Amplitude Of Endplate Potentials Research Articles

Effect of 2-Nitropropane on Chemical Neurotransmission, Spontaneous and Evoked, in the Sartorius Muscle of the Frog

n this project we studied the effect of 2-nitropropane in spontaneous and evoked chemical neurotransmission, using as a model the frog neuromuscular junction, in vitro. The experiments were carried out in the sciatic nerve- sartorius muscle preparation. Our results show that neurotransmission was inhibited, manifesting as a decrease in the amplitude and instantaneous frequency of miniature end-plate potentials and the reduction of end-plate potentials amplitude of the neuromuscular junction, evoked by nerve stimulation. Because 2-nitropropane showed inhibitory effects at peripheral level, we recommend that in work environments where there is exposure to organic solvents, neurological clinical examinations must be performed to detect abnormalities or neurological damage the earlie

Neuromuscular actions of edrophonium in the lateral segmental tail muscle of the rat in vivo

The actions of edrophonium on neuromuscular transmission in vivo have been studied using the lateral segmental tail muscles of the rat. The drug produced an increase in amplitude of the miniature endplate potentials (m.e.p.p.s) without an effect on their frequency and increased the amplitude of the end-plate potentials (e.p.p.s) without increasing quantal release of transmitter. It is concluded that the anticholinesterase action of this drug in vivo facilitates neuromuscular transmission, which confirms many of the findings from in vitro studies.

A selective inhibitor of cAMP-specific phosphodiesterase, Ro 20-1724, has no effect on the quantal release of acetylcholine from the mouse phrenic nerve.

The indirect twitch response of the mouse isolated phrenic nerve-diaphragm preparation, partially paralysed by tubocurarine, was restored only by about 10% by Ro 20-1724 at 2 to 280 microM. The solvent vehicle, dimethylsulphoxide, also showed the same effect to a similar extent. Intracellular recordings with glass microelectrodes revealed that Ro 20-1724 (40 microM) affected neither the resting membrane potential, the amplitude and frequency of miniature endplate potentials nor the amplitude of nerve-impulse evoked endplate potentials recorded in curarized preparations. The result indicates that Ro 20-1724 at a concentration four times the IC50 of phosphodiesterase inhibition has no effect on the quantal release of acetylcholine from a mammalian motor nerve and suggests that cAMP has no modulatory effect on the transmitter release.

Extraneuronal toxicity of Alzheimer's β‐amyloid peptide: Comparative study on vertebrate skeletal muscles

Alzheimer's β-amyloid peptide (βAP) is known to possess a wide range of toxic effects on neurons in vitro and in vivo; however, there is little information available regarding its impact on other excitable tissues such as skeletal muscles, which, apart from brain cells, are thought to also be targets of βAP. Utilizing the combination of electrophysiology and myography, we investigated whether βAP also impairs the functioning of myocytes in frogs and mice. Although application of βAP in the range of 10(-6) to 10(-8) M induced depolarization of muscle fibers in both species, it impaired contractility in frogs but not in mice, by reducing endplate potential amplitude and increasing the threshold potential. Unchanged contractility in the mouse in the presence of βAP is due to a higher safety factor of neuromuscular transmission in mammals compared with amphibians. Possible clinical implications are discussed.

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.

Complexin Facilitates Coupling of Secretory Vesicles and Voltage-Gated Calcium Channels

The SNARE complex mediates fusion of vesicle and plasma membranes in exocytosis. Complexin (Cplx) binds with high affinity and 1:1 stoichiometry to the SNARE complex and is believed to regulate SNARE function. However, how Cplx functions is still controversial. Using mouse neuromuscular junctions (NMJs) and adrenal chromaffin cells, we tested the hypothesis that Cplx, in addition to enlarging the pool of primed vesicles, enhances exocytosis and neurotransmission by increasing the coupling between vesicles and voltage-gated calcium channels (VGCC). Only Cplx 1 is expressed at adult mouse NMJs. Cplx 1 knockout NMJs displayed not only a diminished endplate potential (EPP) amplitude, but also a loss of synchronicity (jitter). Furthermore, whereas in response to high-frequency stimulation, wildtype NMJs displayed marked depression of EPP size upon successive stimulations, knockout NMJs showed facilitation. These observations suggest that in Cplx knockout NMJs, vesicles and calcium channels are uncoupled. To further test this hypothesis, we recorded capacitance measurements from mouse adrenal chromaffin cells. We used short depolarizations to elicit fusion of vesicles close to the calcium channels (IRP) and longer depolarizations to elicit fusion of the remaining primed vesicles (RRP). Cplx 2 KO chromaffin cells have a significantly smaller IRP/RRP ratio, and we can rescue the phenotype by heterologous expression of complexin in KO cells. Our work in two separate model systems supports a role for complexin in coupling vesicles to VGCC.

Myasthenic syndrome caused by plectinopathy

Plectin crosslinks intermediate filaments to their targets in different tissues. Defects in plectin cause epidermolysis bullosa simplex (EBS), muscular dystrophy (MD), and sometimes pyloric atresia. Association of EBS with a myasthenic syndrome (MyS) was documented in a single patient in 1999. To analyze the clinical, structural, and genetic aspects of a second and fatal case of EBS associated with a MyS and search for the genetic basis of the disease in a previously reported patient with EBS-MD-MyS. Clinical observations; histochemical, immunocytochemical, and electron microscopy studies of skeletal muscle and neuromuscular junction; and mutation analysis. An African American man had EBS since early infancy, and progressive muscle weakness, hyperCKemia, and myasthenic symptoms refractory to therapy since age 3 years. Eventually he became motionless and died at age 42 years. At age 15 years, he had a marked EMG decrement, and a reduced miniature endplate potential amplitude. The myopathy was associated with dislocated muscle fiber organelles, structurally abnormal nuclei, focal plasmalemmal defects, and focal calcium ingress into muscle fibers. The neuromuscular junctions showed destruction of the junctional folds, and remodeling. Mutation analysis demonstrated a known p.Arg2319X and a novel c.12043dupG mutation in PLEC1. The EBS-MD-MyS patient reported in 1999 also carried c.12043dupG and a novel p.Gln2057X mutation. The novel mutations were absent in 200 Caucasian and 100 African American subjects. The MyS in plectinopathy is attributed to destruction of the junctional folds and the myopathy to defective anchoring of muscle fiber organelles and defects in sarcolemmal integrity.

Congenital myasthenic syndrome associated with epidermolysis bullosa caused by homozygous mutations in PLEC1 and CHRNE

Mutations in the plectin gene (PLEC1) cause epidermolysis bullosa simplex (EBS), which may associate with muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA). The association of EBS with congenital myasthenic syndrome (CMS) is also suspected to result from PLEC1 mutations. We report here a consanguineous patient with EBS and CMS for whom mutational analysis of PLEC1 revealed a homozygous 36 nucleotide insertion (1506_1507ins36) that results in a reduced expression of PLEC1 mRNA and plectin in the patient muscle. In addition, mutational analysis of CHRNE revealed a homozygous 1293insG, which is a well-known low-expressor receptor mutation. A skin biopsy revealed signs of EBS, and an anconeus muscle biopsy showed signs of a mild myopathy. Endplate studies showed fragmentation of endplates, postsynaptic simplification, and large collections of thread-like mitochondria. Amplitudes of miniature endplate potentials were diminished, but the endplate quantal content was actually increased. The complex phenotype presented here results from mutations in two separate genes. While the skin manifestations are because of the PLEC1 mutation, footprints of mutations in PLEC1 and CHRNE are present at the neuromuscular junction of the patient indicating that abnormalities in both genes contribute to the CMS phenotype.

Quantal release of acetylcholine in mice with reduced levels of the vesicular acetylcholine transporter

Mammalian motor nerve terminals contain hundreds of thousands of synaptic vesicles, but only a fraction of these vesicles is immediately available for release, the remainder forming a reserve pool. The supply of vesicles is replenished through endocytosis, and newly formed vesicles are refilled with acetylcholine through a process that depends on the vesicular acetylcholine transporter (VAChT). During expression of short-term plasticity, quantal release can be increased, but it is unknown whether this reflects enhanced recruitment of vesicles from the reserve pool or rapid recycling. We examined spontaneous and evoked release of acetylcholine at endplates from genetically modified VAChT KD(HOM) mice that express approximately 30% of the normal level of VAChT to determine steps rate-limited by synaptic vesicle filling. Quantal content and quantal size were reduced in VAChT KD(HOM) mice compared with wild-type controls. Although high-frequency stimulation did not reduce quantal size further, the post-tetanic increase in end-plate potential amplitude or MEPP frequency was significantly smaller in VAChT KD(HOM) mice. This was the case even when tetanic depression was eliminated using an extracellular solution containing reduced Ca(2+) and raised Mg(2+). These results reveal the dependence of short-term plasticity on the level of VAChT expression and efficient synaptic vesicle filling.

Mutations in MUSK causing congenital myasthenic syndrome impair MuSK–Dok-7 interaction

We describe a severe congenital myasthenic syndrome (CMS) caused by two missense mutations in the gene encoding the muscle specific receptor tyrosine kinase (MUSK). The identified MUSK mutations M605I and A727V are both located in the kinase domain of MuSK. Intracellular microelectrode recordings and microscopy studies of the neuromuscular junction conducted in an anconeus muscle biopsy revealed decreased miniature endplate potential amplitudes, reduced endplate size and simplification of secondary synaptic folds, which were consistent with postsynaptic deficit. The study also showed a striking reduction of the endplate potential quantal content, consistent with additional presynaptic failure. Expression studies in MuSK deficient myotubes revealed that A727V, which is located within the catalytic loop of the enzyme, caused severe impairment of agrin-dependent MuSK phosphorylation, aggregation of acetylcholine receptors (AChRs) and interaction of MuSK with Dok-7, an essential intracellular binding protein of MuSK. In contrast, M605I, resulted in only moderate impairment of agrin-dependent MuSK phosphorylation, aggregation of AChRs and interaction of MuSK with Dok-7. There was no impairment of interaction of mutants with either the low-density lipoprotein receptor-related protein, Lrp4 (a co-receptor of agrin) or with the mammalian homolog of the Drosophila tumorous imaginal discs (Tid1). Our findings demonstrate that missense mutations in MUSK can result in a severe form of CMS and indicate that the inability of MuSK mutants to interact with Dok-7, but not with Lrp4 or Tid1, is a major determinant of the pathogenesis of the CMS caused by MUSK mutations.

Frontoxins, three-finger toxins from Micrurus frontalis venom, decrease miniature endplate potential amplitude at frog neuromuscular junction

Neurotoxicity is a major symptom of envenomation caused by Brazilian coral snake Micrurus frontalis. Due to the small amount of material that can be collected, no neurotoxin has been fully sequenced from this venom. In this work we report six new three-finger like toxins isolated from the venom of the coral snake M. frontalis which we named Frontoxin (FTx) I–VI. Toxins were purified using multiple steps of RP-HPLC. Molecular masses were determined by MALDI-TOF and ESI ion-trap mass spectrometry. The complete amino acid sequence of FTx II, III, IV and V were determined by sequencing of overlapping proteolytic fragments by Edman degradation and by de novo sequencing. The amino acid sequences of FTx I, II, III and VI predict 4 conserved disulphide bonds and structural similarity to previously reported short-chain α-neurotoxins. FTx IV and V each contained 10 conserved cysteines and share high similarity with long-chain α-neurotoxins. At the frog neuromuscular junction FTx II, III and IV reduced miniature endplate potential amplitudes in a time-and concentration-dependent manner suggesting Frontoxins block nicotinic acetylcholine receptors.

Effects of sepsis on the neuromuscular blocking actions of d-tubocurarine on rat adductor and abductor laryngeal muscles

We evaluated the effects of sepsis on the neuromuscular blocking actions of d-tubocurarine (dTc) in the lateral cricoarytenoid (LCA) and posterior cricoarytenoid (PCA) muscles, an adductor muscle and an abductor muscle of the vocal cords, respectively, in vitro. Sepsis was induced in rats by cecal ligation and puncture (CLP) to elicit panperitonitis. Electromyograms (EMGs) and endplate potentials (EPPs) were recorded from the LCA and PCA muscles of CLP-operated septic rats and sham-operated nonseptic rats, using extracellular and intracellular microelectrodes, respectively. EMG and EPP (amplitude and quantum content) were depressed by dTc, but the dTc-induced neuromuscular blocking effects were attenuated by sepsis. The suppressive effects of dTc on EMG and EPP (amplitude and quantum content) were less intense in the LCA muscle than in the PCA muscle under both sepsis and nonsepsis conditions. Our study shows that sepsis has a depressive effect on dTc-induced neuromuscular blocking actions at both the adductor and abductor muscles of vocal cords in the larynx.

Suppression of mediator secretion in murine neogenic motor synapses with the participation of L-type Ca2+-channels and ryanodine receptors

L-type Ca(2+)-channel blockers, verapamil (5 microM) and nifedipine (10 microM), have increased the quantum composition of endplate potentials (EPP) and the level of induced rhythmic activity of neogenic synapses. L-type Ca(2+)-channel activator BAY 8644 (1 microM) has a decreased mediator secretion level. Nifedipine (10 microM) has not changed the frequency and amplitude of diminutive EPPs in the dormant state or during potassium depolarization. Blocking of the prejunctional ryanodine receptor with ryanodine (10 microM) led to an increase in the single EPP quantum composition that was qualitatively similar to nifedipine and verapamil, but more marked, and also caused the reinforcement of mediator release during the rhythmic EPP salvo. Ryanodine receptor activation with ryanodine (1 microM) resulted in reduction of the quantum composition of single and rhythmic EPPs. This effect was partially prevented with nifedipine (10 microM).

Neuromuscular activity of BaTX, a presynaptic basic PLA 2 isolated from Bothrops alternatus snake venom

We have previously isolated a Lys49 phospholipase A 2 homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007–1.4 µM) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 µM toxin: 58 ± 4 and 24 ± 1 min, respectively; n = 3–8; mean ± S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 µM) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59 ± 4%, n = 12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 µM). In addition, a decrease in the organ bath temperature from 37 °C to 24 °C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity.

Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome

Background:We describe a severe form of congenital myasthenic syndrome (CMS) associated with congenital nephrosis and ocular malformations caused by two truncating mutations in the gene encoding the laminin β2 subunit...

The vesicle cycle in motor nerve endings of the mouse diaphragm

Experiments on the mouse diaphragm muscle using intracellular microelectrode recordings and fluorescence microscopy were performed to study the dynamics of transmitter secretion and synaptic vesicle recycling processes (the exocytosis-endocytosis cycle) in motor nerve endings (NE) during prolonged rhythmic stimulation (20 impulses/sec). During stimulation, there were triphasic changes in the amplitude of endplate potentials (EPP): an initial rapid reduction, followed by prolonged (1-2 min) stabilization of amplitude, i.e., a plateau, and then a further slow decrease. Restoration of EPP amplitude after stimulation for 3 min occurred over a period of several seconds. Loading of synaptic vesicles with the fluorescent endocytic stain FM1-43 showed that rhythmic stimulation led to a gradual (over 5-6 min) decrease in NE fluorescence, demonstrating exocytosis of synaptic vesicles. Quantum analysis of the electrophysiological data and comparison of these data with results from fluorescence studies suggested that mouse NE have a high rate of endocytosis and reutilization of synaptic vesicles (the mean recycling time was about 50 sec), which may support the maintenance of reliable synaptic transmission during prolonged high-frequency activity. The sizes of the release-ready and recycling pools of synaptic vesicles were determined quantitatively. It is suggested that vesicle recycling in mouse NE occurs via a short, rapid pathway with incorporation into the recycling pool. Vesicles of the reserve pool are not used for transmitter secretion in the stimulation conditions used here.

CORRELATION BETWEEN SINGLE FIBRE EMG JITTER AND ENDPLATE POTENTIALS STUDIED IN MILD EXPERIMENTAL BOTULINUM POISONING

To study the correlation between single fibre EMG jitter and endplate potential amplitude we examined neuromuscular transmission in rats paralysed with botulinum toxin type A with single fibre EMG (SFEMG) in vivo and with intracellular microelectrode techniques in vitro. In muscles that were not completely paralysed SFEMG showed an increased neuromuscular jitter on nerve stimulation and in these muscles endplate potentials were of reduced amplitude. When the nerve stimulation frequency increased the jitter decreased. Intravenous injection of drugs that increase the acetylcholine release and endplate potential amplitude markedly reduced the jitter and the frequency dependence disappeared.

Effects of Vitamin B1 Antagonists on Synaptic Transmission in a Striated Muscle of the Mouse

We studied the effects of antagonists of vitamin В 1 , pyrithiamine and oxythiamine, on neuromuscular transmission in the diaphragmatic muscle of the mouse. In isolated phrenico-hemidiaphragmatic preparations obtained from animals i.p. injected with 100 mg/kg pyrithiamine 1.5 h earlier, the amplitudes of miniature end-plate potentials (mEPPs) and evoked end-plate potentials (EPPs), as well as an estimate of the quantum content of EPP, were significantly smaller than in the control. In similar preparations examined 3 or 24 h after subcutaneous injections of 400 mg/kg oxythiamine, the amplitude of EPPs and their quantum content were also smaller than in the control. Such an effect was not found 72 h after injection of oxythiamine.

Effect of Vitamin В1 Alimentary Deficiency on Spontaneous and Evoked Transmitter Release in Murine Neuromuscular Synapses

Using a microelectrode technique, we studied the effects of alimentary vitamin В 1 deficiency on synaptic transmission in isolated phrenico-hemidiaphragmatic murine preparations. Animals of group І (control) were on a standard thiamine-controlled diet (16 mg/kg thiamine) with no limitations. Animals of group II (control with alimentary limitation) were on the same diet, but daily consumption in these animals was limited and made similar to the amount of food consumed by the animals of group ІІІ within idential periods of cage housing (for differentiation of the effects of anorexia related to the thiamine-deficient state in group III and proper effects of В 1 hypovitaminosis). Animals of group ІІІ (thiamine-deficient) were on a standard diet (with no limitations) mostly analogous to that in group І but containing no thiamine. In phrenicohemidiaphragmatic preparations obtained from animals of group ІІІ, the amplitude of end-plate potentials (EPPs) and miniature EPPs (mEPPs) on the 10th day of consumption of the thiamine-defficient diet and the quantum composition of EPPs on the 20th day became significantly (P < 0.01) smaller than in preparations obtained from animals of both groups І and ІІ. The frequency of mEPPs and membrane potential of muscle fibers in group ІІІ remained unchanged. Two processes, a decrease in the dimension of the transmitter quantum (which is observed within rather early stages of the development of thiamine-defficient state) and a decrease in the quantum composition of evoked EPPs (at later stages) underlie a gradual decrease in the amplitude of EPPs related to the development of alimentary vitamin В 1 deficiency.

Effect of PAR1 agonist on acetylcholine secretion in a newly formed neuromuscular synapse in mice

Peptide agonist of PARI in a concentration of 10 microM significantly facilitated neuromuscular transmission in newly formed synapses in mice. The absence of changes in the amplitude of miniature end-plate potentials attests to presynaptic mechanism of the effect of PAR1 agonist. The effect of the peptide was blocked by protein kinase A inhibitor H89 (1 microM). Blockade of inositol-1,4,5-trisphosphate receptors with 2-amino-ethoxydiphenylborate (30 microM) did not prevent the effects of PARI agonist. Inhibition of protein kinase C with bisindolylmaleimide (1 microM) facilitated neuromuscular transmission in newly formed synapses. Protein kinase C inhibition was associated with reversal of the object of PARI agonist: transmission inhibition instead of facilitation.