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

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

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

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

Presynaptic K(+) channels, vesicular Ca(2+)/H (+) antiport--synaptotagmin, and acetylcholinesterase, three mechanisms cutting short the cholinergic signal at neuromuscular and nerve-electroplaque junctions.

In neuromuscular and nerve-electroplaque junctions, nerve impulses can be transmitted at high frequencies. This implies that transmission of individual impulses must be very brief. We describe three mechanisms which curtail the time course of individual impulses at these synapses: (1) opening of presynaptic K(+) channels (delayed rectifier) efficiently curtails the presynaptic action potential. Inhibition of K(+) channel by aminopyridines transforms the normally brief postsynaptic potential (2-3 ms) to a long-lasting "giant" potential (exceeding half a second); (2) a low-affinity Ca(2+)/H(+) antiport ensures rapid Ca(2+) sequestration into synaptic vesicles, curtailing the calcium signal and thereby the duration of transmitter release. Indeed vesicular Ca(2+)/H(+) antiport inhibition by bafilomycin or Sr(2+) prolongs the duration of the postsynaptic potential. We recently showed that synaptotagmin-1 is required for this antiport activity; thus the vesicular Ca(2+)/H(+) antiport might be synaptotagmin itself, or regulated by it; and (3) it is recalled that, in these junctions, acetylcholinesterase is highly concentrated in the synaptic cleft and that anticholinesterases lengthen the endplate time course. Therefore, at three different steps of synaptic transmission, an efficient mechanism curtails the local synaptic signal. When one of these three mechanisms is inhibited, the duration of individual impulses is prolonged, but the synapse loses its faculty to fire at high frequencies.

Reduced expression of the vesicular acetylcholine transporter and neurotransmitter content affects synaptic vesicle distribution and shape in mouse neuromuscular junction.

In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KD(HOM)) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KD(HOM) mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1-43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KD(HOM) neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KD(HOM) exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape.

The effect of severing L6 nerve root of the sacral plexus on lower extremity function: an experimental study in rhesus monkeys.

BACKGROUND: Nerve transfer is a valid surgical procedure for lower-extremity function restoration after lumbosacral plexus avulsion. OBJECTIVE: To evaluate the impact of severing the L6 nerve root on the functions of the healthy limb in rhesus monkeys and the feasibility of using the contralateral L6 nerve root as a donor nerve to repair lumbosacral plexus root avulsion. METHODS: Twenty-four rhesus monkeys were randomly assigned into 2 groups. In the experimental group, the right L6 nerve root was explored and severed, whereas in the control group animals underwent a sham operation. Electrophysiology, muscle mass, histology, and ultrastructure of the target muscles were examined. RESULTS: Three weeks after transection, reduced amplitude and prolonged latency of compound muscle action potential were observed in the medial gastrocnemius, extensor digitorum brevis, peroneus longus, and abductor hallucis muscles of the experimental group, as well as reduction in muscle mass and myofiber cross-sectional area of these muscles. The number of myelinated nerve fibers of the sciatic nerve in the experimental group was significantly less than that of the control group. Abnormal ultrastructure of motor end plates of these muscles was also observed in the experimental group. Eight weeks postoperatively, all of these parameters were similar between the experimental and control groups. CONCLUSION: Severing the L6 nerve root does not damage the healthy limb as far as electrophysiology, muscle mass, histology, and ultrastructure of the target muscles are concerned. L6 (analogous to S1 in humans) nerve root may be used as a donor nerve to repair lumbosacral plexus root avulsion.

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

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

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

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

[Histochemical findings of and fine structural changes in motor endplates in diseases with neuromuscular transmission abnormalities].

We herein review the histochemical findings and fine structural changes of motor endplates associated with diseases causing neuromuscular transmission abnormalities. In anti-acetylcholine receptor (AChR) antibody-positive myasthenia gravis (MG), type 2 fiber atrophy is observed, and the motor endplates show a reduction in the nerve terminal area, simplification of the postsynaptic membrane, decreased number of acetylcholine receptors, and deposition of immune complexes. In anti-MuSK antibody-positive MG, the fine structure shows a decrease in the postsynaptic membrane length, but the secondary synaptic cleft is preserved. There is no decrease in the number of AChRs, and there are no deposits of immune complexes at the motor endplates. Patients with Lambert-Eaton myasthenic syndrome show type 2 fiber atrophy, their motor endplates show a decrease in both the mean postsynaptic area and postsynaptic membrane length in the brachial biceps muscle. Congenital myasthenic syndrome with episodic apnea is characterized only by small-sized synaptic vesicles; the postsynaptic area is preserved. In subjects with congenital myasthenic syndrome with acetylcholinesterase deficiency, quantitative electron microscopy reveals a significant decrease in the nerve terminal size and presynaptic membrane length; further, the Schwann cell processes extend into the primary synaptic cleft, and partially or completely occlude the presynaptic membrane. The postsynaptic folds are degenerated, and associated with pinocytotic vesicles and labyrinthine membranous networks. Patients with slow-channel congenital myasthenia syndrome show type 1 fiber predominance, and their junctional folds are typically degenerated with widened synaptic space and loss of AChRs. Patients with AChR deficiency syndrome caused by recessive mutations in AChR subunits also show type 1 fiber predominance, and while most junctional folds are normal, some are simplified and have smaller than normal endplates. Rapsin and MuSK mutations cause type 1 fiber predominance, and the small postsynaptic area is associated with AChR decrease.

Endplate structure and parameters of neuromuscular transmission in sporadic centronuclear myopathy associated with myasthenia.

Centronuclear myopathy is a pathologically diagnosed congenital myopathy. The disease genes encode proteins with membrane modulating properties (MTM1, DNM2, and BIN1) or alter excitation-contraction coupling (RYR1). Some patients also have myasthenic symptoms but electrodiagnostic and endplate studies in these are limited. A sporadic patient had fatigable weakness and a decremental EMG response. Analysis of centronuclear myopathy disease- and candidate-genes identified no mutations. Quantitative endplate electron microscopy studies revealed simplified postsynaptic regions, endplate remodeling with normal nerve terminal size, normal synaptic vesicle density, and mild acetylcholine receptor deficiency. The amplitude of the miniature endplate potential was decreased to 60% of normal. Quantal release by nerve impulse was reduced to 40% of normal due to a decreased number of releasable quanta. The safety margin of neuromuscular transmission is compromised by decreased quantal release by nerve impulse and by a reduced postsynaptic response to the released quanta.

Immunocytochemical demonstration of M(1) muscarinic acetylcholine receptors at the presynaptic and postsynaptic membranes of rat diaphragm endplates.

M(1)-muscarinic acetylcholine (ACh) receptors (M(1)R) were directly demonstrated immunocytochemically in electronmicroscopic images of rat diaphragm neuromuscular junctions (NMJ). Specific electron-dense granules were located at presynaptic nerve ending membranes and in the sarcolemma in the depths of postsynaptic folds. This first visualization of M(1)R on both sides of the NMJ is in agreement with previous pharmacological data on the regulatory role of M(1)R in quantal and non-quantal ACh release.

Motor endplate remodeling in some cases with congenital myasthenic syndrome.

The architecture of motor endplates in three cases with congenital myasthenic syndrome (CMS) was compared with ultrastructure of the normal control neuromuscular junction (NMJ). The remodeling of postsynaptic region was observed in all three individuals. The most conspicuous abnormalities seen in the slow channel syndrome was the vacuolization and disorganization of secondary synaptic clefts which extended for beyoned the border of NMJ. Degenerated postsynaptic nuclei and junctional sarcoplasm were an additional feature of presented syndromes. The quite different feature of NMJ was observed in the DOK-7 deficient syndrome. The appearance of small, pale terminal axons, poorly developed postsynaptic membrane with the sparse secondary synaptic clefts and degenerated postsynaptic nuclei suggested impairment of postsynaptic region maturation. The conjunction of postsynaptic membrane paucity and its degeneration was a specific structural feature observed in the third syndrome with no established genetic defects.

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

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

Rab3 dynamically controls protein composition at active zones.

Synaptic transmission requires the localization of presynaptic release machinery to active zones. Mechanisms regulating the abundance of such synaptic proteins at individual release sites are likely determinants of site-specific synaptic efficacy. We now identify a role for the small GTPase Rab3 in regulating the distribution of presynaptic components to active zones. At Drosophila rab3 mutant NMJs, the presynaptic protein Bruchpilot, calcium channels, and electron-dense T bars are concentrated at a fraction of available active zones, leaving the majority of sites devoid of these key presynaptic release components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting Brp to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the composition of the presynaptic release machinery. While previous studies of Rab3 have focused on its role in the synaptic vesicle cycle, these findings demonstrate an additional and unexpected function for Rab3 in the localization of presynaptic proteins to active zones.

Glutamatergic reinnervation and assembly of glutamatergic synapses in adult rat skeletal muscle occurs at cholinergic endplates.

After denervation of adult rat abdominal muscles, the postsynaptic apparatus of neuromuscular junctions (NMJs) retains its original architecture and clustering of acetylcholine receptors (AChRs). When descending fibers of the spinal cord are surgically diverted to this muscle by a nerve grafting procedure, supraspinal glutamatergic neurons can innervate muscle fibers and restore motor function; the newly formed NMJs switch from a cholinergic to a glutamatergic-type synapse. We show here that regenerating nerve endings contact the fibers in an area occupied by cholinergic endplates. These NMJs are morphologically indistinguishable from those in controls, but they differ in the subunit composition of AChRs. Moreover, by immunofluorescence and immunoelectron microscopy, new NMJs express glutamatergic synapse markers. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 partially colocalizes with AChRs, and vesicular glutamate transporter 2 is localized in the presynaptic compartment. Immunoprecipitation analysis of membranes from reinnervated muscle showed that AMPA receptor subunits GluR1 and GluR2 coimmunoprecipitate with rapsyn, the AChR-anchoring protein at the NMJ. Taken together, these results indicate that cholinergic endplates can be targeted by new glutamatergic projections and that the clustering of AMPA receptors occurs there.

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

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

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

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

Influence of location, fluid flow direction, and tissue maturity on the macroscopic permeability of vertebral end plates.

STUDY DESIGN: We implemented a pilot study in a growing animal model. The macroscopic permeability of the vertebral endplates was measured. The influence of location, tissue maturity, and fluid flow direction was quantified. OBJECTIVE: We hypothesized that the macroscopic permeability of vertebral endplate may decrease with maturity of the vertebral segment. SUMMARY OF BACKGROUND DATA: The alternation of loading induced by the diurnal cycle generates convective flux into the vertebral segment with the dominant flow path through the vertebral endplates. The alteration of mass transport at the disc-vertebrae interface may interrupt the mechanobiologic balance, and have an effect such as degenerative changes or scoliosis. METHODS: A previously validated method for measuring permeability, based on the relaxation pressure caused by a transient-flow rate was used. Three specimens were extracted from each L1 to L5 endplate. Seventy-one specimens were frozen, and 64 were stored fresh in a standard culture media. A microscopic analysis completed the biomechanical analysis. RESULTS: At 2, 4, and 6 months, the mean permeability (10(-14) m/N x s, flow-in/flow-out) of the central zone was respectively: 1.23/1.66, 1.03/1.29, and 0.792/1.00. Laterally, it was 1.03/1.19, 1.094/1.001, and 0.765/0.863. For all groups, cartilage endplate and growth plate were both thinner in the center of the plate. Weak differences of the vascular network were detected, except for a small increase of vascular density in the central zone. CONCLUSION: The results from this animal study showed that the central zone of the vertebral endplate was more permeable than the periphery and the flow-out permeability was up to 35% greater than the flow-in permeability. Increase of permeability with decrease of cartilage thickness was noticed within the same age group. We also found a statistically significant decrease of the macroscopic permeability correlated with the tissue maturity.

Ultrastructural visualization of the transmembranous and cytomatrix-related part of nicotinic acetylcholine receptor of frog motor endplate by means of an immunochemical avidity of IgG for d-tubocurarine.

In the present study, a fine ultrastructural localization of nicotinic acetylcholine receptor (nAChR) was attempted, using d-tubocurarine (d-TC), a quaternary ammonium compound binding to nAChR. The localization was based on the binding avidity of immunoglobulin G (IgG) for acetylcholine (ACh) and other quaternary ammonium compounds, such as d-TC. d-TC was applied to the frog neuromuscular preparation and caused a blockade of neuromuscular transmission. Then, d-TC was rendered insoluble in situ by silicotungstic acid (STA), a precipitating agent of soluble proteins and quaternary ammonium compounds. After tissue fixation, a normal rabbit serum was applied to the fine precipitate of the insoluble salt of d-TC silicotungstate (quaternary ammonium radical of d-TC) to form the immunochemical complex d-TC- rabbit IgG at ACh binding sites. The IgG of the complex was revealed by means of the conventional immunoperoxidase procedure used for ultrastructural localization. Under the electron microscope, fine diaminobenzidine (DAB) precipitates appeared as regular rod-like structures oriented to cytoplasmic side of the horizontal part (crest) of the postsynaptic membrane (between the junctional folds) which is known to be endowed with nAChR. The rod-like precipitates were not observed in the postsynaptic junctional folds which are devoid of nAChR. The distance separating the rods each other was rather constant (12 - 15 nm), while the length of the rods was variable and exceeded the usual length of nAChR. The present work indicates that the rod-like structures, already observed in association with sarcoplasmic side of the postsynaptic membrane, did correspond to the intramembranous and intracytoplasmic part of nAChR and related proteins. These cytochemical results confirm that d-TC binds to ACh binding sites in the pore of nAChR, and raise the question of DAB staining of cytoskeletal proteins related to the nAChR complex.

Local stabilization of microtubule assembly improves recovery of facial nerve function after repair.

Within a recent study on the recovery of vibrissae motor performance after facial nerve repair in blind (strain SD/RCS) and sighted (strain SD) rats, we found that, despite persisting myotopic disorganization in the facial nucleus, the blind animals fully restored vibrissal whisking. Searching for the morphological substrates of this improved recovery, we compared the amount of cytoskeletal proteins in the leading edge of elongating axons between both strains. Since our results showed an enhanced expression of neuronal class III beta-tubulin in the blind rats, we wondered whether this was due to an increased synthesis or to a delayed turnover of microtubules. In the present report, we approached this question applying established pharmacological agents to the transected buccal branch of the facial nerve in sighted Wistar rats perturbing either microtubule assembly towards stabilization (enhanced polymerization with 10 microg/ml taxol) or towards increased synthesis (challenged by destabilization with 100 microg/ml nocodazole and 20 microg/ml vinblastine). Evaluation of the effect(s) 2 months later included estimation of (i) vibrissae motor performance by video-based motion analysis, (ii) the degree of collateral axonal branching by double retrograde neuronal labeling with crystals of Fluoro-Gold and DiI and (iii) the pattern of motor end-plate reinnervation (proportions of mono- and poly-reinnervated) in the largest extrinsic vibrissal muscle, the m. levator labii superioris. We found that only stabilization of microtubules with 10 microg/ml taxol reduced intramuscular axonal sprouting and polyinnervation of the motor end-plates, which was accompanied by improved restoration of function.

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.