Acetylcholine receptor distribution and synapse elimination at the developing neuromuscular junction of mdx mice

The pattern of innervation of the vertebrate neuromuscular junction is established during early development, when junctions go from multiple to single innervation in the phenomenon of synapse elimination, suggesting that changes at the molecular level in the postsynaptic cell lead to the removal of nerve terminals. The mdx mouse is deficient in dystrophin and associated proteins that are part of the postsynaptic cytoskeleton. We used rhodamine-alpha-bungarotoxin and anti-neurofilament IgG-FITC to stain acetylcholine receptors and nerve terminals of the sternomastoid muscle during postnatal development in mdx and control C57BL/10 mice. Using fluorescence confocal microscopy, we observed that, 7 days after birth, 86.7% of the endplates of mdx mice were monoinnervated (n = 200) compared with 41.4% in control mice (n = 200). By the end of the second postnatal week, all endplates were innervated singly (100% mdx and 94.7% controls, n = 200 per group). These results show that dystrophic fibers achieve single innervation earlier, perhaps because dystrophin or a normal cytoskeletal complex is implicated in this phenomenon.     

Colchicine-induced sprouting of the neuromuscular junction in the pigeon extensor digitorum longus muscle

Colchicine-induced motor endplate sprouting in the extensor digitorum longus muscle of the pigeon was examined. Ten days after the drug application sprouting from the endplate arborizations and nodes of Ranvier were observed. No concomitant changes in endplate surface area or in the degree of terminal branching could be demonstrated. Similarities between the sprouting patterns of the pigeon endplate and the mammalian endplate are discussed.     

On the mechanism of action of muscle fibre activity in synapse competition and elimination at the mammalian neuromuscular junction

Activity-dependent competition plays a crucial role in the refinement of synaptic connections in the peripheral and central nervous system. The reduction in number of axons innervating each neuromuscular junction during development, i.e. synapse elimination, appears to be one such competitive activity-driven event. Recently, we showed that asynchronous firing of competing presynaptic terminals is a key player in synapse elimination. Although some previous studies suggested that activity of the postsynaptic cell may be an intermediary in the disposal of redundant presynaptic inputs, the mechanism involved remains unknown. In the present study, in order to assess the role of evoked muscle activity in this process, we inhibited the generation of postsynaptic action potentials in muscle fibers in vivo , through the overexpression of inwardly rectifying Kir2.1 and Kir2.2 channels, via electroporation of the soleus muscle in the mouse hindlimb. Electrophysiological and morphological data show that overexpression of potassium channels in the endplate region of neonatal muscle fibres induces membrane hyperpolarization and an increase in conductance, inhibition of the action potential mechanism and prolonged persistence of polyneuronal innervation. These changes are not seen in muscle fibres with overexpression of a non-conducting Kir2.1 mutant. Our results are compatible with the interpretation that the block of action potential generation, even in single endplates, can inhibit synapse elimination through local signalling.     

Effect of age and muscle type on regeneration of neuromuscular synapses in mice

A silver stain was used to investigate the regeneration of nerve terminals in mouse diaphragm, superior gluteus and tensor fasciae latae (TFL) muscles following nerve crush injury at different ages (9-530 days). The development of myelinated terminal branches and the development and elimination of terminal sprouts were little affected by age or muscle type following reinnervation. However, multiple axonal innervation developed on up to 50% of the gluteus and TFL muscle fibres, and this was subsequently eliminated only in the youngest animals.     

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.     

Capsaicin delays regeneration of the neuromuscular junction of rat extensor digitorum longus muscle after ischemia

Trauma or the tourniquet used in orthopedic surgery is often associated with ischemia-reperfusion (I/R) injury with a consequent decrease of muscle power. To explore whether components of the neuromuscular junction (NMJ) are involved in this muscle dysfunction, NMJs were ultrastructurally characterized in the extensor digitorum longus muscle of rats at reperfusion times of 1, 24, 72, and 168 h after a 120-min arterial occlusion. Disorganization of the presynaptic membrane and mitochondrial injury was noted at 1 h, followed by fragmentation and partial engulfment of nerve terminals by Schwann cells at 24 and 72 h. The magnitude of degenerative changes declined at 168 h, suggesting the commencement of regeneration. The postsynaptic membrane remained intact throughout the whole period. In our previous study, deafferentation with pretreatment of the sciatic nerve with capsaicin, which reduces neurogenic inflammation and has a selective effect on nociceptive fibers, improved functional recovery of the muscle after I/R. The present results document a significantly delayed structural regeneration of the motor nerve terminals after combined capsaicin and I/R treatment. Since capsaicin treatment alone had no discernible effect on the structure of NMJs, the findings point to a possibly indirect effect of capsaicin on the motor nerves, which may predispose them to increased susceptibility unmasked only by a subsequent injury. The mismatch between the enhanced functional improvement of the muscle and delayed regeneration of the nerve after capsaicin pretreatment questions the efficient use of such deafferentation to protect the integrity of neuromuscular junctions in I/R injury.     

The effect of streptomycin on the neuromuscular junction of the frog

The neuromuscular block produced by streptomycin was studied in the frog sartorius muscle by means of intracellular recording and iontophoretic application of acetylcholine. Decreases of the end-plate potentials (e.p.p.s) and acetylcholine potentials (ACh potentials) were observed following the application of streptomycin. At a concentration of 10⁻⁴ M< the e.p.p. was36.8 ± 5.5%(mean ± S.E.) (n = 12), and the ACh potential was53.7 ± 3.8% (n= 12) of the control size. The resting membrane potential and membrane conductance of the muscle fibers were not affected by streptomycin. Streptomycin did not significantly alter the spontaneous prejunctional activity, but the amplitude of the miniature end-plate potentials (m.e.p.p.s) was decreased. After the application of higher concentrations of streptomycin, the quantum content decreased from the control value. It is suggested that the reduction of the e.p.p. amplitude produced by streptomycin is mainly due to a decrease in the sensitivity of the end-plate membrane. In addition, at higher concentrations, streptomycin reduced the amount of transmitter released from the motor nerve in response to a nerve volley.     

Synaptic efficacy at singly- and dually-innervated neuromuscular junctions in the frog, rana pipiens

Electrophysiological investigation was performed on excised, curarized cutaneous pectoris and sartorius muscles of the frog. Sixteen percent of cutaneous pectoris and 7% of sartorius end-plates received dual innervation from two different axons. The resting membrane potential and input resistance of muscle fibers at single synapses were similar to those at dual junctions. The e.p.p.s. generated by individual axons in dual synapses were, on average, smaller in amplitude than e.p.p.s recorded from singly-innervated junctions. However, the total e.p.p. amplitude (sum of the two component e.p.p.s) at dually-innervated end-plates was similar to that of end-plates innervated by a single axon. Our results at polyneuronally-innervated end-plates are consistent with a model of simple competition between motor axons for a limited synaptic space.     

Seasonal comparison of the neuromuscular junction morphology of Bufo marinus

At mammalian neuromuscular junctions (NMJs), prolonged inactivity leads to muscle denervation and atrophy. By contrast, amphibian NMJs do not show such degeneration even though they can remain in a state of drought-imposed dormancy (hibernation) for many years. We have previously reported that during the dry season, toad (Bufo marinus) NMJs display decreased sensitivity to extracellular calcium-dependent neurotransmitter release, which leads to minimal neuromuscular transmission. In the present study, we examined and compared NMJ morphology of toads obtained from the wild during the wet season (February–March) when these toads are active, to toads obtained from dry season (October–November) when toads are inactive. Iliofibularis muscles were isolated and prepared for immunostaining with anti-SV2, a monoclonal antibody that labels synaptic vesicle glycoprotein SV2. The corresponding postsynaptic acetylcholine receptors were stained using Alexa Fluro-555 conjugated α-bungarotoxin. Confocal microscopy and three-dimensional reconstructions were then used to examine the pre-and postsynaptic morphology of toads NMJs from the dry (inactive) and wet (active) seasons. Total axon branch number, the percentage of axon branches with discontinuous distributions of synaptic vesicles, and further the Pearson value of colocalization of pre and postsynaptic elements in each NMJs from both the dry and wet season were compared. While our previous studies on dry toads revealed a significant reduction in evoked neurotransmission, our present findings show that the structure of the NMJs suffered limited level of remodeling, suggesting a mechanism utilized by NMJs in dry season toads to support quick recover from their dormant state after the heavy rain in wet season.     

Barbiturates and transmitter release at the frog neuromuscular junction

The effects of the convulsant and anticonvulsant barbiturates, 5(2-cyclohexylidine ethyl)-5-ethyl barbituric acid (CHEB) an phenobarbital (PHB) were studied at the frog neuromuscular junction. Spontaneous transmitter liberation was augmented by both drugs irrespective of the external Ca2+ concentration, but CHEB was considerably more active than PHB. Miniature end plate potential amplitude was equally reduced by both drugs. The net effect of these agents on end plate potential (e.p.p.) amplitude varied depending on the external Ca2+ concentration. In Ca2+-deficient Ringer's solution both drugs increased the quantal content and the e.p.p. amplitude (CHEB more than PHB). In Ringer's solution containing a normal concentration of Ca2+ ions, both drugs depressed the evoked response and failed to alter the quantal content significantly.     

Facilitation of acetylcholine secretion at a mouse neuromuscular junction

Facilitation of transmitter secretion from motor nerve terminals following one or more conditioning stimuli was examined in mouse sternomastoid muscles. Following a single conditioning stimulus at 20 degrees C, facilitation decayed exponentially during the first 70-80 msec with a time constant of 60 msec. After 70--80 msec, a small slower component of facilitation was apparent. Initial facilitation (obtained by extrapolation back to 'zero' time) had a value of approximately 0.5. Following more than one conditioning stimulus (2-6), initial facilitation was greater but the pattern of decay was similar, the slower component becoming more obvious as the number of conditioning stimuli was increased. The slow decay phase also appeared exponential. The pattern of decay of facilitation could be well fitted by the sum of two exponentials, F1 (0)exp(--t/tau 1) + F2(0)exp(--t/tau 2). After a single stimulus at 20 degrees C, tau 1 and tau 2 had mean values of 35 and 163 msec. The main effect of increasing the number of conditioning stimuli was to increase (F1(0) and F2(0) with little change in tau 1 or tau 2. Changing temperature from 30 degrees C to 10 degrees C increased F1(0) and tau 2 but had relatively little effect on F2(0) and tau 1.     

Inhibitory action of ibotenic acid on the crayfish neuromuscular junction

The effect of ibotenic acid on the crayfish neuromuscular junction was investigated. Ibotenate reduced dose-dependently the amplitude of the excitatory junctional potentials (EJPs) elicited by repetitive stimulation of the excitatory axons. Ibotenate did not affect facilitation of successive EJPs. The decrease in the EJP amplitude caused by ibotenate was almost completely blocked by picrotoxin. A quantum analysis of extracellularly recorded EJPs demonstrated that the mean quantum content was reduced by ibotenate without remarkable change in unit size. Ibotenate increased the conductance change induced by ibotenate was observed even if the glutamate receptor was completely desensitized by the prolonged application of glutamate. From an analysis of the dose-response curve of GABA with or without ibotenate, it is suggested that ibotenate acts on the GABA receptor in the crayfish neuromuscular junction.     

Presynaptic effects of sodium bisulfite at the frog neuromuscular junction

Both spontaneous and evoked transmitter release from the frog neuromuscular junction can be modified by application of sodium bisulfite, a reagent specific for disulfide bonds. An increase in miniature endplate frequency is produced that is not dependent on external calcium, sodium, or presynaptic terminal depolarization. The increased release can be halted by application of the sulfhydryl oxidizing agent DTNB. The response of bisulfite can be prevented by prior treatment of the endplate with acetylcholine or an anticholinesterase. It is concluded that bisulfite produces its effects by acting on a protein in the presynaptic membrane that is involved in regulation of transmitter release.     

Clinical relevance of terminal Schwann cells: An overlooked component of the neuromuscular junction

The terminal Schwann cell (tSC), a type of nonmyelinating Schwann cell, is a significant yet relatively understudied component of the neuromuscular junction. In addition to reviewing the role tSCs play on formation, maintenance, and remodeling of the synapse, we review studies that implicate tSCs in neuromuscular diseases including spinal muscular atrophy, Miller–Fisher syndrome, and amyotrophic lateral sclerosis, among others. We also discuss the importance of these cells on degeneration and regeneration after nerve injury. Knowledge of tSC biology may improve our understanding of disease pathogenesis and help us identify new and innovative therapeutic strategies for the many patients who suffer from neuromuscular disorders and nerve injuries.     

Qualitative and quantitative changes in acetylcholine receptor distribution at the neuromuscular junction following free muscle transfer

The qualitative and quantitative changes in acetylcholine receptor distribution were studied in the gracilis muscle of the Wistar rat following free neurovascular transfer. Even at 30 weeks after transfer, the morphology of the neuromuscular junction failed to return to the presurgical state. The number of acetylcholine receptors at the reinnervated neuromuscular junction also remained lower than the control. The persistent weakness following free neurovascular muscle transfer may be attributed to these qualitative and quantitative changes at the neuromuscular junction.     

Origin of quantal size variation and high‐frequency miniature postsynaptic currents at the Caenorhabditis elegans neuromuscular junction

The neuromuscular junction (NMJ) of Caenorhabditis elegans has proved to be a very useful model synapse for investigating molecular mechanisms of synaptic transmission. Intriguingly, miniature postsynaptic currents (minis) at this synapse occur at an unusually high frequency (50–90 Hz in wild‐type worms) and show large variation in quantal size (from <10 pA to >200 pA). It is important to understand the cellular and molecular bases for these properties of minis in order to interpret electrophysiological data from this synapse properly. Existing data suggest that several factors may contribute to the high frequency and quantal size variation, including 1) the establishment of multiple NMJs with each body‐wall muscle cell, 2) diversity of postsynaptic receptors (two acetylcholine receptors and one GABA receptor), 3) association of one presynaptic site with several body‐wall muscle cells, 4) effects of Ca²⁺ at the presynaptic site, and 5) a possibly elevated (less negative) resting membrane potential in motoneurons. Neither the frequency nor the quantal size of minis is affected by electrical coupling of body‐wall muscle cells. Furthermore, quantal size variation is not due to synchronized multivesicular release. Analyses of the C. elegans NMJ may lead to a better understanding of the mechanisms controlling the frequency and quantal size of minis of other synapses as well. © 2010 Wiley‐Liss, Inc.     

Oscillations in evoked transmitter release at the crayfish neuromuscular junction

A time series analysis of excitatory junction potentials recorded extracellularly and focally at the crayfish neuromuscular junction indicated that, under the proper experimental conditions, periodic oscillations in evoked transmitter release occur. These low amplitude periodicities are superimposed on the otherwise random pattern of release.     

Molecular architecture of the neuromuscular junction

The neuromuscular junction (NMJ) is a complex structure that serves to efficiently communicate the electrical impulse from the motor neuron to the skeletal muscle to signal contraction. Over the last 200 years, technological advances in microscopy allowed visualization of the existence of a gap between the motor neuron and skeletal muscle that necessitated the existence of a messenger, which proved to be acetylcholine. Ultrastructural analysis identified vesicles in the presynaptic nerve terminal, which provided a beautiful structural correlate for the quantal nature of neuromuscular transmission, and the imaging of synaptic folds on the muscle surface demonstrated that specializations of the underlying protein scaffold were required. Molecular analysis in the last 20 years has confirmed the preferential expression of synaptic proteins, which is guided by a precise developmental program and maintained by signals from nerve. Although often overlooked, the Schwann cell that caps the NMJ and the basal lamina is proving to be critical in maintenance of the junction. Genetic and autoimmune disorders are known that compromise neuromuscular transmission and provide further insights into the complexities of NMJ function as well as the subtle differences that exist among NMJ that may underlie the differential susceptibility of muscle groups to neuromuscular transmission diseases. In this review we summarize the synaptic physiology, architecture, and variations in synaptic structure among muscle types. The important roles of specific signaling pathways involved in NMJ development and acetylcholine receptor (AChR) clustering are reviewed. Finally, genetic and autoimmune disorders and their effects on NMJ architecture and neuromuscular transmission are examined.     

Vitamin D and/or calcium deficient diets may differentially affect muscle fiber neuromuscular junction innervation

Introduction: There is evidence that supports a role for Vitamin D (Vit. D) in muscle. The exact mechanism by which Vit. D deficiency impairs muscle strength and function is not clear. Methods: Three‐week‐old mice were fed diets with varied combinations of Vit. D and Ca²⁺ deficiency. Behavioral testing, genomic and protein analysis, and muscle histology were performed with a focus on neuromuscular junction (NMJ) ‐related genes. Results: Vit. D and Ca²⁺ deficient mice performed more poorly on given behavioral tasks than animals with Vit. D deficiency alone. Genomic and protein analysis of the soleus and tibialis anterior muscles revealed changes in several Vit. D metabolic, NMJ‐related, and protein chaperoning and refolding genes. Conclusions: These data suggest that detrimental effects of a Vit. D deficient or a Vit. D and Ca²⁺ deficient diet may be a result of differential alterations in the structure and function of the NMJ and a lack of a sustained stress response in muscles. Muscle Nerve 54 : 1120–1132, 2016     

Endotoxin alters spontaneous transmitter release at the frog neuromuscular junction.

The direct neurotoxic effects of E. coli endotoxin (ETX) on spontaneous transmitter release were tested at the frog sartorius muscle neuromuscular junction. Spontaneous transmitter release was monitored by intracellularly recording miniature end-plate potentials (MEPPs). Junctions were continuously exposed to standard concentrations of 10 microgram/ml of 3 ETX samples, 2 of which produced a significant elevation of MEPP frequency followed by a decline of frequency to very low rates. The third ETX sample, known to have a decreased canine lethality, was without effect on MEPP frequency. No significant changes in MEPP amplitude were evident. The rate of change in MEPP frequency, but not the peak frequency, was reduced by lowering ETX concentrations. Alterations in MEPP frequency induced by ETX were prevented by removing Ca++ and antagonized by high [K+]out. The results suggest that ETX alters ion conductance channels, particularly those for Ca++, in the presynaptic terminal membrane.