Extracorporeal shock wave treatment can selectively destroy end plates in neuromuscular junctions.

INTRODUCTION: This study assesses the effect of radial extracorporeal shock wave (rESW) exposure on neuromuscular transmission and neuromuscular junction (NMJ) morphology. METHODS: We applied 2,000 rESWs at 0.18 mJ/mm2 and a frequency of 15 Hz to the right calf of male rats, measured the compound muscle action potential (CMAP), and examined NMJ morphology using electron microscopy. Left calf muscles were used as controls. RESULTS: rESW exposure significantly reduced CMAP amplitude without delayed latency in exposed muscles compared with controls. All rESW-exposed muscles exhibited NMJs with irregular end plates. Mean interjunctional fold interval was significantly increased compared with controls. However, axon terminals and muscle fibers surrounding NMJs with irregular end plates were unchanged. DISCUSSION: This localized destruction of end plates may be caused by differences in acoustic impedance induced by the density of acetylcholine receptors. These results provide a possible mechanism for the effectiveness of rESW treatment for spasticity and dystonia. Muscle Nerve 57: 466-472, 2018.

A new mouse model for the slow-channel congenital myasthenic syndrome induced by the AChR εL221F mutation.

We have generated a new mouse model for congenital myasthenic syndromes by inserting the missense mutation L221F into the ε subunit of the acetylcholine receptor by homologous recombination. This mutation has been identified in man to cause a mild form of slow-channel congenital myasthenic syndrome with variable penetrance. In our mouse model we observe as in human patients prolonged endplate currents. The summation of endplate potentials may account for a depolarization block at increasing stimulus frequencies, moderate reduced muscle strength and tetanic fade. Calcium and intracellular vesicle accumulation as well as junctional fold loss and organelle degeneration underlying a typical endplate myopathy, were identified. Moreover, a remodeling of neuromuscular junctions occurs in a muscle-dependent pattern expressing variable phenotypic effects. Altogether, this mouse model provides new insight into the pathophysiology of congenital myasthenia and serves as a new tool for deciphering signaling pathways induced by excitotoxicity at peripheral synapses.

[Neurofilament immune fluorescence staining: a new method for observing the morphology of the motor endplate].

OBJECTIVE: To investigate the effect of neurofilament (NF) immune fluorescence staining on observing morphology of the motor endplate. METHODS: Six SPF rats were used and their bilateral soleus muscles harvested under anesthesia. Then the samples were fixed with polyphosphate formaldehyde, and made frozen sections. Finally the neurofilament immunofluorescence staining was performed. The morphology of the motor endplate was observed under fluorescence microscope and laser scanning confocal fluorescence microscope. RESULTS: The claw-shape motor endplate was seen clearly under the fluorescent microscope and laser scanning confocal fluorescence microscope, and the images observed used to reconstruct the three-dimensional structure of motor endplate. CONCLUSION: Neurofilament immune fluorescence staining is a useful method for the morphology study of the motor endplate.

Early effects of carbachol on the morphology of motor endplates of mammalian skeletal muscle fibers.

Long-term disturbance of the calcium homeostasis of motor endplates (MEPs) causes necrosis of muscle fibers. The onset of morphological changes in response to this disturbance, particularly in relation to the fiber type, is presently unknown. Omohyoid muscles of mice were incubated for 1-30 minutes in 0.1 mM carbachol, an acetylcholine agonist that causes an inward calcium current. In these muscles, the structural changes of the sarcomeres and the MEP sarcoplasm were evaluated at the light- and electron-microscopic level. Predominantly in type I fibers, carbachol incubation resulted in strong contractures of the sarcomeres underlying the MEPs. Owing to these contractures, the usual beret-like form of the MEP-associated sarcoplasm was deformed into a mushroom-like body. Consequently, the squeezed MEPs partially overlapped the adjacent muscle fiber segments. There are no signs of contractures below the MEPs if muscles were incubated in carbachol in calcium-free Tyrode's solution. Carbachol induced inward calcium current and produced fiber-type-specific contractures. This finding points to differences in the handling of calcium in MEPs. Possible mechanisms for these fiber-type-specific differences caused by carbachol-induced calcium entry are assessed.

Biological activities of a new crotamine-like peptide from Crotalus oreganus helleri on C2C12 and CHO cell lines, and ultrastructural changes on motor endplate and striated muscle.

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 µM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.

Disruption of the midkine gene (Mdk) delays degeneration and regeneration in injured peripheral nerve.

Midkine (MK) is a growth factor implicated in the development and repair of various tissues, especially neural tissues. MK acts as a reparative neurotrophic factor in damaged peripheral nerves. A postulated role of MK in the degeneration and regeneration of sciatic nerves was explored by comparing wild-type (Mdk(+/+)) mice with MK-deficient (Mdk(-/-)) mice after freezing injury. In the Mdk(-/-) mice, a regenerative delay was observed, preceded by a decelerated Wallerian degeneration (WD). The relative wet weight of the soleus muscle slowly declined, and recovery was delayed compared with that in the Mdk(+/+) mice. In the regenerating nerve, unmyelinated axons were unevenly distributed, and some axons contained myelin-like, concentrically lamellated bodies. In the endplates of soleus muscles, nerve terminals containing synaptic vesicles disappeared in both mice. In Mdk(-/-) mice, the appearance of nerve terminals was delayed in synaptic vesicles of terminal buttons after injury. The recovery of evoked electromyogram was delayed in Mdk(-/-) mice compared with Mdk(+/+) mice. Our results suggested a delay in axonal degeneration and regeneration in Mdk(-/-) mice compared with Mdk(+/+) mice, and the delayed regeneration was associated with a delayed recovery of motor function. These findings show that a lack of MK following peripheral nerve injury is a critical factor in degeneration and regeneration, and manipulation of the supply of MK may offer interesting therapeutic options for the treatment of peripheral nerve damage.

Topographical segregation of old and new acetylcholine receptors at developing ectopic endplates in adult rat muscle.

We have used radioautographic methods to examine the topography of addition and removal of acetylcholine receptors (AChRs) within receptor clusters at developing ectopic synapses in adult rat soleus muscle. After AChRs within a cluster had been pulse-labeled with 125I-alpha-bungarotoxin (125I-alpha-BuTx), the area that they occupied within the cluster shrank with time. Thus the old receptors at new endplates occupy a continually decreasing area of the growing receptor cluster. To localize newly added AChRs, we pretreated the muscles with unlabeled alpha-BuTx, thus blocking the old receptors, and then labeled newly added receptors with 125I-alpha-BuTx 1 or 2 d later. In radioautographs, AChR clusters from these muscles appeared as annuli or "doughnuts," unlike control (unpretreated) clusters, which were more nearly uniformly labeled. This visual impression was confirmed by analyzing the radial grain density distribution. Thus growth and turnover of AChR clusters at ectopic endplates takes place by the addition of receptors at the periphery of the clusters. Our data are most consistent with a model in which receptor removal occurs by endocytosis randomly throughout the cluster.

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n average of 6.4 +/- 2.1 X 10(7) DFP-binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP-binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.

Density and distribution of alpha-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle.

Acetylcholine receptors (AChR) are organized in a discrete and predictable fashion in the postsynaptic regions of vertebrate skeletal muscle. When muscle is damaged, nerves and myofibers including muscular elements of the endplate degenerate, but the connective tissue elements survive. Muscle fibers regenerate within the basal lamina of the original myofiber. Postsynaptic differentiation in regenerated mammalian skeletal muscle can occur in different ways: (a) at the site of the original endplate in the presence or absence of the nerve, or (b) at ectopic regions of the regenerated myofiber in the presence of the nerve when the original endplate is not present. The present study used (125)I-alpha- bungarotoxin ((125)I-alpha-BuTX) and EM autoradiography to examine the density and distribution of AChR in postsynaptic structures regenerated at the site of the original endplate in the absence of the nerve and at ectopic sites of the myofiber in the presence of the nerve when the original endplate was removed. In regenerated myofibers, the density of alpha-BuTX-binding sites fell within the range of densities observed in uninjured muscle whether postsynaptic differentiation occurred at the site of the original endplate in the absence of the nerve or at an originally ectopic position of the regenerated myofiber. In addition, the distribution of alpha-BuTX-binding sites within the regenerated postsynaptic regions closely resembled the distribution of apha-BuTX- binding sites in uninjured muscle. Morphometric analysis was performed on postsynaptic structures formed at the site of the original endplate in the absence of the nerve or at an ectopic position of the regenerated myofiber by interaction of the nerve and muscle. Although variation in the depth of the primary cleft occurred, there was little difference between the overall structure of regenerated postsynaptic structures and that of endplates of uninjured muscles.

Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle.

Tritiated diisopropylfluorophosphate (DFP) was used to phosphorylate acetylcholinesterase (AChase) in the motor end plate of mouse sternomastoid muscle, and its distribution within the end plate was evaluated quantitatively by electron microscope radioautography. With the use of emulsion layers whose sensitivity to tritium had been calibrated, the density of AChase in different components of the end plate was calculated. The AChase was primarily localized (85%) in the junctional fold region. The concentration of AChase there was more than 20,000 active sites per cubic micron of tissue. The resolution of the technique was not sufficient to determine whether there was some AChase in the nerve end bulb; however, if there is any there, the concentration must be less than 10% of that at the junctional fold region.

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca2+, and 10(-4) M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze-fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of less than 1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15 degrees K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5 - 10 micron, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was approximately 3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released. Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed.

P29: a novel tyrosine-phosphorylated membrane protein present in small clear vesicles of neurons and endocrine cells.

A novel membrane protein from rat brain synaptic vesicles with an apparent 29,000 Mr (p29) was characterized. Using monospecific polyclonal antibodies, the distribution of p29 was studied in a variety of tissues by light and electron microscopy and immunoblot analysis. Within the nervous system, p29 was present in virtually all nerve terminals. It was selectively associated with small synaptic vesicles and a perinuclear region corresponding to the area of the Golgi complex. P29 was not detected in any other subcellular organelles including large dense-core vesicles. The distribution of p29 in various subcellular fractions from rat brain was very similar to that of synaptophysin and synaptobrevin. The highest enrichment occurred in purified small synaptic vesicles. Outside the nervous system, p29 was found only in endocrine cell types specialized for peptide hormone secretion. In these cells, p29 had a distribution very similar to that of synaptophysin. It was associated with microvesicles of heterogeneous size and shape that are primarily concentrated in the centrosomal-Golgi complex area. Secretory granules were mostly unlabeled, but their membrane occasionally contained small labeled evaginations. Immunoisolation of subcellular organelles from undifferentiated PC12 cells with antisynaptophysin antibodies led to a concomitant enrichment of p29, synaptobrevin, and synaptophysin, further supporting a colocalization of all three proteins. P29 has an isoelectric point of approximately 5.0 and is not N-glycosylated. It is an integral membrane protein and all antibody binding sites are exposed on the cytoplasmic side of the vesicles. Two monoclonal antibodies raised against p29 cross reacted with synaptophysin, indicating the presence of related epitopes. P29, like synaptophysin, was phosphorylated on tyrosine residues by endogenous tyrosine kinase activity in intact vesicles.

Developmental regulation of membrane traffic organization during synaptogenesis in mouse diaphragm muscle.

In innervated adult skeletal muscles, the Golgi apparatus (GA) displays a set of remarkable features in comparison with embryonic myotubes. We have previously shown by immunocytochemical techniques, that in adult innervated fibers, the GA is no longer associated with all the nuclei, but appears to be concentrated mostly in the subneural domain under the nerve endings in chick (Jasmin, B. J., J. Cartaud, M. Bornens, and J.-P. Changeux. 1989. Proc. Natl. Acad. Sci. USA. 86:7218-7222) and rat (Jasmin, B. J., C. Antony, J.-P. Changeux, and J. Cartaud. 1995. Eur. J. Neurosci. 7:470-479). In addition to such compartmentalization, biochemical modifications take place that suggest a functional specialization of the subsynaptic GA. Here, we focused on the developmental regulation of the membrane traffic organization during the early steps of synaptogenesis in mouse diaphragm muscle. We investigated by immunofluorescence microscopy on cryosections, the distribution of selected subcompartments of the exocytic pathway, and also of a representative endocytic subcompartment with respect to the junctional or extrajunctional domains of developing myofibers. We show that throughout development the RER, the intermediate compartment, and the prelysosomal compartment (mannose 6-phosphate receptor-rich compartment) are homogeneously distributed along the fibers, irrespective of the subneural or extrajunctional domains. In contrast, at embryonic day E17, thus 2-3 d after the onset of innervation, most GA markers become restricted to the subneural domain. Interestingly, some Golgi markers (e.g., alpha-mannosidase II, TGN 38, present in the embryonic myotubes) are no longer detected in the innervated fiber even in the subsynaptic GA. These data show that in innervated muscle fibers, the distal part of the biosynthetic pathway, i.e., the GA, is remodeled selectively shortly after the onset of innervation. As a consequence, in the innervated fiber, the GA exists both as an evenly distributed organelle with basic functions, and as a highly differentiated subsynaptic organelle ensuring maturation and targeting of synaptic proteins. Finally, in the adult, denervation of a hemidiaphragm causes a burst of reexpression of all Golgi markers in extrasynaptic domains of the fibers, hence showing that the particular organization of the secretory pathway is placed under nerve control.

Asymmetry of the acetylcholine channel revealed by quaternary anesthetics.

Tissue-cultured rat myoballs were examined electrophysiologically with a suction pipette, which was used for voltage clamping and internal perfusion. The lidocaine derivative QX-314 caused a time- and membrane potentia-dependent block of acetylcholine-induced current only when applied from the extracellular membrane surface. The same compounds caused a use-dependent block of the sodium channel only from the intracellular membrane surface. These experiments demonstrate a fundamental asymmetry of the acetylcholine receptor-channel complex.

Schwann cell processes guide regeneration of peripheral axons.

Terminal Schwann cells overlying the neuromuscular junction sprout elaborate processes upon muscle denervation. We show here that motor axons use these processes as guides/substrates during regeneration; in so doing, they escape the confines of endplates and grow between endplates to generate polyneuronal innervation. We also show that Schwann cells in the nerve provide similar guidance. Axons extend from the cut end of a nerve in association with Schwann cell processes and appear to navigate along them. The processes extend from axotomized nerves at the same rate and in the same manner as they do from axon-containing nerves. The rate of process extension limits the rate at which axons regenerate. Thus, Schwann cell processes lead and guide peripheral regeneration.

Nerve sprouting in muscle is induced and guided by processes extended by Schwann cells.

Partial denervation or paralysis with botulinum toxin, manipulations that induce sprouting of nerve terminals in muscle, also induced terminal Schwann cells to extend processes. These processes were associated with every nerve sprout and in some cases were longer than the sprouts that appeared to be growing along them. Following partial denervation, more than 70% of the nerve sprouts that grew to innervate nearby denervated endplates were associated with Schwann cell processes that had extended from the denervated endplates, i.e., in the direction opposite to nerve growth. Implantation of Schwann cells into an innervated muscle induced sprouting upon contact of an axon or nerve terminal by Schwann cell processes. These observations show that Schwann cells induce and guide axonal sprouting in muscle.

Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle.

In adult vertebrate skeletal muscle acetylcholine receptors are localized to the neuromuscular junction. Upon denervation, this distribution changes, with new receptors appearing in extrajunctional regions of the muscle fiber. The location of acetylcholine receptors in innervated or denervated muscle may result, in part, from the distribution of their RNAs. This was tested by assaying for receptor RNAs in junctional and extrajunctional regions of innervated and denervated rat soleus muscle using in situ hybridization and RNAase protection assays. These experiments showed alpha, beta, and delta subunit RNAs concentrated beneath the endplates of innervated muscle fibers. Following denervation, there was an unequal distribution of receptor RNAs along the muscle fiber, with highest levels occurring in extrajunctional regions near the endplate. These data are consistent with a nonuniform pattern of gene expression in adult skeletal muscle fibers.

Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor epsilon subunit.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome caused by heteroallelic mutations of the acetylcholine receptor (AChR) epsilon subunit gene. The mutations are an in-frame duplication of six residues in the long cytoplasmic loop (epsilon1254ins18) and a cysteine-loop null mutation (epsilonC128S). The epsilon1254 ins18 mutation causes mode switching in the kinetics of receptor activation in which three modes activate slowly and inactivate rapidly. The epsilon1245ins18-AChR at the endplate shows abnormally brief activation episodes during steady state agonist application and appears electrically silent during the synaptic response to acetylcholine. The phenotypic consequences are endplate AChR deficiency, simplification of the postsynaptic region, and compensatory expression of fetal AChR that restores electrical activity at the endplate and rescues the phenotype.

Functional compensative mechanism of upper limb with root avulsion of C(5)-C(6) of brachial plexus after ipsilateral C(7) transfer.

OBJECTIVE: To investigate the compensative mechanism of no further impairment of the upper limb after ipsilateral C(7) transfer for treatment of root avulsion of C(5)-C(6) of the brachial plexus. METHODS: Sixty Sprague Dawley (SD) rats were randomly divided into a C7-transection group and a control group, 30 rats each. In the C(7)-transection group, the left forelimbs of the animals underwent transection of ipsilateral C(7) nerve root while C(5) and C(6) nerve roots were avulsed. In the control group, the left forelimbs only underwent C(5) and C(6) root avulsion. The representative muscles of C(7) (innervated mainly by C(7)) including latissimus dorsi, triceps, extensor carpi radialis brevis and extensor digitorum communis were evaluated with neurophysiological investigation, muscular histology and motor end plate histomorphometry 3, 6 and 12 weeks after operation. The right forelimbs of all rats were taken as the control sides. RESULTS: Three weeks after operation, the recovery rates of amplitudes of compound muscle action potential (CMAP) and CMAP latency, muscular wet weight and cross-sectional area of muscle fibers, and area of postsynaptic membranes of those four representative muscles in the C(7)-transection group were significantly lower than those of the control group (P less than 0.05 or P less than 0.01). Six weeks postoperatively, the recovery rates of CMAP amplitude and latency of the triceps showed no significant difference between the C(7)-transection group and the control group (P larger than 0.05). For the extensor carpi radialis brevis and the extensor digitorum communis, the recovery rates of the cross-sectional area of muscle fibers, the amplitude and latency of CMAP and the area of postsynaptic membranes showed no significant difference between the two groups (P larger than 0.05), while the rest parameters were still significantly different between the two group (P less than 0.05 or P less than 0.01). As far as the ultramicrostructure was concerned in the C(7)-transection group, more motor end plates of four representative muscles were observed and their ultramicrostructure also had a tendency to mature as compared with those of 3 weeks postoperatively. Twelve weeks after operation, all parameters of the C(7)-transection group were not significantly different from those of the control group (P >0.05). In the C7-transection group, the motor end plates were densely distributed and their ultramicrostructure in four representative muscles appeared to be mature as compared with those of the control group. CONCLUSIONS: After ipsilateral C(7) transfer for treatment of root avulsion of C(5)-C(6) of the brachial plexus, the nerve fibers of the lower trunk can compensatively innervate fibers of C(7)-representative muscles by means of motor end plate regeneration, so there is no further impairment on the injured upper limb.

Triple knock-out of CNTF, LIF, and CT-1 defines cooperative and distinct roles of these neurotrophic factors for motoneuron maintenance and function.

Members of the ciliary neurotrophic factor (CNTF)-leukemia inhibitory factor (LIF) gene family play an essential role for survival of developing and postnatal motoneurons. When subunits of the shared receptor complex are inactivated by homologous recombination, the mice die at approximately birth and exhibit reduced numbers of motoneurons in the spinal cord and brainstem nuclei. However, mice in which cntf, lif, or cardiotrophin-1 (ct-1) are inactivated can survive and show less motoneuron cell loss. This suggests cooperative and redundant roles of these ligands. However, their cooperative functions are not well understood. We generated cntf/lif/ct-1 triple-knock-out and combinations of double-knock-out mice to study the individual and combined roles of CNTF, LIF and CT-1 on postnatal motoneuron survival and function. Triple-knock-out mice exhibit increased motoneuron cell loss in the lumbar spinal cord that correlates with muscle weakness during early postnatal development. LIF deficiency leads to pronounced loss of distal axons and motor endplate alterations, whereas CNTF-and/or CT-1-deficient mice do not show significant changes in morphology of these structures. In cntf/lif/ct-1 triple-knock-out mice, various degrees of muscle fiber type grouping are found, indicating that denervation and reinnervation had occurred. We conclude from these findings that CNTF, LIF, and CT-1 have distinct functions for motoneuron survival and function and that LIF plays a more important role for postnatal maintenance of distal axons and motor endplates than CNTF or CT-1.