Endplate Region Research Articles

Spatial characterization of the motor neuron columns supplying the rat forelimb

Rats can generate a rich array of forepaw and forelimb movements that are similar, although not as complex, to those produced by human and non-human primates. When reaching for food for instance, rats display skilled movements of the forelimb and the paw, therefore, making them attractive models to validate strategies aimed at the recovery of fine motor control. Surprisingly however, few anatomical studies have been performed on the central control of forelimb movements in the rat. The current series of experiments examined the details of the segmental arrangement of motor neurons that supply the rat forelimb. The distribution of motor end plates across the rat forelimb was first visualized by means of acetylcholinesterase histochemistry, and this information was used to create a motor end plate map of the forelimb muscles. This map was subsequently used as a guide for multiple injections of retrograde tracers along the motor end plate regions of 11 forelimb muscles. The entire cervical region of the spinal cord was subsequently analyzed under epifluorescence. This tract-tracing analysis confirmed that motor neurons innervating the rat forelimb are arranged in columns within the cervical segments of the spinal cord. This anatomical investigation also supports the previous observation that, although discrete, some of the motor neuron columns lying in the cervical aspect of the rat spinal cord are inter-mingled. The length of these columns, and hence the overlap between them, appears to be greater than previously reported, particularly within the uppermost segments of the brachial plexus.

An analysis of the relationships between subthreshold electrical properties and excitability in skeletal muscle

Skeletal muscle activation requires action potential (AP) initiation followed by its sarcolemmal propagation and tubular excitation to trigger Ca2+ release and contraction. Recent studies demonstrate that ion channels underlying the resting membrane conductance (GM) of fast-twitch mammalian muscle fibers are highly regulated during muscle activity. Thus, onset of activity reduces GM, whereas prolonged activity can markedly elevate GM. Although these observations implicate GM regulation in control of muscle excitability, classical theoretical studies in un-myelinated axons predict little influence of GM on membrane excitability. However, surface membrane morphologies differ markedly between un-myelinated axons and muscle fibers, predominantly because of the tubular (t)-system of muscle fibers. This study develops a linear circuit model of mammalian muscle fiber and uses this to assess the role of subthreshold electrical properties, including GM changes during muscle activity, for AP initiation, AP propagation, and t-system excitation. Experimental observations of frequency-dependent length constant and membrane-phase properties in fast-twitch rat fibers could only be replicated by models that included t-system luminal resistances. Having quantified these resistances, the resulting models showed enhanced conduction velocity of passive current flow also implicating elevated AP propagation velocity. Furthermore, the resistances filter passive currents such that higher frequency current components would determine sarcolemma AP conduction velocity, whereas lower frequency components excite t-system APs. Because GM modulation affects only the low-frequency membrane impedance, the GM changes in active muscle would predominantly affect neuromuscular transmission and low-frequency t-system excitation while exerting little influence on the high-frequency process of sarcolemmal AP propagation. This physiological role of GM regulation was increased by high Cl− permeability, as in muscle endplate regions, and by increased extracellular [K+], as observed in working muscle. Thus, reduced GM at the onset of exercise would enhance t-system excitation and neuromuscular transmission, whereas elevated GM after sustained activity would inhibit these processes and thereby accentuate muscle fatigue.

Calcitonin receptor-like receptor expression in rat skeletal muscle fibers

The calcitonin gene related peptide (CGRP) pathway is important in many processes including several in the central and peripheral nervous systems. CGRP is present in motor neurons and in sensory tracts, with its expression likely regulated by its use. This is supported by the fact that axotomy results in increased CGRP production in the nerve cell body. The target CGRP receptor, produced in part from the calcitonin receptor-like receptor (Calcrl) gene, has been thought to be present in multiple forms based on kinetic studies; however, understanding of the regulation of the expression of the Calcrl gene remains incomplete. CALCRL is an important factor in aging and associated disorders. This study focused on the neuromuscular system where it has been unclear whether different proteins are initially translated and whether higher levels of CALCRL are localized to the endplate regions. Rat gracilis muscle neuromuscular junctions were examined by isolating endplate enriched and non-endplate regions identified by staining for acetylcholine esterase or conjugated α-bungarotoxin binding. The CALCRL protein was detected at approximately 60kDa by Western immunoblotting and, in the isolated extracts, we found that the Calcrl mRNA level was elevated 6 fold in the muscle endplate regions and that there were two distinct Calcrl messages present. Sequence analysis showed that the two different Calcrl forms were due to alternative splicing but in a non-coding region of the transcript such that only one translation product would be generated. This indicates that previously identified pharmacologic heterogeneity is most likely due to post-translational modifications and interactions.

Desensitization and models of receptor‐channel activation

Desensitization and models of receptor‐channel activation

Remodeling of the neuromuscular junction precedes sarcopenia related alterations in myofibers

Several mechanisms contributing to the etiology of sarcopenia (age-related loss of muscle size) have been postulated. One of these attributes the loss of muscle mass to a preceding age-related denervation of myofibers. The aim of this study was to determine if signs of denervation were apparent at the neuromuscular junction (NMJ) before fiber atrophy, or fiber type conversion could be documented, and to reveal if a muscle’s activity level impacts its sensitivity to age-related denervation. Plantaris and soleus muscles were obtained from young adult (10 months) and early aged (21 months) rats. Pre- and post-synaptic NMJ morphology was quantified with cytofluorescent staining of nerve terminal branches and endplate regions, respectively. Myofiber profiles (fiber size and fiber type composition) were assessed with histochemical procedures. Results show that in the lightly recruited plantaris, significant ( P < 0.05) signs of denervation were noted in aged rats, while the same muscles displayed no change in myofiber profile. In the heavily recruited soleus, however, there was little evidence of denervation, and again no alterations in myofiber profile. These results indicate that age-related denervation occurs before myofiber atrophy, and that high amounts of neuromuscular activity may delay the onset of age-related denervation and sarcopenia.

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.

A Study on the Structural Properties of the Lumbar Endplate

Histologic experiments and biomechanical tests were performed in human cadaveric lumbar spine models. To determine (1) the anatomic structures of lumbar endplates, (2) the relationship between bone mineral density (BMD) and biomechanical properties of lumbar endplates, and (3) the influence of spinal level on the failure loads of lumbar endplates. Previous works have shown that the posterolateral corners of the lumbar endplates are stronger than the anterior and central regions. Information on the microstructures of lumbar endplates and the effects of both BMD and spinal level on biomechanical properties of lumbar endplates would be valuable for spine surgeons and implant designers to avoid subsidence. Twenty fresh human cadaver lumbar vertebrae specimens were collected for HE staining and the sections through the specimens were examined under light microscope. The areas of the pore structures in the lumbar endplates were measured and analyzed by statistical methods. Sixty-five cadaver lumbar vertebrae were evaluated with dual energy radiograph absorptiometry and according to their BMD, all vertebrae were divided into 3 groups. Indentation tests were performed at 27 standardized test sites in endplates of these vertebrae using a 1.5-mm-diameter, hemispherical indenter with a rate of 12 mm/min. The failure load at each test site was determined using the load-displacement curve. Spearman analysis was used to evaluate the correlation between BMD and failure loads of lumbar endplates. Factorial analysis of variance was performed to reveal the effects of both BMD and spinal level on failure load distribution on the lumbar endplates. (1) The peripheral regions were thicker than the central regions of lumbar endplates. The central regions were porous, as the fused trabeculae and peripheral regions had fewer and smaller pore structures. (2) The tests showed that there were significant differences in failure load between 3 groups (P < 0.01). BMD was positively correlated with the failure load of the lumbar endplate. (3) The strongest position of the lumbar endplate was the posterolateral region closest to the pedicles and the failure loads tended to increase from anterior to posterior regions of the lumbar endplates. (4) The failure load distribution did not change with the BMD decrease. (5) The differences of the failure loads between the lumbar segments were significant (P < 0.05). The failure loads of the lumbar endplates showed an increasing tendency from L1-L5 segments. The differences in the anatomic structures of different regions are the histologic foundation of biomechanical properties of lumbar endplates. It is necessary that BMD should be measured before operation and implants should be placed in posterior-lateral regions of lumbar endplates so that subsidence syndrome can be reduced as much as possible. In addition, the fact that the endplates of the upper lumbar segments with the lower strength have higher risk of subsidence should be concerned about by spine surgeons and implant designers.

Resistance to d-Tubocurarine of the Rat Diaphragm as Compared to a Limb Muscle

The diaphragm is resistant to competitive neuromuscular blocking agents, as compared to peripheral muscles. The basis of this difference may be a higher concentration of acetylcholine released or higher number of postsynaptic nicotinic acetylcholine receptors in diaphragmatic neuromuscular junctions. Nerve-evoked twitch-tension was measured in rat hemidiaphragm as was Extensor digitorum longus (EDL) nerve-muscle preparation to determine the effective D-tubocurarine concentration that decreased twitch responses by 50%. The mean quantal content of endplate potentials was determined in single junctions in a low-Ca(2+), high-Mg(2+) Krebs-Ringer medium. Strips of hemidiaphragm and EDL muscle, containing the endplate regions, were used to determine the number of nAChR nicotinic acetylcholine receptor binding sites with the aid of radiolabeled [(125)I]alpha-bungarotoxin. The effective D-tubocurarine concentration that decreased twitch responses by 50% (median [interquartile range]) was seven-fold higher in the hemidiaphragm than in the EDL (1.82 microm [1.43-2.20] vs. 0.26 microm [0.23-0.29], P < 0.01). The median of the mean quantal content was higher in the hemidiaphragm than in the EDL (0.57 [0.44-0.84] vs. (0.14 [0.11-0.19], P < 0.01). The number of specific [(125)I]alpha-bungarotoxin binding sites to junctional nicotinic acetylcholine receptors was higher in the diaphragm than in the EDL (1.15 fmol/mg [0.48-1.70] vs. 0.55 fmol/mg [0.23-0.70 ], P < 0.05). The current study indicates that the resistance of the diaphragm to neuromuscular blocking agents can be explained by both a higher mean quantal content of endplate potentials and a higher number of nicotinic acetylcholine receptor binding sites than in the peripheral EDL muscle.

Effects of age on training‐induced adaptations of the neuromuscular junction

Unlike skeletal muscle, the effect of aging on training induced adaptations of the neuromuscular junction (NMJ) has received scant attention. This study aimed to determine whether this important synapse exhibits age‐specific adaptations to endurance training. Twenty young adult (8 mo) and 20 aged (24 mo) male Fischer 344 rats were assigned to either exercise trained or sedentary control groups (4 groups, N=10/group). All trained rats participated in the same 10 week program of treadmill running (5days/wk). Following the 10 week intervention, all rats were euthanized and soleus muscles were removed and quickly frozen at resting length. Muscles were sectioned and stained with cytofluorescent procedures to visualize pre‐synaptic nerve terminal branches, and post‐synaptic acetylcholine receptors. Pre‐ and post‐synaptic images were collected with confocal microscopy and used to quantify various parameters of NMJ morphology. Results showed that young, but not aged rats demonstrated significant (P&lt;0.05) training‐induced increases in pre‐synaptic nerve terminal branch number, total branch length, and branching complexity. Similarly, only the NMJs of young rats displayed significant training‐induced increments in perimeter length around endplate regions, and expanded areas occupied by acetylcholine receptors. Our data show that aging blunts the ability of the NMJ to adapt to endurance exercise training.Supported by the National Institute on Aging, and The Foundation for Aging Studies and Exercise Science Research.

Autoradiographic visualization of extrajunctional acetylcholine receptors in whole human biceps brachii muscle Changes in amyotrophic lateral sclerosis

The morphological distribution of acetylcholine receptors in the whole biceps brachii muscle from a patient with amyotrophic lateral sclerosis (ALS) and from a control patient was studied by in vitro autoradiography with 3H-alpha-bungarotoxin (alpha-Btx). In ALS, 3H-alpha-Btx binding occurred over the entire muscle, while in the control case the binding was restricted to the motor end-plate region.

Characterization of urotensin II, distribution of urotensin II, urotensin II‐related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction

Urotensin II (UII) and UII-related peptide (URP) are paralog neuropeptides whose existence and distribution in mouse have not yet been investigated. In this study, we showed by HPLC/RIA analysis that the UII-immunoreactive molecule in the mouse brain corresponds to a new UII(17) isoform. Moreover, calcium mobilization assays indicated that UII(17) and URP were equally potent in stimulating UII receptor (UT receptor). Quantitative RT-PCR and in situ hybridization analysis revealed that in the CNS UII and URP mRNAs were predominantly expressed in brainstem and spinal motoneurons. Besides, they were differentially expressed in the medial vestibular nucleus, locus coeruleus and the ventral medulla. In periphery, both mRNAs were expressed in skeletal muscle, testis, vagina, stomach, and gall bladder, whereas only URP mRNA could be detected in the seminal vesicle, heart, colon, and thymus. By contrast, the UT receptor mRNA was widely expressed, and notably, very high amounts of transcript occurred in skeletal muscle and prostate. In the biceps femoris muscle, UII-like immunoreactivity was shown to coexist with synaptophysin in muscle motor end plate regions. Altogether these results suggest that (i) UII and URP may have many redundant biological effects, especially at the neuromuscular junction; (ii) URP may more specifically participate to autonomic, cardiovascular and reproductive functions.

Position of Interbody Spacer in Transforaminal Lumbar Interbody Fusion: Effect on 3-dimensional Stability and Sagittal Lumbar Contour

Biomechanical study. To test 2 different intervertebral positions of a semilunar cage and their effects on 3-dimensional stability and segmental lordosis in a model of transforaminal lumbar interbody fusion (TLIF). In his original TLIF description, Harms recommended decortication of endplates, followed by placement of mesh cages in the middle-posterior intervertebral third. Subsequent studies presented conflicting recommendations: anterior placement of the spacer-cage for better load-sharing versus placement on the stronger posterolateral endplate regions. Six human lumbar spinal functional units were first tested intact. TLIF was performed using a semilunar poly-ether-ether-ketone cage randomly inserted in the anterior (TLIF-A) or posterior (TLIF-P) disc space. Pedicle screws and rods were added. Unconstrained pure moments in axial-torsion, lateral-bending (LB), and flexion-extension (FE) were applied under 0.05 Hz and +/-5 Nm sinusoidal waveform. Segmental motions were recorded. Range of motion (ROM) and neutral zone (NZ) were calculated. Pairwise comparisons were made using nonparametric Wilcoxon-matched pairs signed rank sum test with statistical significance set at P<0.05. TLIF-A and TLIF-P significantly decreased ROM (P<0.05) of the intact spinal functional unit, in FE and LB. In axial-torsion, decrease of ROM after TLIF procedures was not significant (P>0.05). Delta-ROM between TLIF-A and TLIF-P was not significant (P>0.05). TLIF-A and TLIF-P significantly decreased NZ in LB (P<0.05). In FE, TLIF-P significantly decreased NZ (P<0.05); TLIF-A showed a trend toward significance (P=0.09). Delta-NZ between TLIF-A and TLIF-P was not significant (P>0.05). Segmental lordosis of TLIF-A and TLIF-P on C-arm views showed angle differences within the range of measurement error of Cobb angles. Difference in ROM and NZ between anterior (TLIF-A) or posterior (TLIF-P) positions was not statistically significant. Similarly, both positions did not influence segmental lordosis.

Early‐onset degeneration of the intervertebral disc and vertebral end plate in mice deficient in type IX collagen

Type IX collagen is an important component of the intervertebral disc extracellular matrix. Mutations in type IX collagen are associated with premature disc degeneration in mice and a predisposition to disc disorders in humans. The aim of this study was to assess the prevalence and timeline of intervertebral disc degeneration in mice homozygous for an inactivated Col9a1 gene. Intact spine segments were harvested from wild-type (WT) and type IX collagen-knockout (Col9a1(-/-)) mice at 3, 6, and 12 months of age. Sagittal spine sections were evaluated for evidence of histologic changes, by 2 blinded graders, using a semiquantitative grading method. There was evidence of more degeneration of the disc and end plate in the spines of Col9a1(-/-) mice compared with those of WT controls, at most time points. These findings were significant for the disc region at 3 and 6 months (P<0.01) and at 12 months (P<0.10) and for the end plate region only at 6 months (P<0.10). Degenerative changes in the disc consisted of cellular changes and mucous degeneration. Degeneration in the end plates was associated with more cell proliferation, cartilage disorganization, and new bone formation. A deletion mutation for type IX collagen is associated with connective tissue changes characteristic of musculoskeletal degeneration in bony and cartilaginous tissue regions. Some of the observed changes were similar to cartilage changes in osteoarthritis, while others were more similar to disc degenerative changes in humans. The finding of premature onset of intervertebral disc degeneration in this mouse model may be useful in studies of the pathology and treatment of human disc degeneration.

Myasthenia gravis.

Myasthenia gravis (MG) is a rare, autoimmune neuromuscular junction disorder. Contemporary prevalence rates approach 1/5,000. MG presents with painless, fluctuating, fatigable weakness involving specific muscle groups. Ocular weakness with asymmetric ptosis and binocular diplopia is the most typical initial presentation, while early or isolated oropharyngeal or limb weakness is less common. The course is variable, and most patients with initial ocular weakness develop bulbar or limb weakness within three years of initial symptom onset. MG results from antibody-mediated, T cell-dependent immunologic attack on the endplate region of the postsynaptic membrane. In patients with fatigable muscle weakness, the diagnosis of MG is supported by: 1. pharmacologic testing with edrophonium chloride that elicits unequivocal improvement in strength; 2. electrophysiologic testing with repetitive nerve stimulation (RNS) studies and/or single-fiber electromyography (SFEMG) that demonstrates a primary postsynaptic neuromuscular junctional disorder; and 3. serologic demonstration of acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK) antibodies. Differential diagnosis includes congenital myasthenic syndromes, Lambert Eaton syndrome, botulism, organophosphate intoxication, mitochondrial disorders involving progressive external ophthalmoplegia, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), motor neuron disease, and brainstem ischemia. Treatment must be individualized, and may include symptomatic treatment with cholinesterase inhibitors and immune modulation with corticosteroids, azathioprine, cyclosporine, and mycophenolate mofetil. Rapid, temporary improvement may be achieved for myasthenic crises and exacerbations with plasma exchange (PEX) or intravenous immunoglobulin (IVIg). Owing to improved diagnostic testing, immunotherapy, and intensive care, the contemporary prognosis is favorable with less than five percent mortality and nearly normal life expectancy.

Fatigue analysis of interference EMG signals obtained from biceps brachii during isometric voluntary contraction at various force levels

The aim of the present study was to test the applicability of indices of muscle fatigue to interference EMG signals detected at various distances from the end-plate region during isometric voluntary contractions at different force levels. Bar electrode with 12 leading off surfaces and 5 mm inter-pole distance was used to detect EMG from human m. biceps brachii. The sensitivity of the new spectral indices to detect muscle fatigue was higher than that of mean or median frequencies. Considerable variations in the characteristic frequencies and values of spectral indices that could reflect recruitment and/or rotation of MUs were found under submaximal efforts. The increase of the indices was considered as a sign of peripheral muscle fatigue while their decreasing could be a sign of de-recruitment of fatigued or/and recruitment of new MUs reflecting central fatigue. The sensitivity of the indices to fatigue depended on the electrode arrangement and its longitudinal position in respect of the end-plate region and ends of the muscle fibres. It was larger for the electrodes placed in the middle of the semi-fibre. To overcome the problem with inappropriate position of the electrode, one could use an electrode whose longitudinal dimension would cover the entire semi-length of the analyzed fibres.

Distinct patterns of motor nerve terminal sprouting induced by ciliary neurotrophic factor vs. Botulinum toxin

Both diffusible and surface-bound molecules are thought to induce sprouting of motor nerve terminals in response to paralysis. Here we report that the sprouting induced by ciliary neurotrophic factor (CNTF) is qualitatively different from the sprouting induced by botulinum toxin (BoTX). We show first that subcutaneous application of CNTF to levator auris longus muscles of adult mice evokes sprouting from nearly all nerve terminals. Surprisingly, however, most terminal sprouts remain within the boundaries of the endplate region and rarely grow extrasynaptically even if CNTF is administered chronically. In contrast, terminal sprouts induced by BoTX extend vigorously along the extrasynaptic muscle surface. The different patterns of sprout elongation are attributable in part to different patterns of initiation: whereas CNTF-induced sprouts emerge randomly from the surface of terminal branches, BoTX-induced sprouts emerge exclusively along the perimeter of terminal branches in direct apposition to muscle fiber membranes. Combined treatment with CNTF and BoTX produces exceptionally robust extraterminal sprouting with little if any intrasynaptic growth of terminal sprouts. We interpret these results as showing that paralysis induces sprouting primarily by muscle-associated, surface-bound molecules rather than by diffusible factors. Our findings may be useful in defining the physiological role of the numerous candidate sprouting-inducers and in promoting compensatory sprouting after nerve injury for therapeutic benefit.

Quantitative Proteomics Profiling of Sarcomere Associated Proteins in Limb and Extraocular Muscle Allotypes

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04±0.06 ms in wild-type junctions and 5.4 ms±0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.

Overexpression of Rapsyn in Rat Muscle Increases Acetylcholine Receptor Levels in Chronic Experimental Autoimmune Myasthenia Gravis

The primary autoantigen in myasthenia gravis, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the neuromuscular junction by rapsyn. Previously, we found that overexpression of rapsyn by cDNA transfection protects AChRs in rat muscles from antibody-mediated loss in passive transfer experimental autoimmune myasthenia gravis (EAMG). Here, we determined whether rapsyn overexpression can reduce or even reverse AChR loss in muscles that are already damaged by chronic EAMG, which mimics the human disease. Active immunization against purified AChR was performed in female Lewis rats. Rapsyn overexpression resulted in an increase in total muscle membrane AChR levels, with some AChR at neuromuscular junctions but much of it in extrasynaptic membrane regions. At the ultrastructural level, most endplates in rapsyn-treated chronic EAMG muscles showed increased damage to the postsynaptic membrane. Although rapsyn overexpression stabilized AChRs in intact or mildly damaged endplates, the rapsyn-induced increase of membrane AChR enhanced autoantibody binding and membrane damage in severe ongoing disease. Thus, these results show the complexity of synaptic stabilization of AChR during the autoantibody attack. They also indicate that the expression of receptor-associated proteins may determine the severity of autoimmune diseases caused by anti-receptor antibodies.

LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction

Low-density lipoprotein receptor-related protein 4 (Lrp4) is a member of a family of structurally related, single-pass transmembrane proteins that carry out a variety of functions in development and physiology, including signal transduction and receptor-mediated endocytosis. Lrp4 is expressed in multiple tissues in the mouse, and is important for the proper development and morphogenesis of limbs, ectodermal organs, lungs and kidneys. We show that Lrp4 is also expressed in the post-synaptic endplate region of muscles and is required to form neuromuscular synapses. Lrp4-mutant mice die at birth with defects in both presynaptic and postsynaptic differentiation, including aberrant motor axon growth and branching, a lack of acetylcholine receptor and postsynaptic protein clustering, and a failure to express postsynaptic genes selectively by myofiber synaptic nuclei. Our data show that Lrp4 is required during the earliest events in postsynaptic neuromuscular junction (NMJ) formation and suggest that it acts in the early, nerveindependent steps of NMJ assembly. The identification of Lrp4 as a crucial factor for NMJ formation may have implications for human neuromuscular diseases such as myasthenia syndromes.