NMDA receptors at the endplate of rat skeletal muscles: precise postsynaptic localization

In this study we demonstrate expression of the N-methyl-D-aspartate receptor NR1 subunit in the rat neuromuscular junction of skeletal muscles of different functional types (extensor digitorum longus, soleus, and diaphragm muscles) using fluorescence immunocytochemistry. Electron microscopic immunocytochemistry has shown that the NR1 subunit is localized solely on the sarcolemma in the depths of the postsynaptic folds. These findings suggest participation of the glutamatergic signaling system in functioning of the adult mammalian neuromuscular junction.     

Nitric oxide synthase is concentrated at the skeletal muscle endplate

NO performs a wide array of cell signaling functions. Neuronal NO synthase (nNOS) immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NDP) activity, a marker of nNOS, were concentrated at adult rat neuromuscular junctions and persisted in denervated muscle indicating the localization of the enzyme to the postsynaptic surface. The concentration of nNOS at the muscle endplate suggests NO could serve as a messenger pre- and postsynapticly.     

Congruity of acetylcholine receptor,acetylcholinesterase, and Dolichos biflorus lectin binding glycoprotein in postsynaptic-like sarcolemmal specializations in noninnervated regenerating rat muscles

Noninnervated regenerating muscles are able to form focal postsynaptic-like sarcolemmal specializations either in places of the former motor endplates ( “junctional” specializations) or elsewhere along the muscle fibers (extrajunctional specializations). The triple labeling histochemical method was introduced to analyse the congruity of focalization in such specializations of 3 synaptic components: acetylcholinesterase (AChE), acetylcholine receptor (AChR), and a specific synaptic glycoprotein which binds Dolichos biflorus lectin (DBAR). Noninnervated regenerating soleus and extensor digitorum longus (EDL) muscles of the rat were examined and compared with denervated muscles of neonatal and adult rats. All junctional sarcolemmal specializations in noninnervated regenerating muscles accumulated AChE and AChR. Localization of the 2 components was identical within the limits of resolution of the method. DBAR could not be demonstrated in junctional specializations in 17-day-old regenerating muscles. It seems that an agrin-like inducing substance in the former junctional basal lamina invariably triggers the accumulation of both AChE and AChR in the underlying sarcolemma of the regenerating muscle fiber. However, accumulation of DBAR would probably require the presence of the motor nerve. In most of the extrajunctional sarcolemmal specializations in 8-day-old regenerating soleus and EDL muscles, both AChE and AChR accumulated. However, about 10 percent of AChE accumulations lacked AChR and about 35% of AChR accumulations lacked AChE. Even greater variability was observed in 17-day-old regenerating muscles. The presence of DBAR in the extrajunctional postsynaptic-like sarcolemmal specializations could not be demonstrated. Similar extrajunctional sarcolemmal specializations were observed in denervated postnatal rat muscles. About 70% contained both AChE and AChR, and 30% contained only AChR, but none contained DBAR. In denervated mature muscles, sparse extrajunctional AChR accumulations did not contain detectable amounts of AChE. The ability to form complex postsynaptic-like sarcolemmal specializations in the absence of nerve, which is probably inherent to noninnervated immature muscle fibers, may be reduced with muscle maturation. Variable accumulation of individual components in the postsynaptic-like specializations indicates that different triggering factors may be involved in their accumulation or, at least, the mechanisms of their accumulation can function relatively independently. © 1993 Wiley-Liss, Inc.     

Juvenile and adult rat neuromuscular junctions: density,distribution, and morphology

Anatomical and physiological differences in neuromuscular junctions (NMJs) between juvenile and adult muscle may partially explain the variability in clinical results following chemodenervation with botulinum toxin or nerve repair in children and adults. We evaluated NMJ density, distribution, and morphometry in biceps brachii and gastrocnemius muscles from juvenile and adult rats. Motor endplates were stained with Karnovsky-Roots methods, and NMJ density (number/gram muscle tissue) was determined. The NMJ morphometry was quantitated with alpha-bungarotoxin labeling using confocal microscopy. Juvenile rats had a greater NMJ density in both muscles compared with adult rats. Juveniles and adult rats had a similar NMJ distribution in both muscles. In juvenile muscles NMJs occupied approximately 50% of the surface area and 70% of the length, width, circumference, and gutter depth compared with adult muscles. Our study demonstrates that although NMJs are smaller, juvenile muscles have a higher NMJ density than do adult muscles with similar distributions. If these age-dependent differences in NMJ density are obtained in humans, they may account, at least in part, for the better recovery that occurs in children than adults after nerve repair and also suggest that the dosage of botulinum toxin (units per kg) for chemodenervation may need to be increased in juveniles.     

Incomplete recovery of endplate potential amplitude while intermittently activating rat soleus neuromuscular junctions in situ

Studies dealing with neuromuscular transmission efficacy typically employ continuous patterns of activation to demonstrate decrements in endplate potential (epp) amplitude. Recent evidence from rat diaphragm muscle has shown that including periods of quiescence to the stimulation protocol allows epp amplitude to recover between series of contractions. Whether similar recovery occurs in rat hindlimb muscle is unknown. In this study, we have measured declines in epp amplitude in rat soleus muscle during trains of stimulation evoked either continuously (10 s) or intermittently (400 ms repeated every second), using an in situ approach. As in diaphragm, we found that rest periods within the intermittent trains significantly improved neuromuscular transmission efficacy. However, unlike the diaphragm, epp amplitude recovery was incomplete even by the second train in the intermittent protocols, recovery being frequency-dependent and ranging from 40 to 50%. The results suggest that the kinetics of epp amplitude rundown and recovery may be muscle-specific, and should be considered when evaluating situations in which neuromuscular transmission efficacy may be altered.     

Regulation of Motoneuronal Calcitonin Gene–related Peptide (CGRP) During Axonal Growth and Neuromuscular Synaptic Plasticity Induced by Botulinum Toxin in Rats

The aim of this study was to examine whether changes in rat motoneuronal calcitonin gene–related peptide (CGRP) can be correlated with axonal growth and plasticity of neuromuscular synapses. Nerve terminal outgrowth was induced by local paralysis with botulinum toxin. Normal adult soleus and tibialis anterior did not show detectable CGRP content at the motor endplates. Following botulinum toxin injection there was a progressive, transient and bimodal increase in CGRP in both motoneuron cell bodies which innervated poisoned muscles and their motor endplates. CGRP content was moderately increased 1 day after paralysis and, after an initial decline, reached a peak 20 days after injection. This was followed by a gradual decrease and a return to normal levels at the 200th day. CGRP changes in intoxicated endplates were less evident in the tibialis anterior than in the soleus muscle. The CGRP content in motoneurons was positively correlated with the degree of intramuscular nerve sprouting found by silver staining. In situ hybridization revealed an increase in CGRP mRNA in spinal cord motoneurons 20 days after toxin administration. We conclude that motoneurons regulate their CGRP in situations in which peripheral synapse remodelling and plasticity occur.     

Endplate dysfunction in healthy muscle following a period of disuse

The objective of this study was to test the hypothesis that muscle disuse can result in abnormal neuromuscular transmission. Six healthy volunteers (2 females, mean age = 33 years) participated in the study. Cast immobilization of one leg, for a period of 4 weeks, was used as a model of disuse; the contralateral leg was used as a control. Three testing sessions, consisting of stimulation single fiber electromyography of the soleus muscle bilaterally, were done before, immediately after, and 4 weeks after cast removal. The main outcome measure was neuromuscular jitter, measured as the mean of the consecutive differences (MCD) of single muscle fiber potentials. A mean MCD was calculated for each muscle, and the data from all subjects was pooled to calculate a sample mean MCD. The control side showed good reproducibility of results between testing sessions. The experimental side showed a significant increase of the sample mean MCD after casting when compared to: preimmobilization values (P < 0.001), and to the control side (P < 0.001). After 4 weeks of recovery, the sample mean MCD returned to preimmobilization values. This study presents evidence that muscle disuse, without any neuromuscular disease, can result in a reversible dysfunction of neuromuscular transmission. © 1996 John Wiley & Sons, Inc.     

Differential effects of NT-3 on reinnervation of the fast extensor digitorum longus (EDL) and the slow soleus muscle of rat

Previous studies of gastrocnemius muscle reinnervation showed specific normalization of the proportion and diameter of fast type 2b muscle fibres following NT-3 delivery to the proximal stump of the cut sciatic nerve. Here, we investigate if normalization was related to greater improvement of muscle reinnervation of fast (extensor digitorum longus; EDL) than slow (soleus) motor units. NT-3-impregnated (NT-3 group) or plain fibronectin (FN group) mats were inserted into a sciatic nerve gap. Neuromuscular junctions (NMJs) labelled with TRITC-alpha-bungarotoxin were colabelled with calcitonin gene-related peptide (CGRP) or 4E2 antisera and imaged using confocal microscopy. CGRP and 4E2 were used as markers for newly reinnervated and structurally mature NMJs, respectively. At 40 days postsurgery, denervated NMJs in EDL and soleus muscles of both groups presented a 50% decrease of surface area due to decreased width. At day 80 in EDL, more NMJs were reinnervated by CGRP-immunoreactive terminals in the NT-3 (7.1%) than in the FN group (4.2%); there was no difference between groups for soleus. At 120 days, 4E2-immunoreactive NMJs were more numerous in EDL of the NT-3 (40.0%) than in the FN group (7.3%), unlike in soleus (NT-3, 1. 6%; FN, 1.8%), and presented a partial size recovery. These results indicate that NT-3 preferentially improves reinnervation of fast muscles over slow muscle, although the mechanism of this improvement is still unclear.     

Coated vesicles from developing and adult rat skeletal muscles contain multiple molecular forms of acetylcholinesterase

The main purpose of this work was to determine which of the multiple isoforms of acetylcholinesterase (AChE) are associated with clathrin-coated vesicles (CVs) from developing and adult rat skeletal muscles. CV-enriched preparations were obtained by subcellular fractionation/equilibrium sedimentation and further purified by immunoadsorption to anti-clathrin IgG-coated Staphylococcus aureus cells. Analysis of individual AChE isoforms by velocity sedimentation ultracentrifugation showed that a) while both globular (G-forms) and asymmetric (A-forms) AChE were detected in all subcellular fractions evaluated, the CV-enriched fraction contained a higher proportion of A-forms (mainly the A12 species); b) most of the AChE activity contained in such a CV fraction was recovered following immunoadsorption; c) alkaline extraction conditions (pH 8.5) which depolymerize clathrin were necessary to detect a large proportion of A-forms in both the CV-enriched and immunoprecipitated preparations, while most of the G-forms (especially G1 + G2 AChE) were detected following extraction at pH 6.8; and d) comparison of AChE isoform profiles from neonate and adult muscle CV-enriched fractions showed a greater concentration of A-forms in the former. These data suggest that considerable amounts of A12 and, to a lesser extent, G4 AChE are sequestered within muscle CVs which may be destined for the plasmalemma. Our findings also indicate that the relative proportions of AChE isoenzymes in rat muscle CVs vary according to the extent of muscle development and lend support to the contention that CVs participate in the externalization of functionally important AChE isoenzymes.     

Satellite cell activity is required for hypertrophy of overloaded adult rat muscle

Hypertrophy of extensor digitorum longus muscle, overloaded by the removal of the synergist tibialis anterior (TA) muscle, in growing rats is inhibited if endogenous satellite cells are sterilized by exposure to irradiation. However, normal muscle growth is not eliminated, only diminished. To test whether irradiated, overloaded muscle can hypertrophy in the absence of normal growth-related stimuli, experiments were conducted on mature rats. TA muscle ablation caused hypertrophy of EDL muscle, characterized by a significant increase in muscle mass and the size of type IIx and type IIb fibers, and a proportional increase the number of myonuclei. When ablation was preceded by irradiation hypertrophy did not occur. The results indicate that satellite cell activation, division, and fusion is necessary for compensatory hypertrophy of fully mature muscle, and may be important to the understanding of the limits of recovery of inherited muscle myopathies treated by myogenic cell implantation. © 1994 John Wiley & Sons, Inc.     

Acetylcholinesterase histochemistry in the non-endplate region of skeletal muscles and effect of denervation

We measured acetylcholinesterase (AChE) in the non-endplate region of rat muscle, documenting its intrinsic activity within muscle fibers, as well as the extrinsic level in the capillaries and endomysium. When each muscle was considered as a whole, intrinsic AChE activity detected within the fibers was stronger in the fast-twitch extensor digitorum longus than in the slow-twitch soleus. Analysis of individual muscle fibers also showed the same tendency with a higher value in the fast-twitch type II fibers than in the slow-twitch type I fibers. On the average, 73% of the fibers showed intermediate or strong enzymatic activity in the fast-twitch muscle, whereas 56% of the slow-twitch muscle had only low activity. Sectioning or ligation of the sciatic nerve resulted in nearly complete abolition of the enzyme in the non-endplate region of the denervated muscles within 7 days, suggesting that nerve transmission regulates AChE activity not only in the endplate, as is well known, but also outside this region. Human skeletal muscles showed the same pattern of AChE activity in the non-endplate region as seen in rat muscles.     

Investigation of neuromuscular abnormalities in neurotrophin‐3‐deficient mice

Neurotrophin‐3 (NT‐3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT‐3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency‐induced reduction in NT‐3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1‐43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve‐mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1‐43 were impaired in mature NT‐3‐deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype‐dependent variation in the number of synaptic vesicles near the active zone. NT‐3^+/? mice had normal soleus muscle fiber numbers but their fibers had smaller cross‐sectional areas and were more densely‐packed than wild‐type littermates. Moreover, the muscles of adult NT‐3‐deficient animals were weaker than those of wild‐type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT‐3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.     

Nerve stump length-dependent loss of acetylcholinesterase activity in endplate regions of rat diaphragm

Alterations in acetylcholinesterase (AChE) activity were investigated in middle endplate (MEP), distal endplate (DEP) and non-endplate (NEP) regions of rat diaphragm muscle after phrenic nerve transection either intrathoracically (low, 3–5 mm nerve stump) or in the neck (high, 50–55 mm nerve stump). When the phrenic nerve was severed low, the time of initial loss of AChE activity in MEP and DEP regions was 20 h. In contrast, when the nerve was transected in the neck, the onset time of AChE activity loss was delayed in both MEP and DEP regions to 24 h. Therefore, the onset time of loss of AChE activity was dependent on the nerve stump length. The rate of loss of AChE activity was not significantly different for MEP or DEP regions after either high or low nerve transection and averaged a rate of 1.54%/h. Loss of AChE activity in the NEP region was independent of nerve stump length and enzyme activity decreased at a rate of 0.62–0.63%/h, beginning immediately after nerve transection. AChE activity in phrenic nerve was also measured and was found to contribute significantly to measurements of endplate enzyme activity in diaphragm. Data indicate a nerve length-dependent loss of AChE in MEP and DEP regions of muscle with an apparent velocity of 300 mm/day, suggesting a relationship to fast axoplasmic transport of either AChE itself or a trophic factor.     

Synaptic activity‐related classical protein kinase C isoform localization in the adult rat neuromuscular synapse

Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of α, βI, and βII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCα and PKCβI were located in the nerve terminals, whereas PKCα and PKCβII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity. J. Comp. Neurol. 518:211–228, 2010. © 2009 Wiley‐Liss, Inc.     

Mammalian target of rapamycin complex 1 is involved in differentiation of regenerating myofibers in vivo

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms.     

Innervation of dystrophic muscle after muscle stem cell therapy

Introduction: Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor‐derived muscle fibers are structurally innervated. Methods: Green fluorescent protein (GFP)–expressing MuSCs were transplanted into the tibials anterior of adult dystrophic mdx/mTR mice. Three weeks later the neuromuscular junction was labeled by immunohistochemistry. Results: The percent overlap between pre‐ and postsynaptic immunolabeling was greater in donor‐derived GFP⁺ myofibers, and fewer GFP⁺ myofibers were identified as denervated compared with control GFP^– fibers (P = 0.001 and 0.03). GFP⁺ fibers also demonstrated acetylcholine receptor fragmentation and expanded endplate area, indicators of muscle reinnervation (P = 0.008 and 0.033). Conclusion: It is unclear whether GFP⁺ fibers are a result of de novo synthesis or fusion with damaged endogenous fibers. Either way, donor‐derived fibers demonstrate clear histological innervation. Muscle Nerve 54 : 763–768, 2016     

Matrix metalloproteinases MMP-2 and MMP-9 in denervated muscle and injured nerve

Nerve crush or axotomy results in a transient or long-term denervation accompanied by remodelling in nerve, muscle and neuromuscular junctions. These changes include an increased turnover of several extracellular matrix molecules and proliferation of Schwann cells in injured nerves. Given the role of matrix degrading metalloproteinases MMP-2 and MMP-9 (gelatinases-type IV collagenases) in extracellular matrix remodelling, we investigated their regulation and activation in denervated muscles and injured nerves in mice. For this, immunofluorescence using MMP-2 and MMP-9 antibodies was carried concomitantly with gelatin zymography and quantification of gelatinase activity using [³H]-gelatin substrate. Results show that in normal mouse muscles MMP-2 and MMP-9 are localized at the neuromuscular junctions, in Schwann cells and the perineurium of the intramuscular nerves. In denervated mouse muscles, MMP-2 immunolabelling persists at the neuromuscular junctions but decreases in the nerves whereas MMP-9 immunolabelling persists at the neuromuscular junctions but is enhanced in degenerated intramuscular nerves. Denervated muscles did not show any significant change of gelatinolytic activity or expression pattern, while injured nerves exhibited a transient increase of MMP-9 and activation of MMP-2. In conclusion, this study demonstrates that MMP-2 and MMP-9 are expressed at mouse neuromuscular junctions and that their localization and expression pattern appear not to be modified by denervation. Their modulation in injured nerves suggests they are involved in axonal degeneration and regeneration.     

β‐Catenin regulates membrane potential in muscle cells by regulating the α2 subunit of Na,K‐ATPase

Muscle β‐catenin has been shown to play a role in the formation of the neuromuscular junction (NMJ). Our previous studies showed that muscle‐specific conditional knockout of β‐catenin (HSA‐β‐cat^?/?) results in early postnatal death in mice. To understand the underlying mechanisms, we investigated the electrophysiological properties of muscle cells from HSA‐β‐cat^?/? and control mice, and found that, in the absence of muscle β‐catenin, the resting membrane potential (RMP) depolarised in muscle cells from the diaphragm, gastrocnemius and extensor digitorum longus muscles. Furthermore, in a primary line of mouse myoblasts (C2C12 cells) transfected with small‐interfering RNAs targeting β‐catenin, the RMP was depolarised as well. Finally, the expression levels of the α2 subunit of sodium/potassium adenosine triphosphatase were reduced by β‐catenin knockdown in vitro or deletion in vivo. These results suggest a possible mechanism underlying the depolarised RMP in the absence of muscle β‐catenin, and provide additional evidence supporting a role for β‐catenin in the development of NMJs.