Degeneration of motor endplates (MEPs) in denervated muscle is thought to be a key factor limiting functional regeneration after peripheral nerve injury (PNI) in humans. However, there is currently no paradigm to determine MEP status in denervated human muscle to estimate likelihood of reinnervation success. Here, we present a quantitative analysis of MEP status in biopsies of denervated muscles taken during nerve repair surgery and ensuing functional recovery.

The lack of physiologically relevant and predictive cell-based assays is one of the major obstacles for testing and developing botulinum neurotoxins (BoNTs) therapeutics. Human-induced pluripotent stem cells (hiPSCs)-derivatives now offer the opportunity to improve the relevance of cellular models and thus the translational value of preclinical data.

Motor endplates are composed of a motor neuron terminal and muscle fiber and are distributed in skeletal muscle, causing muscle contraction. However, traditional motor endplate staining methods are limited to the observation of partial skeletal muscle. The procedure was time-consuming due to strict incubation conditions, and usually provided unsatisfactory results. We explored a novel method to label motor endplate rapidly by in vivo injection of fluorescent α-bungarotoxin.

The neuromuscular junction (NMJ) is a prototypic chemical synapse between the spinal motor neuron and the motor endplate. Gene expression profiles of the motor endplate are not fully elucidated. Collagen Q (ColQ) is a collagenic tail subunit of asymmetric forms of acetylcholinesterase and is driven by two distinct promoters. pColQ1 is active throughout the slow-twitch muscle, whereas pColQ1a is active at the motor endplate of fast-twitch muscle. We made a transgenic mouse line that expresses nuclear localization signal (NLS)-attached Cre recombinase under the control of pColQ1a (pColQ1a-Cre mouse). RiboTag mouse expresses an HA-tagged ribosomal subunit, RPL22, in cells expressing Cre recombinase. We generated pColQ1a-Cre:RiboTag mouse, and confirmed that HA-tagged RPL22 was enriched at the NMJ of tibialis anterior (TA) muscle. Next, we confirmed that Chrne and Musk that are specifically expressed at the NMJ were indeed enriched in HA-immunoprecipitated (IP) RNA, whereas Sox10 and S100b, markers for Schwann cells, and Icam1, a marker for vascular endothelial cells, and Pax3, a marker for muscle satellite cells, were scarcely detected. Gene set enrichment analysis (GSEA) of RNA-seq data showed that "phosphatidylinositol signaling system" and "extracellular matrix receptor interaction" were enriched at the motor endplate. Subsequent analysis revealed that genes encoding diacylglycerol kinases, phosphatidylinositol kinases, phospholipases, integrins, and laminins were enriched at the motor endplate. We first characterized the gene expression profile under translation at the motor endplate of TA muscle using the RiboTag technique. We expect that our gene expression profiling will help elucidate molecular mechanisms of the development, maintenance, and pathology of the NMJ.

As mammals age, their neuromuscular junctions (NMJs) gradually change their form, acquiring an increasingly fragmented appearance consisting of numerous isolated regions of synaptic differentiation. It has been suggested that this remodelling is associated with impairment of neuromuscular transmission, and that this contributes to age-related muscle weakness in mammals, including humans. The underlying hypothesis, that increasing NMJ fragmentation is associated with impaired transmission, has never been directly tested. Here, by comparing the structure and function of individual NMJs, we show that neuromuscular transmission at the most highly fragmented NMJs in the diaphragms of old (26-28 months) mice is, if anything, stronger than in middle-aged (12-14 months) mice. We suggest that NMJ fragmentation per se is not a reliable indicator of impaired neuromuscular transmission.

Motor endplate reinnervation is critical for restoring motor function of the denervated muscle. We developed a novel surgical technique called nerve-muscle-endplate band grafting (NMEG) for muscle reinnervation.

Spinal cord injury (SCI) results in muscle atrophy and a shift of slow oxidative to fast glycolytic fibers. Electrical stimulation (ES) at least partially restores muscle mass and fiber type distribution. The objective of this study was to was to characterize the early molecular adaptations that occur in rat soleus muscle after initiating isometric resistance exercise by ES for one hour per day for 1, 3 or 7 days when ES was begun 16 weeks after SCI. Additionally, changes in mRNA levels after ES were compared with those induced in soleus at the same time points after gastrocnemius tenotomy (GA).

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.

1. Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve-diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+-aequorin luminescence. 2. ACh (0.1-3 mM, 20 microl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration-dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 mM) of applied ACh. 3. A pulse of nicotinic agonists, (-)-nicotine (10 mM) and 1,1-dimethyl-4-phenyl-piperazinium (10 mM), but not a muscarinic agonist pilocarpine (10 mM), also elicited a biphasic Ca2+ signal. 4. Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 microg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca2+ component caused by ACh application was observed. 5. These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca2+ signal reflecting Ca2+ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.

In the inherited childhood neuromuscular disease spinal muscular atrophy (SMA), lower motor neuron death and severe muscle weakness result from the reduction of the ubiquitously expressed protein survival of motor neuron (SMN). Although SMA mice recapitulate many features of the human disease, it has remained unclear if their short lifespan and motor weakness are primarily due to cell-autonomous defects in motor neurons. Using Hb9(Cre) as a driver, we selectively raised SMN expression in motor neurons in conditional SMAΔ7 mice. Unlike a previous study that used choline acetyltransferase (ChAT(Cre+) ) as a driver on the same mice, and another report that used Hb9(Cre) as a driver on a different line of conditional SMA mice, we found no improvement in survival, weight, motor behavior and presynaptic neurofilament accumulation. However, like in ChAT(Cre+) mice, we detected rescue of endplate size and mitigation of neuromuscular junction (NMJ) denervation status. The rescue of endplate size occurred in the absence of an increase in myofiber size, suggesting endplate size is determined by the motor neuron in these animals. Real time-PCR showed that the expression of spinal cord SMN transcript was sharply reduced in Hb9(Cre+) SMA mice relative to ChAT(Cre+) SMA mice. This suggests that our lack of overall phenotypic improvement is most likely due to an unexpectedly poor recombination efficiency driven by Hb9(Cre) . Nonetheless, the low levels of SMN were sufficient to rescue two NMJ structural parameters indicating that these motor neuron cell autonomous phenotypes are very sensitive to changes in motoneuronal SMN levels. Our results directly suggest that even those therapeutic interventions with very modest effects in raising SMN in motor neurons may provide mitigation of neuromuscular phenotypes in SMA patients.

Homocysteine (HCY) is a pro-inflammatory sulphur-containing redox active endogenous amino acid, which concentration increases in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). A widely held view suggests that HCY could contribute to neurodegeneration via promotion of oxidative stress. However, the action of HCY on motor nerve terminals has not been investigated so far. We previously reported that oxidative stress inhibited synaptic transmission at the neuromuscular junction, targeting primarily the motor nerve terminals. In the current study, we investigated the effect of HCY on oxidative stress-induced impairment of transmitter release at the mouse diaphragm muscle. The mild oxidant H2O2 decreased the intensity of spontaneous quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl D-aspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS.

The distal end of the spinal cord and neuromuscular junction may develop secondary degeneration and damage following spinal cord injury because of the loss of neural connections. In this study, a rat model of spinal cord injury, established using a modified Allen's method, was injected with basic fibroblast growth factor solution via subarachnoid catheter. After injection, rats with spinal cord injury displayed higher scores on the Basso, Beattie and Bresnahan locomotor scale. Motor function was also well recovered and hematoxylin-eosin staining showed that spinal glial scar hyperplasia was not apparent. Additionally, anterior tibial muscle fibers slowly, but progressively, atrophied. nohistochemical staining showed that the absorbance values of calcitonin gene related peptide and acetylcholinesterase in anterior tibial muscle and spinal cord were similar, and injection of basic broblast growth factor increased this absorbance. Results showed that after spinal cord injury, the distal motor neurons and motor endplate degenerated. Changes in calcitonin gene related peptide and acetylcholinesterase in the spinal cord anterior horn motor neurons and motor endplate then occurred that were consistent with this regeneration. Our findings indicate that basic fibroblast growth factor can protect the endplate through attenuating the decreased expression of calcitonin gene related peptide and acetylcholinesterase in anterior horn motor neurons of the injured spinal cord.

1. The neuronal nicotinic receptor antagonists hexamethonium and methyllycaconitine (MLA) have been used to study the putative prejunctional nicotinic ACh receptors (AChRs) mediating a negative-feedback control of ACh release from motor nerve terminals in voltage-clamped rat phrenic nerve/ hemidiaphragm preparations. 2. Hexamethonium (200 microM), but not MLA (0.4-2.0 microM), decreased the time constant of decay of both endplate currents (e.p.cs) and miniature endplate currents (m.e.p.cs), indicating endplate ion channel block with hexamethonium. However, driving function analysis and reconvolution of e.p.cs and m.e.p.cs indicated that this ion channel block did not compromise the analysis of e.p.c. quantal content. 3. At low frequencies of stimulation (0.5-2 Hz), hexamethonium (200 microM) and MLA (2.0 microM) increased e.p.c. quantal content by 30-40%. At high frequencies (50-150 Hz) neither compound affected e.p.c. quantal content. All effects on quantal content were paralleled by changes in the size of the pool of quanta available for release. 4. The low frequency augmentation of e.p.c. quantal content by hexamethonium was absent when extracellular [Ca2+] was lowered from 2.0 to 0.5 mM. 5. At the concentrations studied, MLA and hexamethonium produced a small (10-20%) decrease in the peak amplitude of m.e.p.cs. 6. Neither apamin (100 nM) nor charybdotoxin (80 nM) had effects on spontaneous or nerve evoked current amplitudes at any frequency of stimulation. Thus the ability of nicotinic antagonists to augment e.p.c. quantal content is not due to inhibition of Ca(2+)-activated K(+)-channels. 7. We suggest that hexamethonium and MLA increase evoked ACh release by blocking prejunctional nicotinic AChRs. These receptors exert a negative feedback control over evoked ACh release and are probably of the alpha-bungarotoxin-insensitive neuronal type.

The mechanism of action of tonically applied choline, the agonist of α7 nicotinic acetylcholine receptors (nAChRs), to the spontaneous and evoked release of a neurotransmitter in mouse motor synapses in diaphragm neuromuscular preparations using intracellular microelectrode recordings of miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) was studied. Exogenous choline was shown to exhibit a presynaptic inhibitory effect on the amplitude and quantal content of EPPs for the activity of neuromuscular junction evoked by single and rhythmic stimuli. This effect was inhibited either by antagonists of α7-nAChRs, such as methyllycaconitine and α-cobratoxin, or by blocking SK-type calcium-activated potassium (KCa) channels with apamin or blocking intraterminal ryanodine receptors with ryanodine. A hypothesis was put forward that choline in mouse motoneuron nerve terminals can activate presynaptic α7-nAChRs, followed by the release of the stored calcium through ryanodine receptors and activation of SK-type KCa channels, resulting in sustained decay of the quantal content of the evoked neurotransmitter release.

Recovery of motor function after peripheral nerve injury requires treatment of the neuromuscular junction (NMJ), as well as the injured nerve and skeletal muscle. The purpose of this study was to examine the effects of ultrasound (US) stimulation on NMJ degeneration after denervation using a rat model of peroneal nerve transection. Twelve-week-old male Wistar rats were randomly assigned to 3 groups: US stimulation, sham stimulation, and intact. US or sham stimulation was performed on the left tibialis anterior (TA) muscle starting the day after peroneal nerve transection for 5 minutes daily under anesthesia. Four weeks later, the number and morphology of the motor endplates were analyzed to assess NMJ in the TA muscle. The endplates were classified as normal, partially fragmented, or fully fragmented for morphometric analysis. In addition, the number of terminal Schwann cells (tSCs) per endplate and percentage of endplates with tSCs (tSC retention percentage) were calculated to evaluate the effect of tSCs on NMJs. Our results showed that endplates degenerated 4 weeks after transection, with a decrease in the normal type and an increase in the fully fragmented type in both the US and sham groups compared to the intact group. Furthermore, the US group showed significant suppression of the normal type decrease and a fully fragmented type increase compared to the sham group. These results suggest that US stimulation inhibits endplate degeneration in denervated TA muscles. In contrast, the number of endplates and tSC and tSC retention percentages were not significantly different between the US and sham groups. Further investigations are required to determine the molecular mechanisms by which US stimulation suppresses degeneration.

Spinal muscular atrophy (SMA) is a childhood motor neuron disease caused by anomalies in the SMN1 gene. Although therapeutics have been approved for the treatment of SMA, there is a therapeutic time window, after which efficacy is reduced. Hallmarks of motor unit pathology in SMA include loss of motor-neurons and neuromuscular junction (NMJs). Following an increase in Smn levels, it is unclear how much damage can be repaired and the degree to which normal connections are re-established. Here, we perform a detailed analysis of motor unit pathology before and after restoration of Smn levels. Using a Smn-inducible mouse model of SMA, we show that genetic restoration of Smn results in a dramatic reduction in NMJ pathology, with restoration of innervation patterns, preservation of axon and endplate number and normalized expression of P53-associated transcripts. Notably, presynaptic swelling and elevated Pmaip levels remained. We analysed the effect of either early or delayed treated of an antisense oligonucleotide (ASO) targeting SMN2 on a range of differentially vulnerable muscles. Following ASO administration, the majority of endplates appeared fully occupied. However, there was an underlying loss of axons and endplates, which was more prevalent following a delay in treatment. There was an increase in average motor unit size following both early and delayed treatment. Together this work demonstrates the remarkably regenerative capacity of the motor neuron following Smn restoration, but highlights that recovery is incomplete. This work suggests that there is an opportunity to enhance neuromuscular junction recovery following administration of Smn-enhancing therapeutics.

In several animal models of motor neuron disease, degeneration begins in the periphery. Clarifying the possible role of Schwann cells remains a priority. We recently showed that terminal Schwann cells (TSCs) exhibit abnormalities in postnatal mice that express mutations of the SOD1 enzyme found in inherited human motor neuron disease. TSC abnormalities appeared before disease-related denervation commenced and the extent of TSC abnormality at P30 correlated with the extent of subsequent denervation. Denervated neuromuscular junctions (NMJs) were also observed that lacked any labeling for TSCs. This suggested that SOD1 TSCs may respond differently than wildtype TSCs to denervation which remain at denervated NMJs for several months. In the present study, the response of SOD1 TSCs to experimental denervation was examined. At P30 and P60, SC-specific S100 labeling was quickly lost from SOD1 NMJs and from preterminal regions. Evidence indicates that this loss eventually becomes complete at most SOD1 NMJs before reinnervation occurs. The loss of labeling was not specific for S100 and did not depend on loss of activity. Although some post-denervation labeling loss occurred at wildtype NMJs, this loss was never complete. Soon after denervation, large cells appeared near SOD1 NMJ bands which colabeled for SC markers as well as for activated caspase-3 suggesting that distal SOD1 SCs may experience cell death following denervation. Denervated SOD1 NMJs viewed 7 days after denervation with the electron microscope confirmed the absence of TSCs overlying endplates. These observations demonstrate that SOD1 TSCs and distal SCs respond abnormally to denervation. This behavior can be expected to hinder reinnervation and raises further questions concerning the ability of SOD1 TSCs to support normal functioning of motor terminals.

This study examined whether activity is a contributing factor to motor terminal degeneration in mice that overexpress the G93A mutation of the SOD1 enzyme found in humans with inherited motor neuron disease. Previously, we showed that overload of muscles accomplished by synergist denervation accelerated motor terminal degeneration in dogs with hereditary canine spinal muscular atrophy (HCSMA). In the present study, we found that SOD1 plantaris muscles overloaded for 2months showed no differences of neuromuscular junction innervation status when compared with normally loaded, contralateral plantaris muscles. Complete elimination of motor terminal activity using blockade of sciatic nerve conduction with tetrodotoxin cuffs for 1month also produced no change of plantaris innervation status. To assess possible effects of activity on motor terminal function, we examined the synaptic properties of SOD1 soleus neuromuscular junctions at a time when significant denervation of close synergists had occurred as a result of natural disease progression. When examined in glucose media, SOD1 soleus synaptic properties were similar to wildtype. When glycolysis was inhibited and ATP production limited to mitochondria, however, blocking of evoked synaptic transmission occurred and a large increase in the frequency of spontaneous mEPCs was observed. Similar effects were observed at neuromuscular junctions in muscle from dogs with inherited motor neuron disease (HCSMA), although significant defects of synaptic transmission exist at these neuromuscular junctions when examined in glucose media, as reported previously. These results suggest that glycolysis compensates for mitochondrial dysfunction at motor terminals of SOD1 mice and HCSMA dogs. This compensatory mechanism may help to support resting and activity-related metabolism in the presence of dysfunctional mitochondria and prolong the survival of SOD1 motor terminals.

The aim of this study was to compare the acute effects of thrombin and brain-derived neurotrophic factor (BDNF) on spontaneous miniature endplate potentials (MEPPs) and multiquantal evoked endplate potentials (EPPs) in mouse neuromuscular junctions (NMJs) of m. diaphragma and m. EDL. Intracellular microelectrode recordings of MEPPs and EPPs were used to evaluate the changes in acetylcholine (ACh) release in mature and newly-formed mouse NMJs. Thrombin (1 nM) increased the amplitude of MEPPs and EPPs by 25-30% in mature and newly-formed NMJs. This effect was due to an enhanced loading of synaptic vesicles with ACh and increase of ACh quantal size, since it was fully prevented by blocking of vesicular ACh transporter. It was also prevented by tropomyosin-related kinase B (TrkB) receptors inhibitor ANA12. Exogenous BDNF (1 nM) mimicked thrombin effect and increased the amplitude of MEPPs and EPPs by 25-30%. It required involvement of protein kinase A (PKA) and mitogen-activated protein kinase (MEK1/2)-mediated pathway, but not phospholipase C (PLC). Blocking A2A adenosine receptors by ZM241385 abolished the effect of BDNF, whereas additional stimulation of A2A receptors by CGS21680 increased MEPP amplitudes, which was prevented by MEK1/2 inhibitor U0126. At mature NMJs, BDNF enhanced MEPPs frequency by 30-40%. This effect was selectively prevented by inhibition of PLC, but not PKA or MEK1/2. It is suggested that interrelated effects of thrombin/BDNF in mature and newly-formed NMJs are realized via enhancement of vesicular ACh transport and quantal size increase. BDNF-induced potentiation of synaptic transmission involves the functional coupling between A2A receptor-dependent active PKA and neurotrophin-triggered MAPK pathway, as well as PLC-dependent increase in frequency of MEPPs.

Myofascial trigger points (MTrPs) are defined as hyperirritable spots in a palpable taut band (TB) of skeletal muscle fibers. Knowing the formation and location of MTrPs is a great help to prevent their development and inactivate existing MTrPs. This study aimed to obtain new evidence that myofascial trigger spots (MTrSs), which are similar to human MTrPs, are found in dysfunctional motor endplates by observing the morphological characteristics of muscles and changes in biochemical substances. A total of 32 male Sprague Dawley rats were randomly divided into four groups: two control groups (i.e., C1 and C2) and two model groups (i.e., M1 and M2). C1 and M1 were used for acetylcholine (ACh) content measurement, while C2 and M2 were utilized for acetylcholinesterase (AChE) staining. In the model groups, blunt striking injury was induced and eccentric exercise was applied to the left gastrocnemius for 8 weeks. After 1 month, spontaneous electrical activity(SEA), AChE optical density (OD), muscle fiber diameter, and ACh content were measured. The results showed that extensive abnormal endplate noise (aEPN), including positive neurons, fibrillation potentials, fasciculation potential, and high amplitude (endplate spikes [EPS]), is present at MTrSs in M1. Quantitative electromyography results showed that the amplitudes of aEPN and frequency of EPS in M1 were significantly higher than those of C1. The ACh content of MTrSs in M1 was significantly higher than that in C1. The AChE OD value of M2 was significantly lower than that of C2. In addition, the diameter of the muscle fibers in the AChE-stained area was longer in M2 than in C2. In conclusion, MTrSs formed at the motor endplate with a larger diameter of muscle fibers. Excessive ACh release and decreased AChE activity at MTrSs stimulated muscle action potential and muscle contraction.