Matrix metalloproteinase 3 deletion preserves denervated motor endplates after traumatic nerve injury.

OBJECTIVE: Traumatic peripheral nerve injuries often produce permanent functional deficits despite optimal surgical and medical management. One reason for the impaired target organ reinnervation is degradation of motor endplates during prolonged denervation. Here we investigate the effect of preserving agrin on the stability of denervated endplates. Because matrix metalloproteinase 3 (MMP3) is known to degrade agrin, we examined the changes in endplate structure following traumatic nerve injury in MMP3 knockout mice. METHODS: After creation of a critical size nerve defect to preclude reinnervation, we characterized receptor area, receptor density, and endplate morphology in denervated plantaris muscles in wild-type and MMP3 null mice. The level of agrin and muscle-specific kinase (MuSK) was assessed at denervated endplates. In addition, denervated muscles were subjected to ex vivo stimulation with acetylcholine. Finally, reinnervation potential was compared after long-term denervation. RESULTS: In wild-type mice, the endplates demonstrated time-dependent decreases in area and receptor density and conversion to an immature receptor phenotype. In striking contrast, all denervation-induced changes were attenuated in MMP3 null mice, with endplates retaining their differentiated form. Agrin and MuSK were preserved in endplates from denervated MMP3 null animals. Furthermore, denervated muscles from MMP3 null mice demonstrated greater endplate efficacy and reinnervation. INTERPRETATION: These results demonstrate a critical role for MMP3 in motor endplate remodeling, and reveal a potential target for therapeutic intervention to prevent motor endplate degradation following nerve injury.

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy.

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.

Synaptic basal lamina-associated congenital myasthenic syndromes.

Proteins associated with the basal lamina (BL) participate in complex signal transduction processes that are essential for the development and maintenance of the neuromuscular junction (NMJ). Most important junctional BL proteins are collagens, such as collagen IV (α3-6), collagen XIII, and ColQ; laminins; nidogens; and heparan sulfate proteoglycans, such as perlecan and agrin. Mice lacking Colq (Colq(-/-)), laminin ß2 (Lamb2(-/-)), or collagen XIII (Col13a1(-/-)) show immature nerve terminals enwrapped by Schwann cell projections that invaginate into the synaptic cleft and decrease contact surface for neurotransmission. Human mutations in COLQ, LAMB2, and AGRN cause congenital myasthenic syndromes (CMSs) owing to deficiency of ColQ, laminin-ß2, and agrin, respectively. In these syndromes the NMJ ultrastructure shows striking resemblance to that of mice lacking the corresponding protein; furthermore, the extracellular localization of mutant proteins may provide favorable conditions for replacement strategies based on gene therapy and stem cells.

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.

Mitochondria in motor nerve terminals: function in health and in mutant superoxide dismutase 1 mouse models of familial ALS.

Mitochondria contribute to neuronal function not only via their ability to generate ATP, but also via their ability to buffer large Ca(2+) loads. This review summarizes evidence that mitochondrial Ca(2+) sequestration is especially important for sustaining the function of vertebrate motor nerve terminals during repetitive stimulation. Motor terminal mitochondria can sequester large amounts of Ca(2+) because they have mechanisms for limiting both the mitochondrial depolarization and the increase in matrix free [Ca(2+)] associated with Ca(2+) influx. In mice expressing mutations of human superoxide dismutase -1 (SOD1) that cause some cases of familial amyotrophic lateral sclerosis (fALS), motor terminals degenerate well before the death of motor neuron cell bodies. This review presents evidence for early and progressive mitochondrial dysfunction in motor terminals of mutant SOD1 mice (G93A, G85R). This dysfunction would impair mitochondrial ability to sequester stimulation-associated Ca(2+) loads, and thus likely contributes to the early degeneration of motor terminals.

Beneficial effects of albuterol in congenital endplate acetylcholinesterase deficiency and Dok-7 myasthenia.

INTRODUCTION: Congenital myasthenic syndromes (CMS) are disabling but treatable disorders. Anticholinesterase therapy is effective in most of them, but is contraindicated in endplate (EP) acetylcholinesterase (AChE) deficiency, the slow-channel syndrome, Dok-7 myasthenia, and ß(2) -laminin deficiency, and is not useful in CMS due to defects in muscle-specific kinase (MuSK), agrin, and plectin. EP AChE, Dok-7, and ß(2)-laminin deficiencies respond favorably to ephedrine, but ephedrine can no longer be prescribed in the USA. METHODS: We used albuterol, another sympathomimetic agent, to treat 3 patients with EP AChE deficiency and 15 with Dok-7 myasthenia. Response to therapy was evaluated by a 9-point questionnaire pertaining to activities of daily life. RESULTS: Comparison of the pre- and posttreatment responses indicated a beneficial response to albuterol (P < 0.001) in both patient groups. The adverse effects of therapy were like those of ephedrine. CONCLUSION: Our observations should spur controlled, prospective clinical trials of albuterol in these as well as other CMS.

Myosin heavy chain isoform profiles remain altered at 7 months if the lacerated medial gastrocnemius is poorly reinnervated: a study in rabbits.

Lacerated skeletal muscles often do not recover full function after repair. Denervated muscles with altered myosin heavy chain isoform (MHC) profiles are known to result in functional impairment. We studied the functional recovery of lacerated muscles, assessing MHC profile changes in association to the involvement of the intramuscular nerve (IM). We tested three lacerated models using the rabbit's medial gastrocnemius where the IM was either cut (NNR), repaired (NR), or preserved intact (NP). Muscles were assessed 7 months after repair for muscle atrophy, isometric contraction (by electrical stimulation), and fibrosis formation at the lesion site. Changes in myofibrillar actomyosin adenosine triphosphatase activity, MHC profile, regenerating myofibers and reinnervation were assessed by Western blot, histology, or immunohistology. Lacerated muscles with a repaired (NR) or an intact (NP) IM showed good recovery, with no significant changes in the MHC profile. Muscles where the IM was not repaired (NNR) resulted in significant scar area at the lesion site (p < 0.05), muscle atrophy (67%, p < 0.05) and loss in contractile properties (63% of the uninjured side, p < 0.05). At 7 months, all muscles were reinnervated. However, the NNR had an inappropriate (polyneural) and poorly distributed reinnervation, the presence of regenerating myofibers, and demonstrated a fast-to-slow MHC transition (71%:29% to 44%:56%, ANOVA, p = 0.018). This was associated to the cut IM when the NNR muscle was lacerated. Poor reinnervation in lacerated skeletal muscles alters the myosin heavy chain profile permanently. This study provides a rationale to also consider biological solutions to improve nerve regeneration and reinnervation in the surgical repair of lacerated muscles.

Evidence of a dosage effect and a physiological endplate acetylcholinesterase deficiency in the first mouse models mimicking Schwartz-Jampel syndrome neuromyotonia.

Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.

Protein kinase C activity affects neurotransmitter release at polyinnervated neuromuscular synapses.

By using intracellular recording, we studied how protein kinase C (PKC) activity affected transmitter release in singly and dually innervated endplates of the Levator auris longus muscle of 5-6-day-old rats during axonal competition in the postnatal synaptic elimination period. In dually innervated fibers, a second endplate potential (EPP) may appear after the first one when the stimulation intensity is increased. The nerve terminals that generate the lowest and the highest EPP amplitudes are designated "small-EPP generating ending" (SEGE) and "large-EPP generating ending" (LEGE), respectively. Blocking PKC with calphostin C, staurosporine, or chelerythrine results in an increased release from SEGE ( approximately 80%), whereas release from LEGE and from endings generating only one EPP (OEGE) is not significantly affected. Blocking PKC also leads to the recruitment of silent synapses (acetylcholine cannot be released before PKC inhibition). The mean number of functional axon terminals per synapse increases by approximately 47%, and these are now designated the "recruited-EPP generating endings" (REGE). This suggests that axonal PKC can modulate postnatal synaptic elimination by favoring the nerve terminal disconnection of certain weak axonal endings (REGE and SEGE). We conclude that a PKC-mediated mechanism should occupy a pivotal place in neonatal synapse elimination, because functional axonal withdrawal can indeed be turned back by PKC block.

Prenatal alcohol exposure and early postnatal changes in the developing nerve-muscle system.

BACKGROUND: Extensive research on prenatal alcohol exposure has proven the potent teratogenicity of this substance of abuse. Children born to alcoholic mothers are often diagnosed with fetal alcohol syndrome (FAS). Those afflicted with FAS often have muscle weakness, muscle wasting, and atrophy. This study assessed the effects of prenatal alcohol exposure on the developing rat neuromuscular system. METHODS: Pregnant Sprague-Dawley rats were injected intraperitoneally with 1.0 ml of 20% ethyl alcohol/100 gm body weight. Unexposed rats served as controls. The offspring were killed 2, 3, 4, and 5 weeks after birth, and their body weights were recorded. The tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were recovered and weighed. The TA muscles were histochemically stained by silver cholinesterase in order to study the pattern of innervation. The EDL muscles were processed and stained by hematoxylin-eosin. The number and size of the EDL muscle fibers was quantified. The sciatic nerve was also removed and stained by Swank and Davenport's method to demonstrate the myelin pattern. RESULTS: Assessment at the neuromuscular junction showed a higher proportion of endplates polyneuronally innervated in the alcohol-exposed rats. The muscle weights, as well as the number and size of the muscle fibers, were significantly reduced in these animals. A light-microscopy examination of the nerve sections revealed alterations in the connectivity of myelin. CONCLUSIONS: The finding that a higher proportion of endplates were polyneuronally innervated in the alcohol-exposed rats indicates that the maturation process of the neuromuscular system was delayed, thus confirming the deleterious effects of alcohol on growth and maturation of the nerve-muscle system.

Partial nicotinic receptor blockade unmasks a modulatory role of nitric oxide on urethral striated neuromuscular transmission.

The objective of this study was to investigate the possible modulatory role of endogenous nitric oxide (NO) production on the urethral striated muscle (USM) function in the sheep urethra. Significant NO synthase (NOS) activity was measured in both the particulate and cytosolic fractions of USM homogenates. NOS activity was calcium-dependent and showed greater inhibition by NOS inhibitors selective of the neural NOS isoform (nNOS). nNOS immunoreactivity was present in intramural nerves as well as in the sarcolemma of some striated fibers, being denser at the neuromuscular junction (NMJ). Double immunolabeling showed co-localization of nNOS with both alpha-bungarotoxin and choline acetyltransferase, at the USM endplates. For the first time, functional data support a role of NO on the USM contractility "in vitro," which became evident following partial nicotinic receptor inactivation with low concentrations of D-tubocurarine. Only under D-tubocurarine (0.25 microM) treatment, different NOS inhibitors, specially N(G)-propyl-L-arginine, as well as the guanylate cyclase inhibitor ODQ, all showed a significant enhancing effect on contractions induced by electrical field stimulation of intrinsic somatic nerves. These data suggest that local production of NO at the urethral NMJ may modulate release and/or action of acetylcholine on motor endplates by cyclic GMP-mediated effects. This modulatory action could be especially relevant when neuromuscular transmission at the USM is impaired.

Neuromuscular organization of the superior longitudinalis muscle in the human tongue. 1. Motor endplate morphology and muscle fiber architecture.

Proper tongue function is essential for respiration and mastication, yet we lack basic information on the anatomical organization underlying human tongue movement. Here we use microdissection, acetylcholinesterase histochemistry, silver staining of nerves, alpha bungarotoxin binding and immunohistochemistry to describe muscle fiber architecture and motor endplate (MEP) distribution of the human superior longitudinalis muscle (SL). The human SL extends from tongue base to tongue tip and is composed of fiber bundles that range from 2.8 to 15.7 mm in length. Individual muscle fibers of the SL range from 1.2 to 17.3 mm in length (1.3-18.2% of muscle length). Seventy-one percent of SL fibers have blunt-blunt terminations; the remainder have blunt-taper terminations. Multiple MEPs are present along SL length and dual MEPs are present on some muscle fibers. These data demonstrate that the human SL is a muscle of "in-series" design. We suggest that SL motor units are organized to innervate specific regions of the tongue body and that activation of SL motor units according to anteroposterior location is one strategy employed by the nervous system to control tongue shape and tongue movement.

Pulsed electromagnetic fields induce peripheral nerve regeneration and endplate enzymatic changes.

An experimental study was carried out in rats with the purpose of demonstrating the capacity of pulsed electromagnetic fields (PEMFs) to stimulate regeneration of the peripheral nervous system (PNS). Wistar and Brown Norway (BN) rats were used. Direct sciatic nerve anastomoses were performed after section or allograft interposition. Treatment groups then received 4 weeks of PEMFs. Control groups received no stimulation. The evaluation of the results was carried out by quantitative morphometric analysis, demonstrating a statistically significant increase in regeneration indices (P < 0.05) in the stimulated groups (9000 +/- 5000 and 4000 +/- 6000) compared to the non-stimulated groups (2000 +/- 4000 and 700 +/- 200). An increase of NAD specific isocitrate dehydrogenase (IDH) activity was found along with an increase in the activity of acetyl cholinesterase at the motor plate. The present study might lead to the search for new alternatives in the stimulation of axonal regenerative processes in the PNS and other possible clinical applications.

Novel assay utilizing fluorochrome-tagged physostigmine (Ph-F) to in situ detect active acetylcholinesterase (AChE) induced during apoptosis.

It was recently reported that acetylcholinesterase (AChE) is expressed in cells undergoing apoptosis and that its presence is essential for assembly of the apoptosome and subsequent caspase-9 activation. To obtain a marker of active AChE that could assay this enzyme in live intact cells and be applicable to fluorescence microscopy and cytometry, the fluorescein-tagged physostigmine (Ph-F), high affinity ligand (inhibitor) reactive with the active center of AChE, was constructed and tested for its ability to in situ label AChE and measure its induction during apoptosis. Ph-F inhibited cholinesterase activity in vitro (IC50 = 10(-6) and 5 x 10(-6) M for equine butyrylcholinesterase and human erythrocyte AChE, respectively) and was a selective marker of cells and structures that were AChE-positive. Thus, exposure of mouse bone marrow cells to Ph-F resulted in the exclusive labeling of megakaryocytes, and of the diaphragm muscle, preferential labeling of the nerve-muscle junctions (end-plates). During apoptosis of carcinoma HeLa cells and leukemic HL-60 or Jurkat cells triggered either by the DNA topoisomerase 1 inhibitor topotecan (TPT) or by oxidative stress (H2O2), the cells become reactive with Ph-F. Their Ph-F derived fluorescence was measured by flow and laser scanning cytometry. The appearance of Ph-F binding sites during apoptosis was preceded by the loss of mitochondrial potential, was concurrent with the presence of activated caspases, and was followed by loss of membrane integrity. At a very early stage of apoptosis, when nucleolar segregation was apparent, the Ph-F binding sites were distinctly localized within the nucleolus and at later stages of apoptosis in the cytoplasm. During apoptosis triggered by TPT, Ph-F binding was preferentially induced in S-phase cells. Our data on megakaryocytes and end-plates indicate that Ph-F reacts with active sites of AChE, and can be used to reveal the presence of this enzyme in live cells and possibly to study its expression in disorders of the neurological cholinergic system. The findings are also compatible with the reports that AChE may be induced during apoptosis. In fact, the simple and rapid Ph-F binding assay may serve as a convenient marker of apoptotic cells. However, the proposed role of active AChE as an essential factor for assembly of the apoptosome and caspase activation is in question because the AChE inhibitors Ph, Ph-F and BW284c51 did not protect the cells from apoptosis induced by TPT or H2O2. Further studies are thus needed to ascertain the induction and role of AChE in apoptosis.

Increased quantal size in transmission at slow but not fast neuromuscular synapses of apolipoprotein E deficient mice.

Uncertainties from the literature concerning the role of apolipoprotein E (apoE) in central cholinergic function prompted us to investigate what effect apoE may have on transmission at the neuromuscular junction. Both spontaneous and evoked release were measured in isolated extensor digitorum longus (edl) and soleus muscles from both wild-type and apoE-deficient mice. Miniature endplate and nerve-evoked endplate potentials (MEPPs and EPPs, respectively) were indistinguishable in edl muscles in both groups of mice; however, MEPP amplitudes in soleus muscles were significantly larger (by an average of 23%) in apoE-deficient mice compared with 5- to 7-week-old age-matched wild-type mice. The EPP amplitudes were also larger in soleus muscles in the mutant mice, but this was a reflection of the larger quantal size in this muscle because quantal content, determined from the ratio of the average EPP amplitude to average MEPP amplitude, was unchanged from normal in the mutant mice. The MEPP frequency and the percent of nerve stimulations failing to produce an EPP were unchanged from normal in both muscle types in the mutant mice. The difference in quantal size in soleus muscle transmission between mutant and wild-type mice was abolished in the presence of neostigmine, an acetylcholinesterase inhibitor. The results suggest that apoE normally associates with acetylcholinesterase in the synaptic cleft of slow muscles, modulating the activity of the enzyme and therefore quantal size.

Comparison of changes in AChE activity in the brain of the laboratory rat after soman and tabun intoxication.

The aim of this study was to compare changes in activity of acetylcholinesterase (AChE) in the brain and motor endplates of rat after administration of soman and tabun. We took brain and diaphragm from laboratory rats administered a median lethal dose (LD(50)) of soman or tabun. Enzyme activity of AChE was studied in selected structures of brain and in motor endplates in the diaphragm. Histochemical detection of AChE by Karnovski and Roots with simultaneous histochemical detection of alkaline phosphatase in case of brain sections was used. The highest activity of AChE in the control group was found in the striatum, amygdaloid nuclei, substantia nigra, superior colliculi, and motor nuclei of cranial nerves V, X a XII. LD(50) of both nerve agents dramatically decreased the activity of AChE in the structures studied--both brain and diaphragm. After intoxication by either agent, activity in above mentioned nuclei was characterized as low or focally moderate. Very low activity was seen in some structures (CA3 field of hippocampus, some nuclei of the tegmentum and cerebellar cortex). We found minimal differences in the histochemical picture of soman or tabun intoxication, apart from the striatum and the superior colliculi which showed stronger inhibition by tabun.

Presynaptic failure of neuromuscular transmission and synaptic remodeling in EA2.

OBJECTIVE: To further investigate the basis of abnormal neuromuscular transmission in two patients with congenital myasthenic syndrome associated with episodic ataxia type 2 (EA2) using stimulated single fiber EMG (SFEMG) and in vitro microelectrode studies. METHODS: Two patients with genetically characterized EA2 previously shown to have abnormal neuromuscular transmission by voluntary SFEMG were studied with stimulated SFEMG and anconeus muscle biopsy with microelectrode studies and electron microscopy of the neuromuscular junction. RESULTS: In vivo stimulated SFEMG showed signs of presynaptic failure, with jitter and blocking that improved with increased stimulation frequency. Additional evidence of presynaptic failure was provided by the in vitro microelectrode studies, which showed marked reduction of the end plate potential quantal content in both patients. Of note, the end plate potentials showed high sensitivity to N-type blockade with omega-conotoxin not seen in controls. The ultrastructural studies revealed some evidence of small nerve terminals apposed to normal or mildly overdeveloped postsynaptic membranes, suggesting an ongoing degenerative process. CONCLUSIONS: The authors demonstrated presynaptic failure of neurotransmission in patients with heterozygous nonsense mutations in CACNA1A. The contribution of non-P-type calcium channels to the process of neurotransmitter release in these patients likely represents a compensatory mechanism, which is insufficient to restore normal neuromuscular transmission.

Two novel mutations in the COLQ gene cause endplate acetylcholinesterase deficiency.

Congenital myasthenic syndromes with endplate acetylcholinesterase deficiency are very rare autosomal recessive diseases, characterized by onset of the disease in childhood, general weakness increased by exertion, ophthalmoplegia and refractoriness to anticholinesterase drugs. To date, all reported cases are due to mutations within the gene encoding ColQ, a specific collagen that anchors acetylcholinesterase in the basal lamina at the neuromuscular junction. We identified two new cases of congenital myasthenic syndromes with endplate acetylcholinesterase deficiency. The two patients showed different phenotypes. The first patient had mild symptoms in childhood, which worsened at 46 years with severe respiratory insufficiency. The second patient had severe symptoms from birth but improved during adolescence. In both cases, the absence of acetylcholinesterase was demonstrated by morphological and biochemical analyses, and heteroallelic mutations in the COLQ gene were found. Both patients presented a novel splicing mutation (IVS1-1G-->A) affecting the exon encoding the proline-rich attachment domain (PRAD), which interacts with acetylcholinesterase. This splicing mutation was associated with two different mutations, both of which cause truncation of the collagen domain (a known 788insC mutation belonging to one patient and a novel R236X to the other) and may impair its trimeric organization. The close similarity of the mutations of these two patients with different phenotypes suggests that other factors may modify the severity of this disease.