
Rubinstein NA,
and
Kelly AM.
Slow myosin in developing rat skeletal muscle.
J Cell Biol
104:
447-459,
1987[Abstract].
54.
Oppenheim, RW,
Prevette D,
Yin QW,
Collins F,
and
MacDonald J.
Control of embryonic motoneuron survival in vivo by ciliary neurotrophic factor.
Science
251:
1616-1618,
1991[ISI][Medline].
55.
Parry, DJ,
and
Wilkinson RS.
The effect of reinnervation on the distribution of muscle fibre types in the tibialis anterior muscle of the mouse.
Can J Physiol Pharmacol
68:
596-602,
1990[ISI][Medline].
56.
Sakuma, K,
Watanabe K,
Sano M,
Uramoto I,
Nakano H,
Li YJ,
Kaneda S,
Sorimachi Y,
Yoshimoto K,
Yasuhara M,
and
Totsuka T.
A possible role for BDNF, NT-4 and TrkB in the spinal cord and muscle of rat subjected to mechanical overload, bupivacaine injection and axotomy.
Brain Res
907:
1-19,
2001[ISI][Medline].
57.
Sanusi, J,
Greensmith L,
Lowrie MB,
and
Vrbova G.
Motoneurones that innervate the rat soleus muscle mature later than those to the tibialis anterior and extensor digitorum longus muscles.
Dev Neurosci
20:
546-551,
1998[ISI][Medline].
58.
Schiaffino, S,
Gorza L,
Sartore S,
Saggin L,
Ausoni S,
Vianello M,
Gundersen K,
and
Lomo T.
Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.
J Muscle Res Cell Motil
10:
197-205,
1989[ISI][Medline].
59.
Schmalbruch, H.
Growth and denervation response of skeletal muscle fibers of newborn rats.
Muscle Nerve
13:
421-432,
1990[ISI][Medline].
60.
Schmalbruch, H,
and
Rosenthal A.
Neurotrophin-4/5 postpones the death of injured spinal motoneurons in newborn rats.
Brain Res
700:
254-260,
1995[ISI][Medline].
61.
Sendtner, M,
Gotz R,
Holtmann B,
Escary JL,
Masu Y,
Carroll P,
Wolf E,
Brem G,
Brulet P,
and
Thoenen H.
Cryptic physiological trophic support of motoneurons by LIF revealed by double gene targeting of CNTF and LIF.
Curr Biol
6:
686-694,
1996[ISI][Medline].
62.
Sendtner, M,
Gotz R,
Holtmann B,
and
Thoenen H.
Endogenous ciliary neurotrophic factor is a lesion factor for axotomized motoneurons in adult mice.
J Neurosci
17:
6999-7006,
1997
63.
Sendtner, M,
Holtmann B,
Kolbeck R,
Thoenen H,
and
Barde YA.
Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section.
Nature
360:
757-759,
1992[ISI][Medline].
64.
Sendtner, M,
Kreutzberg GW,
and
Thoenen H.
Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy.
Nature
345:
440-441,
1990[ISI][Medline].
65.
Sendtner, M,
Stockli KA,
and
Thoenen H.
Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration.
J Cell Biol
118:
139-148,
1992[Abstract].
66.
Silos-Santiago, I,
Fagan AM,
Garber M,
Fritzsch B,
and
Barbacid M.
Severe sensory deficits but normal CNS development in newborn mice lacking TrkB and TrkC tyrosine protein kinase receptors.
Eur J Neurosci
9:
2045-2056,
1997[ISI][Medline].
67.
Simon, M,
Mann D,
Coulton G,
and
Terenghi G.
Differential tyrosine kinase C mRNA distribution in extensor digitorum longus and soleus motoneurons in adult rats: effect of axotomy and neurotrophin-3 treatment.
Neurosci Lett
320:
9-12,
2002[ISI][Medline].
68.
Sterne, GD,
Coulton GR,
Brown RA,
Green CJ,
and
Terenghi G.
Neurotrophin-3-enhanced nerve regeneration selectively improves recovery of muscle fibers expressing myosin heavy chains 2b.
J Cell Biol
139:
709-715,
1997
69.
Thomas, PE,
and
Ranatunga KW.
Factors affecting muscle fiber transformation in cross-reinnervated muscle.
Muscle Nerve
16:
193-199,
1993[ISI][Medline].
70.
Tissenbaum, HA,
and
Parry DJ.
The effect of partial denervation of tibialis anterior (TA) muscle on the number and sizes of motorneurons in TA motornucleus of normal and dystrophic (C57BL dy2j/dy2j) mice.
Can J Physiol Pharmacol
69:
1769-1773,
1991[ISI][Medline].
71.
Vejsada, R,
Sagot Y,
and
Kato AC.
Quantitative comparison of the transient rescue effects of neurotrophic factors on axotomized motoneurons in vivo.
Eur J Neurosci
7:
108-115,
1995[ISI][Medline].
72.
Wang, T,
Xie K,
and
Lu B.
Neurotrophins promote maturation of developing neuromuscular synapses.
J Neurosci
15:
4796-4805,
1995[Abstract].
73.
Wang, XH,
and
Poo MM.
Potentiation of developing synapses by postsynaptic release of neurotrophin-4.
Neuron
19:
825-835,
1997[ISI][Medline].
74.
Wells, DG,
McKechnie BA,
Kelkar S,
and
Fallon JR.
Neurotrophins regulate agrin-induced postsynaptic differentiation.
Proc Natl Acad Sci USA
96:
1112-1117,
1999
75.
Wong, V,
Arriaga R,
Ip NY,
and
Lindsay RM.
The neurotrophins BDNF, NT-3 and NT-4/5, but not NGF, up-regulate the cholinergic phenotype of developing motor neurons.
Eur J Neurosci
5:
466-474,
1993[ISI][Medline].
76.
Yamamoto, Y,
Livet J,
Pollock RA,
Garces A,
Arce V,
deLapeyriere O,
and
Henderson CE.
Hepatocyte growth factor (HGF/SF) is a muscle-derived survival factor for a subpopulation of embryonic motoneurons.
Development
124:
2903-2913,
1997
77.
Yan, Q,
Elliott JL,
Matheson C,
Sun J,
Zhang L,
Mu X,
Rex KL,
and
Snider WD.
Influences of neurotrophins on mammalian motoneurons in vivo.
J Neurobiol
24:
1555-1577,
1993[ISI][Medline].
78.
Zurn, AD,
Winkel L,
Menoud A,
Djabali K,
and
Aebischer P.
Combined effects of GDNF, BDNF, and CNTF on motoneuron differentiation in vitro.
J Neurosci Res
44:
133-141,
1996[ISI][Medline].