Amplitude Of Miniature Endplate Potentials Research Articles

Two types of miniature endplate potentials in Xenopus nerve-muscle cultures

The frequency distribution of miniature endplate potential (mepp) amplitudes was examined in Xenopus nerve-muscle cultures. In the early days of nerve-muscle co-culture (1-2 days) the mepp amplitude histogram was skewed in the majority of muscle cells and had a peak approximately at 0.6 mV. Innervated muscle cells in older cultures (3-4 days), however, often had two peaks. The distribution at the smaller amplitude was skewed and the peak was approximately at 0.6 mV, whereas the peak at the larger amplitude was more-or-less symmetrical and about 18 times greater than the small one. The time-to-peak value of mepps in the peak at the smaller amplitude was slightly smaller than those in the peak at the larger amplitude. Small mepps were not due to electrotonic spread of mepps generated at distant sites nor due to focal development of acetylcholinesterase. Two peaks were also observed in the amplitude histogram of extracellularly recorded miniature endplate currents. Therefore, these two types of mepps must be generated practically at the same site. Mepps in the peak at the smaller amplitude had similar properties as sub-miniature endplate potentials (s-mepps) described in the adult animal. The second symmetrical peak in older cultures may correspond to classical mepps (c-mepps). The age-dependent shift from the amplitude distribution with one peak to that with two peaks has also been reported in developing and regenerating neuromuscular junctions. Therefore, I suggest that during development in culture, mepps appear at first as s-mepps and subsequently c-mepps develop, forming the second peak in the amplitude histogram.

Electrophysiological analysis of the cholinergic effects of cimetidine and ranitidine

The study takes the form of an analysis of the effects of cimetidine and ranitidine (2 x 10(-4) M) on 9 electrophysiological parameters, using a single frog sciatic nerve-sartorius muscle preparation. The parameters evaluated were: (i) mean end-plate potential (e.p.p.) amplitude, and mean 50%-corrected e.p.p. amplitude, (ii) mean rise time (RT), (iii) mean half-decay (HD), (iv) mean miniature end-plate potential (m.e.p.p.) amplitude, (v) mean resting membrane potential (RMP), (vi) mean m.e.p.p. frequency (f), (vii) mean quantal content (m), (viii) quantum release probability (p), and (ix) number of active sites (n) in the nerve terminal. Marquardt analysiswas used in order to determine the effects of cimetidine and raniditine on end-plate potential kinetics. At the concentration assayed only ranitidine showed a marked blocking effect at postsynaptic level as well as a less pronounced presynaptic effect. The time course of the e.p.p.'s is also more affected by raniditine, though the kinetic behaviour observed is not a unique feature of this drug alone.

A study of the action of tetanus toxin at rat soleus neuromuscular junctions.

Tetanus toxin (TeTX) inhibits the evoked release of acetylcholine (ACh) at rat soleus end-plates. The effects of various procedures which evoke ACh release by raising the level of free intracellular calcium have been investigated at various stages of tetanus intoxication. At all stages studied TeTX has little or no effect on either the frequency or the amplitude of spontaneous miniature end-plate potentials (m.e.p.p.s). After TeTX poisoning, e.p.p. latency is more variable than normal and the slope of the relationship between ln m (quantal content) and ln [Ca]o is reduced from the control value of about 4. Plots of m-1/n against 1/[Ca]o, for n = 1-4, suggest that mmax, the maximum number of quanta releasable by nerve stimulation, is reduced at intoxicated end-plates. Blocking delayed rectification with 3-aminopyridine (1-5 mM) increases m, but has little or no effect on either the slope of ln m-ln [Ca]o plots or estimates of mmax. Several treatments which raise m.e.p.p. rate (high [Ca]o, hyperosmotic medium, addition of lanthanum) are less effective after TeTX poisoning. Some of the tested agents increase m.e.p.p. frequency by a mechanism which is thought to involve a mobilization of calcium from intracellular stores. The decline in m.e.p.p. rate after a period of high-frequency nerve stimulation is different at normal and TeTX-treated end-plates. At tetanus-intoxicated end-plates, the decline differs from that expected if TeTX acted simply to block calcium entry into the terminal. In addition, the increase in m.e.p.p. frequency observed with a high rate of nerve stimulation suggests that considerable amounts of calcium can enter the terminal with each action potential. It is concluded that TeTX blocks transmitter release by acting at a step between calcium influx to the terminal and transmitter release such that the mechanism for ACh release shows a reduced sensitivity to intracellular calcium. The possibility of an additional effect on the presynaptic calcium conductance cannot be excluded. Some differences between the properties of end-plates poisoned with TeTX and botulinum toxin are discussed.

Pre- and post-synaptic actions of McN-A-343 in the neuromuscular junction: Myographic and electrophysiologic studies in vitro

1. 1. McN-A-343 (McN) concentrations up to 10 μg/ml increased the amplitude of the indirectly elicited muscular contractions of the isolated rat diaphragm. 2. 2. The effect was accompanied by an increase in both frequency and amplitude of miniature endplate potentials. 3. 3. The latter effects were abolished by tetrodotoxin, thus suggesting that McN increases the permeability of presynaptic membrane to sodium ions. 4. 4. Larger doses of McN block the neurotransmission probably acting at post-synaptic level.

Analysis of factors restricting the use of binomial statistics for studying transmitter release in neuromuscular junctions

Binomial parameters of transmitter secretion were calculated on the basis of analysis of synaptic potentials in the frog sartorius muscle. Negative values of the parameter p were found in some synapses. This happened most often in low Ca2+ concentrations and with low amplitude of miniature end-plate potentials. The results were interpreted in terms of a model assuming spatial heterogeneity of probability of transmitter quantum release at different release points. Simulation of transmitter secretion by computer showed that the appearance of negative values of the parameter p and incorrect estimates of n experimentally are connected with the form of distribution of probability of transmitter quantum release in the synapse and with the amplitude of miniature potentials.

Effects of the sea anemone Anemonia sulcata toxin II on skeletal muscle and on neuromuscular transmission

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na + channel specific action of ATX II. Na + current inactivation is slowed with the time constant τ h increasing towards positive membrane potentials. The steady state inactivation curve h oo was shifted to more positive potentials and its slope reduced.

The effects of myasthenic immunoglobulins on neuromuscular transmission and thymus histology in mice.

An autoimmune reaction to the endplate acetylcholine receptor (AChR) is thought to be responsible for the muscular weakness of myasthenia gravis (MG). However, the significance of antithymic antibodies and the thymic AChR-like protein is still uncertain. We transferred immunoglobulins (Igs) from patients with MG to mice. Thymitis was observed in 12 of 14 mice, and miniature endplate potential amplitudes were reduced in 8. Control mice showed none of these abnormalities. Immunofluorescence examination failed to reveal the binding of human IgG, IgM, or IgA to the thymic tissue. Our findings support the hypothesis that antithymic antibodies may alter thymic histology.

Microelectrode recording of the effects of agonists and antagonists on alpha-adrenoceptors on rat somatic nerve terminals.

The effects of apomorphine, catechol, clonidine, isoprenaline, (-)-and (+/-)-noradrenaline, phenylephrine, pyrogallol and xylazine were investigated on the frequency and amplitude of miniature endplate potentials (m.e.p.ps) and, with the exception of apomorphine, catechol and pyrogallol, on the amplitude of endplate potentials (e.p.ps) in the rat phrenic nerve diaphragm preparation. Clonidine, (-)-noradrenaline, phenylephrine and xylazine (each at 1.5 X 10(-5)M) increased m.e.p.p. frequency but not amplitude. The other drugs were ineffective, except isoprenaline (1.5 X 10(-5)M) which enhanced m.e.p.p. amplitude but not frequency. The increase in m.e.p.p. frequency was inhibited by phentolamine, prazosin and yohimbine (each 1.5 X 10(-9)M). Prazosin and yohimbine alone each reduced m.e.p.p. frequency but failed to abolish m.e.p.ps even at high concentrations (10(-3)M). Clonidine, (-)-noradrenaline, phenylephrine and xylazine (each 3 X 10(-6)M) enhanced e.p.p. amplitude; this enhancement was blocked by prazosin and by yohimbine (each 3 X 10(-6)M). In preparations fatigued by prolonged continuous nerve stimulation (5 Hz, 0.05 ms for 30 min), (-)-noradrenaline (3.3 X 10(-4)M) restored m.e.p.p. frequency. The results indicate that adrenoceptors on somatic nerve terminals interact with both alpha 1- and alpha 2-agonists and antagonists and show different characteristics from those at autonomic neuroeffector junctions. The alpha-adrenoceptors on somatic nerve terminals may have an ancilliary physiological role in influencing but not controlling transmitter release.

Effect of lanthanum ions on the amplitude distributions of miniature endplate potentials and on synaptic vesicles in frog neuromuscular junctions

Miniature endplate potential (MEPP) amplitude distributions, MEPP frequencies and percentages of small MEPPs were determined as well as synaptic vesicle diameters and numbers in the frog neuromuscular junction during La 3+ treatment. MEPP frequencies initially increased by two orders of magnitude and then fell to very low values. La 3+ treatment had an initial postsynaptic effect making the MEPPs larger. Prolonged treatment had a variable effect on MEPP amplitudes. There were considerable variations in MEPP frequencies in adjacent junctions so single junctions on edge muscle fibers were recorded for the duration of many experiments and later identified in the electron microscope. Therefore, the physiological and morphological conditions of a given identified junction could be compared. There was a loss of synaptic vesicles and no change in mean diameter during depletion. During high MEPP frequencies infoldings occurred on the axolemma and these disappeared when MEPP frequencies decreased towards the end of the La 3+ treatment. After 3–4 h of La 3+ treatment, the overall frequency of MEPPs dropped and many were composed of a small class of MEPPs. It is suggested that the morphological correlate of small MEPPs, as well as the classical bell-MEPPs is likely to be synaptic vesicles.

Neuromuscular blocking properties of suxamethonium and decamethonium in normal and myasthenic rat muscle

Patients with myasthenia gravis (MG) have increased tolerance to the neuromuscular blocking properties of suxamethonium (SCh) and decamethonium (C10) and exhibit a reversal of the C10-induced block by neostigmine. The effects of these drugs were compared in forelimb flexor digitorum longus muscle from normal rats and from rats with experimental autoimmune myasthenia gravis (EAMG) to investigate the similarity of EAMG to MG. The depolarization induced by 1, 5, 10 and 25 μM SCh or C10 at the motor end-plates was significantly higher in normal than in EAMG muscle. However, both normal and EAMG end-plates responded in a similar qualitative manner to each drug. The depolarization produced by SCh was typically maintained until the drug was washed from the bath. The depolarization produced by C10 tended to decrease after reaching its peak despite continued application of the drug. With both drugs, miniature end-plate potential (MEPP) amplitude reduction is maintained until a saline wash. Neostigmine interaction with SCh and C10 in normal and EAMG muscle was compared by measuring isometric twitch tension in vitro. Neostigmine potentiated the neuromuscular block produced by either SCh or C10 in both normal and EAMG muscle. Thus muscle from rats with EAMG shares with MG an increased tolerance to SCh and C10 when compared to normal muscle but does not exhibit the qualitatively different interaction of C10 affected muscle with neostigmine that is found in MG patients. This and other studies comparing EAMG and MG indicate that EAMG is an appropriate model of MG but differences such as we have noted should be considered when extrapolating data from EAMG to the human disease.

Effects of kava on neuromuscular transmission and muscle contractility

The effects of kava, a native drink from Oceania, on neuromuscular transmission and muscle contractility have been examined in mouse phrenic nerve-hemidiaphragm and frog sartorius muscle preparations using twitch tension and intracellular recording techniques. The extent of muscle paralysis induced by kava was similar in both directly and indirectly stimulated mouse hemidiaphragms. The neuromuscular blockade produced was poorly reversed by calcium and by neostigmine. Intracellular recordings from frog sartorius muscles showed that kava depressed the amplitude of both miniature end-plate potentials (mepps) and end-plate potentials (epps) but had no effect on the frequency of mepps. Kava greatly prolonged the duration of mepps and epps and also slowed and depressed directly elicited muscle action potentials. It is concluded that kava causes paralysis by mechanisms similar to local anaesthetics.

Polymodality of distribution of miniature end-plate potential amplitudes due to the action of spatially separate areas of transmitter release

Polymodality of distribution of miniature end-plate potential amplitudes due to the action of spatially separate areas of transmitter release

Reduction of miniature end-plate potential amplitude in extraocular and limb muscles in an animal model of myasthenia gravis

Miniature end-plate potential (MEPP) amplitudes of the superior oblique extraocular muscle (EOM) and forelimb flexor digitorum longus muscle (FDL) of rats with experimental autoimmune myasthenia gravis (EAMG) were expressed as reductions from mean MEPP amplitudes of the respective muscles of pair-matched control animals. Postjunctional blockade of neuromuscular transmission at EOM end-plates was no greater than at limb muscle end-plates. In contrast, the amount of reduction measured in EAMG-affected FDLs (52.3%) was marginally, but significantly, greater than that in EOMs (38.3%). A weak correlation was present between the MEPP reduction observed in an animal's FDL and that recorded in its EOM.

Effects of low temperature and terminal membrane potential on quantal size at frog neuromuscular junction.

1. Two mechanisms proposed for the quantal release of acetylcholine (ACh) are: (i) that the quanta are pre-packaged in vesicles and released by exocytosis and (ii) that the ACh is released from the cytoplasm of the nerve terminal by the opening of an ACh channel. Our experiments were designed to test aspects of these hypotheses.2. Miniature end-plate currents (m.e.p.c.s) were reversibly decreased in amplitude and increased in duration as the temperature was decreased between 15 and 6 degrees C. The amplitude decreased with a Q(10) of 2.4 between 15 and 11 degrees C, and then with a Q(10) of 3 between 11 and 6 degrees C. The half-decay time increased with a Q(10) between 4 and 5 over the entire temperature range.3. The effect of temperature on the end-plate current (e.p.c.) in response to ionophoretically applied ACh was also studied. The e.p.c. in response to a set, sustained dose of ACh was 30% larger at 11 degrees C than at 15 degrees C, and about 10% larger at 6 degrees C than at 15 degrees C.4. The difference in the end-plate response to brief pulses of ACh (m.e.p.c.s) and to sustained application of ACh was analysed by Dionne & Stevens (1975). The amplitude of the sustained response depends on both the number of channels opened in the end-plate, and the length of time they stay open. When our temperature data are analysed in this way, it appears that the amount of ACh/quanta acting on the end-plate is altered by less than 25% over the temperature range 15-6 degrees C.5. From the experiments in which the nerve terminal membrane potential was shifted by external currents it was concluded that miniature end-plate potential amplitude was independent of the terminal membrane potential (Cooke & Quastel, 1973). This conclusion was confirmed by measuring m.e.p.c.s in low Ca(2+) Ringer solution containing 2.0 and 22.0 mM-KCl; there was no consistent change in m.e.p.c. amplitude.6. The effects of both temperature and terminal membrane potential are more readily interpreted by the vesicle than by the channel hypothesis, since for the channel hypothesis the duration of channel opening should be temperature-sensitive and the efflux of ACh(+) through the terminal membrane should be potential-dependent.

Effects of serotonin (5-hydroxytryptamine) on amphibian neuromuscular junction

A study of the effects of serotonin transmission was carried out on the frog neuromuscular junction by means of microelectrode methods. Serotonin was employed in concentrations of 5–100 μM. Serotonin did not affect membrane characteristics or the resting potential whether at non-neuronal (muscular fiber) or endplate segments of the junction. While serotonin did not affect the frequency of the miniature endplate potentials (MEPPs), it significantly decreased evoked release of acetylcholine. Serotonin significantly decreased, in a dose-dependent fashion, the amplitude of acetylcholine potentials, endplate currents (EPCs), endplate potentials (EPPs) and MEPPs. Also, serotonin shortened significantly the EPC time course and half-decay time, and caused loss of membrane voltage sensitivity of the half-decay time. While it did not affect the null potential, serotonin changed the voltage-EPC relationship from linear to non-linear, and markedly attenuated the dependence of EPC amplitude on membrane potential. These results demonstrate that serotonin induces depressant effects at both pre- and post-synaptic sites of amphibian neuromuscular junction and that its post-synaptic action is directed at the receptor-channel macromolecule rather than at either the channel or the receptor alone.

Electrophysiological effects of myasthenic serum factors studied in mouse muscle.

Miniature end-plate potential (mepp) amplitudes were investigated in mouse diaphragm exposed in vitro to different serum fractions from seven patients with myasthenia gravis who had elevated serum anti-acetylcholine receptor antibody levels and from controls. The mepp amplitudes were significantly reduced by whole myasthenic sera, restored by washing, and not reduced by heated (56 degrees C) myasthenic sera, which would inactivate complement but not antireceptor antibody. Immunoglobulin G (IgG)-depleted myasthenic sera also significantly reduced mepp amplitudes, while the IgG fraction alone or with normal serum did not. The results indicate that in vitro reduction of mepp amplitudes in mouse muscle by myasthenic sera is not dependent on the IgG fraction alone, and requires a heat-sensitive factor.

Biochemical and electrophysiological properties of antibodies against pure acetylcholine receptor from vertebrate muscles and its subunits from Torpedo in relation to experimental myasthenia

Immunization of rabbits with homogeneous preparations of acetylcholine receptor from denervated muscle of cat and chicken, which contained single or multiple sizes of polypeptides respectively, induced myasthenic-like symptoms. One of the resultant antisera, and the IgG fraction thereof, reduced significantly and irreversibly the amplitude of miniature endplate potentials in murine muscle; the effect was not abolished by heat inactivation of complement. This antiserum also retarded the binding of α-bungarotoxin to a solubilised extract of denervated muscle containing homologous receptor. The other five antibody preparations were unable to affect these miniature potentials but many of them did reduce the binding of α-bungarotoxin to denervated muscle receptor in solution and, in some cases, decreased the effectiveness of the latter in blocking neuromuscular transmission. Although inoculation with each of the four individual subunits from the receptor of Torpedo marmorata electroplax did not produce muscle weakness in rabbits, antibodies to α- or β-polypeptides lowered, to a significant extent, the amplitude of spontaneous synaptic potentials in mouse diaphragm muscle. It is concluded that antibodies with direct blocking actions on the receptor-ion channel complex are not common in such immunized animals and their presence cannot be correlated readily with the induction of physical disability. The majority of the antibody species bind to loci distant from the acetylcholine recognition site. Antiserum from one of the immunized rabbits reacted preferentially with receptor from denervated rather than innervated cat and rat muscle, indicating some dissimilarity.

The presynaptic effect of fractions isolated from the sponge Tedania ignis

Two fractions from the sponge Tedania ignis which have presynaptic effects were isolated. In neuromuscular junctions of Rana pipiens, the crude fraction decreases the amplitude of the evoked endplate potential and increases the frequency of miniature endplate potentials (MEPP), without effects on their amplitude or shape. Elution with 1 M acetic acid through Sephadex G15 produces three peaks, only one of which contains the biological activity. The compounds in this peak possess a molecular weight close to 900. Elution with 1M acetic acid through BioGel P2 produces 9 peaks, only 2 of which are biologically active. One of these fractions (fα) increases the frequency of MEPP and another fraction (fβ) inhibits the evoked release of neurotransmitter. None of the fractions change the amplitude or shape of MEPP, nor do they modify the resting membrane or action potentials in frog muscle. The action of fα occurs in low (15 μM) Ca 2+, while the effect of fβ is antagonized by raising the extracellular Ca 2+ concentration above 1.8 MM. Fraction β antagonizes the increase of acetylcholine release produced by the venom of the black widow spider Latrodectus mactans mactans. This antagonistic action of fβ is reversible and the effect of the spider venom reappears if the sponge toxin is washed out with normal Ringer's solution.

The presynaptic neuromuscular blocking effect and phospholipase A 2 activity of textilotoxin, a potent toxin isolated from the venom of the Australian brown snake, Pseudonaja textilis

In the presence of Ca 2+ and deoxycholate, textilotoxin has, on a molar basis, about the same phospholipid-splitting activity as β-bungarotoxin and other presynaptically acting toxins. Sr 2+ inhibited the enzyme activity in the presence of Ca 2+ by about 90%. Textilotoxin blocked neuromuscular transmission without significant change of muscle contractility. As with taipoxin, the mouse diaphragm was more susceptible than the chick biventer cervicis and the rat diaphragm preparations. After complete neuromuscular blockade, the amplitude of spontaneous miniature endplate potentials (m.e.p.p.) and the membrane potential of the mouse diaphragm remained unchanged, as did the contractile response of the chick biventer cervicis muscle to bath-applied acetylcholine. Textilotoxin caused an immediate facilitation of transmitter release in the mouse diaphragm lasting for about 4 min, followed by depression lasting for about 15 min and then a second phase of facilitation lasting for about 25 min, before causing final inhibition. The frequency of m.e.p.p.s was first depressed, then increased twice before the final depression. The time-course, however, was slower than the change of evoked transmitter release. Like all other presynaptic phospholipase A neurotoxins, textilotoxin bound rapidly to the nerve terminal and its blocking effect was antagonized by high Mg 2+ and Ca 2+ media, but was markedly enhanced on increasing the stimulus frequency from 0.1 Hz to 1.0 Hz. Sr 2+ strongly antagonized the neuromuscular blocking effect of textilotoxin only at 1.0 Hz stimulation, but not at 0.1 Hz. It is concluded that textilotoxin acts mainly on the presynaptic nerve terminal, with multiphasic changes of transmitter release. The final blockade is due to its phospholipase A activity acting inside the terminal axolemma.

Changes in MEPP and EPP amplitude distributions in the mouse diaphragm during synapse formation and degeneration.

Miniature end-plate potential (MEPP) and end-plate potential (EPP) amplitude histograms were examined in the mouse diaphragm during degeneration, deterioration, re-innervation and neonatal development. MEPPs and EPPs were recorded with conventional electrophysiological techniques. Control MEPP amplitude distributions from mice 21-30 days old showed two classes of MEPPs. The larger class composed 80-90% of the MEPPs and formed a bell-shaped distribution (bell-MEPPs). The smaller class (skew-MEPPs) formed a skewed distribution with a peak 1/7 to 1/15 that of bell-MEPPs. Usually, MEPP amplitude distributions did not change during the course of nerve degeneration or during deterioration in the bath. MEPP amplitude distributions from newly re-innervated fibers were composed mainly of skew-MEPPs. At later stages of re-innervation the relative numbers of skew-MEPPs decreased. Many fibers from neonatal mice (2-3 days old) also showed mainly skew-MEPPs. Rise time vs amplitude plots were constructed from neonatal and re-innervating preparations. The skew-MEPP time-to-peak measurements fell on or below the regression line calculated from the time-to-peak data of the bell-MEPPs. This indicates that the skew-MEPPs originated from the same site as the bell-MEPPs. Unitary EPPs were recorded from neonatal and re-innervating preparations by reducing the evoked response with cobalt ions (4 mM). Distributions of unitary EPPs were similar to those of bell-MEPPs. It is concluded that there are two classes of spontaneous quanta. The skew-MEPP class dominates MEPP amplitude distributions during the early stages of re-innervation and early neonatal preparations. In all stages of development the unitary evoked EPPs have the same mean amplitude and time-to-peak as the bell-MEPPs. The data suggest that the skew class is not available for evoked release.