Amplitude Of Miniature Endplate Potentials Research Articles

Effects of 2,4-dithiobiuret treatment in rats on cholinergic function and metabolism of the extensor digitorum longus muscle

Effects of 2,4-dithiobiuret (DTB) treatment in rats on neuromuscular transmission and the disposition of cholinergic substances, acetylcholine (ACh) and choline (Ch), were examined in a combined electrophysiological/biochemical study using an in vitro extensor digitorum longus (EDL) muscle-peroneal nerve preparation. EDL muscle preparations isolated from rats teated with DTB (1 mg/kg/day × 5 days, ip) displayed a 49% depression in the frequency of miniature end-plate potentials (MEPPs) and a 21% depression in mean MEPP amplitude. Statistical analysis of evoked end-plate potentials (EPPs) measured in curarized preparations indicated that the mean quantal content ( m) was significantly depressed in EDL muscles from DTB-treated rats. At stimulation rates of 1, 10, 20, and 50 Hz the estimated values of m in EDL preparations from DTB-treated rats were, respectively, 21, 25, 45, and 51% of that in control preparations. Biochemical determinations of ACh and Ch revealed a significant DTB-induced increase in endogenous ACh and Ch content in EDL preparations fixed for extraction of ACh and Ch immediately after dissection from the treated rats. In vitro, however, there were negligible changes in overall ACh synthesis since the total (tissue and medium) tracer ACh ( 2H 4-ACh) synthesized from tracer Ch ( 2H 4-Ch; 10 μ m) supplied in the perfusion medium was similar in EDL preparations from DTB-treated and control rats. Also, in EDL muscles from DTB-treated rats the resting release of ACh was not affected, but when exogenous Ch ( 2H 4-Ch) was not supplemented in the medium the evoked release (via peroneal nerve stimulation) of ACh was depressed. Thus, decreases in spontaneous quantal ACh release, as detected in the electrophysiological experiments, were not reflected by changes in the biochemically determined ACh resting release. The biochemical determination of evoked ACh release, however, correlated with the decrease in quantal content detected in the electrophysiological analysis of evoked EPPs when exogenous Ch was not supplemented in the perfusion medium. Significant and consistent increases (two to three times) in both Ch content and efflux occurred in the EDL muscles from DTB-intoxicated rats. These results indicate that DTB induces a prejunctional impairment of neuromuscular transmission that is not specifically directed at ACh synthesis. Rather those processes by which ACh is incorporated into or released from vesicles appear to be altered.

The effect of chronic neostigmine treatment on channel properties at the rat skeletal neuromuscular junction

We have studied the effects of chronic neostigmine treatment on single channel properties at the rat skeletal neuromuscular junction. Rats received 0.86 mg kg-1 neostigmine (s.c.) daily for 9-11 days. Microelectrode recordings were then made from the extensor digitorum longus muscle. The amplitude of miniature endplate potentials was significantly reduced in muscles from neostigmine-treated rats as compared with controls. Acetylcholine (2-5 microM) applied in the bath produced a depolarization and associated channel opening frequency (from voltage noise analysis) which were significantly reduced in neostigmine-treated muscles with respect to controls. The depolarization resulting from the opening of a single channel (from voltage noise analysis) and single channel open time and conductance (from current noise analysis) were not significantly changed by chronic neostigmine treatment. It is concluded that chronic neostigmine treatment causes an adaptive reduction in the number of functional acetylcholine receptors at the endplate without otherwise affecting single channel properties themselves.

A Microelectrode Study of the Effects of Atracurium on Neuromuscular Transmission

Using standard microelectrode recording techniques, we studied the effects of atracurium on neuromuscular transmission in a concentration range between 10(-13) and 10(-5) M in the rat diaphragm. Both intact and cut diaphragm preparations were used. Atracurium produced no significant alteration of miniature endplate potential (MEPP) frequency (P greater than 0.05). Increasing concentrations of atracurium (10(-6)-10(-5) M) caused a linear decrease in MEPP amplitude from 70.5 +/- 2.3% to 28.0 +/- 2.5% of baseline levels (P less than 0.05). In the cut diaphragm preparation, atracurium increased the degree of rundown and decreased quantum content of the endplate potential (EPP). The above observations suggest that atracurium interferes with neuromuscular transmission by, first of all, producing cholinergic receptor block and secondly, producing frequency-dependent inhibition of release of acetylcholine from the nerve terminal.

Variability of quantal events in control solution and after cholinesterase blockade in frog

Amplitudes of miniature end-plate currents ( A MEPCs) as well as their time constants of decay ( T MEPCs) contribute to the amplitudes of miniature end-plate potentials and hence determine their mean values and variability. Larger mean amplitudes of miniature end-plate potentials observed after cholinesterase blockade result from both greater mean A MEPCs and greater mean T MEPCs. However, enhanced variability of amplitudes of miniature end-plate potentials is a result mainly of greater variability of T MEPCs. This occurs almost exclusively because the amplitude dependence of T MEPCs becomes markedly enhanced. Although the scatter of T MEPCs about T MEPCs vs A MEPCs curve is also larger, it does not contribute much to the increased variability of T MEPCs. The marked amplitude dependence of T MEPCs, indicates that repetitive binding strongly depends on the quantal size—the number of acetylcholine molecules released per quantum, and suggests that if a quantum is the result of a release of several vesicles, then they should be released very close to each other. Marked amplitude dependence of T MEPCs also shows that, with cholinesterase inhibited, the quantal size can be estimated not only from A MEPCs, but also from T MEPCs. Greater scatter of A MEPCs, from the T MEPC vs A MEPC curve is of more obscure origin and probably results because repetitive binding occurs over enlarged “critical areas” with more variable density of acetylcholine receptors.

Luteinizing hormone releasing hormone modulates the cholinergic transmission in frog neuromuscular junction.

The effects of luteinizing hormone releasing hormone (LHRH) on cholinergic transmission were studied at the neuromuscular junction of the frog. Brief application of LHRH produced a prolonged increase in the amplitude of end-plate potentials (e.p.p.s), which lasted 20 to 30 min after removal of LHRH. LHRH (0.4-1 microM) increased in the quantal content of the e.p.p. dose-dependently, while having no effect on the quantal size. LHRH (0.4-1 microM) did not affect the frequency and the amplitude of miniature end-plate potential (m.e.p.p.). At a high concentration (8 microM), however, LHRH consistently produced an increase in the frequency and a decrease in the amplitude of m.e.p.p. The acetylcholine-induced end-plate current (ACh current) produced by iontophoretic application of ACh was reversibly and dose-dependently reduced by LHRH (4.6-46 microM). An analysis with a dose-response curve of the ACh current revealed that LHRH decreased the sensitivity of the nicotinic receptor in a noncompetitive manner. These results suggest that LHRH at low concentrations facilitates neuromuscular transmission by increasing ACh-release from the presynaptic nerve terminals, while at higher concentrations it depresses transmission post-synaptically. Possible mechanisms of these LHRH actions are discussed.

Action of thiamine on neuromuscular transmission in the frog

The action of thiamine on neuromuscular transmission in the frog sartorius muscle was investigated. It was found that thiamine at a concentration of 1×10−14 to 1×10−4 M increases transmitter secretion at the nerve endings. This is demonstrated by the increased frequency, amplitude, and quantal content of miniature endplate potentials, and is due to the enhanced likelihood of transmitter release. The role of thiamine in regulating synaptic transmission and the mechanism of its interaction with thiamine-sensitive receptors are examined.

Detection of sub-miniature endplate potentials by harmonic analysis

In phrenic nerve-diaphragm preparations from young rats and mice, long-term recordings of miniature endplate potentials (MEPPs) from single synapses were performed. Amplitude histograms obtained from these experiments were analysed with respect to the presence of multiple peaks which are believed to reflect quantal transmitter release. This was accomplished by subjecting MEPP amplitude histograms as well as matched random number histograms to Fourier analysis; harmonics in the MEPP amplitude distribution were considered valid only if they exceeded those of the random number distribution. In 9 out of 11 experiments, substantial evidence for the existence of preferred amplitudes was obtained; in two cases, however, it was completely missing. Applying our method to a series of promising MEPP amplitude histograms selected from the literature, we found a similar relationship between positive and negative results. We also noted a fairly constant relationship between the size of the elementary sub-MEPPs and the mean amplitude of the bell-MEPPs, named so according to the Gaussian shape of their amplitude distribution.

Inhibition of non-quantal acetylcholine leakage by 2(4-phenylpiperidine)cyclohexanol in the mouse diaphragm

The drug 2(4-phenylpiperidine)cyclohexanol (AH 5183) caused hyperpolarization by 1.8 ± 0.6 mV in an end-plate zone of mouse diaphragm fibers without any change in the amplitude of miniature end-plate potentials. This supports the idea that the drug inhibits the non-quantal leakage from motor nerve terminals, probably at those parts of the nerve terminals which were incorporated into the terminal membrane after vesicle exocytosis.

Synergistic interaction of 4-aminopyridine with neostigmine at the neuromuscular junction

The pre- and postsynaptic events at the neuromuscular junction were studied in vitro in rat skeletal muscle exposed to clinically significant concentrations of 4-aminopyridine (4-AP), neostigmine or combinations of the two drugs. Simultaneous application of 4-AP and neostigmine produced increases in the amplitudes of nerve-evoked end-plate potentials which were significantly greater than the summed effects of the drugs applied individually. Such synergism was present at the junctions where transmission was blocked either postsynaptically by d-tubocurarine or presynaptically by low [Ca 2+] 0 and high [Mg 2+ 0. Quantal content analysis in the latter preparation indicated that the evoked release of acetylcholine was potentiated significantly more than the amplitude of spontaneous miniature end-plate potentials, suggesting that the site of synergism is predominantly presynaptic. For symptomatic relief and long-term management of the neuromuscular junction disorders, we propose a combined medication of aminopyridine and anticholinesterase at reduced dosages. Such therapy would minimize adverse effects and be particularly effective in the treatment of such presynaptic disorders as the Lambert-Eaton myasthenic syndrome and botulism.

Miniature endplate potential amplitudes corrected for spatial decay are not normally distributed

Miniature endplate potential (MEPPs) were recorded simultaneously with two intracellular electrodes placed in the muscle fiber near the ends of the frog neuromuscular junction (nmj). The MEPP amplitudes are different in each electrode and are not normally distributed. A method is proposed to correct MEPP amplitudes for spatial decay. The MEPP amplitudes corrected for spatial decay are also not normally distributed. When the class interval of the distribution is small, multiple peaks are observed.

Miniature endplate potential frequency and amplitude determined by an extension of Campbell's theorem

A method based upon an extension of Campbell's theorem is used to measure the amplitude, waveform, and frequency of occurrence of miniature endplate potentials (mepps) at rapidly secreting neuromuscular junctions of frog cutaneous pectoris muscles. Measurements of the variance, skew, and power spectrum of the fluctuations in membrane potential are used to deduce the mepp parameters. These estimates of mepp amplitude and frequency are insensitive to slow drifts in membrane potential that preclude the conventional application of Campbell's theorem, which uses the mean and variance. The new method becomes unreliable at high mepp frequencies because the distribution of the values of membrane potential approaches a Gaussian thereby reducing the accuracy of skew measurements. Frequencies approaching 10(4) s-1 can be measured, however, if the data are high-pass filtered. The method has been tested with computer simulated data and applied to junctions exposed to La3+; the effects of Ca2+ on the La3+-induced secretion have been explored. Some muscles were fixed after treatment with La3+, and changes in nerve terminal ultrastructure were assessed by morphometric analysis of electron micrographs. Horseradish peroxidase was used to obtain information about vesicle recycling.

Postsynaptic block of frog neuromuscular transmission by conotoxin GI

Conotoxin GI, a peptide neurotoxin contained in the venom of the marine snail Conus geographus, was applied to the cutaneous pectoris muscle of the frog, and the effects on the postsynaptic response to acetylcholine were examined. Conotoxin GI reversibly blocked nerve-evoked muscle contractions at concentrations greater than or equal to 3 to 4 microM. Micromolar concentrations of conotoxin GI significantly reduced the amplitude of miniature endplate potentials and membrane depolarizations produced by ionophoretic application of acetylcholine, suggesting that the toxin reduced the postsynaptic sensitivity to acetylcholine. The reduction in the sensitivity of the muscle to acetylcholine was not due to changes in muscle fiber resting membrane potential or input resistance. Conotoxin GI reduced the amplitudes but did not affect the rates of decay of focal, extracellularly recorded endplate currents or miniature endplate currents, suggesting that the toxin did not affect the lifetime of ion channels opened by acetylcholine. Miniature endplate currents decay five to six times more slowly than normal when acetylcholinesterase is blocked with neostigmine methyl sulfate due to repeated binding of acetylcholine to receptors as it diffuses from the synaptic cleft. Conotoxin GI reduced the amplitude and increased the rate of decay of miniature endplate currents recorded in the presence of neostigmine methyl sulfate, suggesting that the toxin reduced the binding of acetylcholine to endplate receptors. These results are consistent with the hypothesis that conotoxin GI blocks neuromuscular transmission at the frog endplate by reducing the binding of acetylcholine to receptors.

Electrophysiological studies of myotoxin a, isolated from prairie rattlesnake ( Crotalus viridis viridis) venom, on murine skeletal muscles

S. J. Hong and C. C. Chang. Electrophysiological studies of myotoxin a, isolated from prairie rattlesnake ( Crotalus viridis viridis) venom, on murine skeletal muscles. Toxicort 23, 927–937, 1985. — Myotoxin a reduced the resting membrane potential of mouse and rat diaphragms from about −80 mV to −60 mV, induced spontaneous repetitive firing and enhanced the contractile force in response to single stimulations. The depolarizing effect was reversed noncompetitively by tetrodotoxin, local anesthetics or low Na + solution, but was augmented by ouabain or low Cl − solution while being unaffected by high K + solution or electrical stimulation of the muscle. The duration of muscle action potential was prolonged by only 20–30%, whereas the rate of rise (d V/ d t) was unaffected. About a 40% increase of membrane conductance was observed, which could be abolished by the Na +-channel blocker tetrodotoxin. By contrast, K + conductance was unaffected. Effects on caffeine-induced contracture, quantal release of neurotransmitter and the amplitude of miniature endplate potential were not appreciably affected. These effects of myotoxin a indicate that the toxin affects the muscle, but not the nerve, by acting specifically on the Na +-channel of the sarcolemma or T-tubule, like crotamine, rather than on the sarcoplasmic reticulum. The effects of sea anemone toxin II on the Na +-channel (marked depolarization and prolongation of action potential) could not be prevented by saturating the muscle with myotoxin a. On the other hand, the effect of veratridine, a member of another group of toxins acting on the Na +-channel, was enhanced. These results suggest that myotoxin a acts on the Na −-channel at a site which is discrete from those of tetrodotoxin, veratridine and sea anemone toxin II.

Location and functioning of transmitter release sites in frog neuromuscular junction

End-plate potentials (EPP) and miniature EPP (MEPP) were recorded in a single neuromuscular synapse of the frog sartorius muscle by means of two microelectrodes with a resistance of 0.5–2.0 Mθ. Groups of signals (fields), reflecting transmitter secretion in spatially distinct release sites were identified by extracellular recording on MEPP amplitude scatter diagrams. Release sites in the nerve ending were found to be unevenly distributed, to be grouped in certain areas, and to differ in their probability of secretion of a quantum of transmitter. Comparison of fields on MEPP and uniquantal EPP amplitude scatter diagrams in solution with low Ca++ concentration (0.2–0.4 mM) showed that ability to induce evoked and spontaneous transmitter release at the release site differs, and that sometimes a release site does not participate in evoked secretion. The results of simultaneous recording of synaptic potentials using extra- and intracellular electrodes indicate that transmitter secretion in spatially distinct groups of release sites leads to the appearance of polymodality in the distribution of amplitudes of intracellularly recorded MEPP and uniquantal EPP.

Congenital canine myasthenia gravis: I. Deficient junctional acetylcholine receptors.

The neuromuscular junction was studied during growth in two breeds of dog with a congenital familial form of myasthenia gravis (CMG): the Jack Russell terrier and the springer spaniel. Light microscopy revealed no difference in endplate size or nerve terminal morphology in age-matched CMG dogs and unaffected littermates. At all ages there was in individual muscle fibers of CMG dogs an approximately 75% lower postsynaptic membrane density of acetylcholine receptors (AChR). There was no evidence that this abnormality had an autoimmune basis. Measurements of miniature endplate potential amplitude in relation to fiber diameter revealed that the low density of AChR in the postsynaptic membrane of CMG dogs remained constant and did not change with the marked progression of muscle weakness during growth. Antigenic determinants of AChR were deficient in CMG muscle to the same extent as alpha-bungarotoxin (alpha BT) binding sites. Thus, a low density of AChR in the muscle's postsynaptic membrane appears to be the principal abnormality of CMG. The data suggest that the normal increase in number of acetylcholine (ACh) quanta released by nerve impulse may not fully compensate for the reduction in depolarization produced by a single quantum as the muscle fiber diameter increases. This could cause progression of weakness during growth. In addition to being a useful animal model of a rare form of CMG that resembles one form of human CMG, canine CMG offers a unique model for investigating events of synaptogenesis at the neuromuscular junction.

Changes in miniature end‐plate potentials after brief nervous stimulation at the frog neuromuscular junction.

The amplitude of miniature end-plate potentials (m.e.p.p.s), recorded at the frog neuromuscular junction in a normal ionic environment and in absence of drugs, was examined following 10-450 nerve impulses using conventional electrophysiological techniques and on-line computational analysis. In both contracting preparations and non-contracting preparations pre-treated with glycerol, 100 or more nerve impulses resulted in a maximal fall in mean amplitude of about 20% with recovery apparent over the next 10-20 min. In an altered ionic environment with a lowered Ca and raised Mg concentration, 450 nerve impulses did not produce a decrease in mean amplitude but a similar reduction was seen following a larger number of impulses. The reduction in amplitude was estimated to follow the release of the order of 5000-10000 quanta at end-plates in a normal ionic environment and on average 17000 quanta in the presence of a lowered Ca and raised Mg concentration. Changes in the mean size of the spontaneous quantal response is considered to be a presynaptic event and to reflect the loss and slow recovery of larger packets of transmitter from a vesicular store that is readily released by nerve impulses.

The action of thallium acetate on spontaneous transmitter release in the rat neuromuscular junction.

Frequencies and amplitudes of miniature endplate potentials (MEPP's) were recorded from neuromuscular junctions of the rat phrenic nerve-diaphragm preparation in vitro. Superfusion of the preparations with Ringer solution containing thallium acetate (Tlac) gradually increased the frequency of MEPP's by a factor of 10 within 30 min (1 X 10(-3) mol/l Tlac) and 180 min (5 X 10(-4) mol/l Tlac), whereas the amplitude of MEPP's remained unchanged. Frequency of MEPP's fitted a Poisson-distribution which persisted during superfusion with Tl+-Ringer. Sub-MEPP amplitudes remained unaffected by the action of thallium. It is concluded that thallium interferes presynaptically with spontaneous transmitter release.

Distribution of spontaneous release along the frog neuromuscular junction

A new method, called the spatial decay method, is described. This method permits to localize the site of occurrence of the spontaneous release producing each miniature end-plate potential (MEPP) recorded at the frog neuromuscular junction (nmj). The method requires simultaneous intracellular recording at both distal ends of the nmj. The electrodes are positioned using Nomarski optics. The assumptions that the MEPP amplitude is maximum in front of its release site and decays exponentially toward the recording electrodes placed distally, permit the calculation of the position of the release site producing each MEPP recorded intracellularly. The spatial decay method indicates that the occurrence of spontaneous release is not uniformly distributed along the frog nmj.

Reinnervation of normal and dystrophic skeletal muscle

A comparative study was conducted of resting membrane potential (RMP), extrajunctional acetylcholine (ACh) sensitivity, spontaneous and neurally evoked transmitter release, and directly and indirectly elicited action potentials in posterior latissimus dorsi (PLD) muscles of normal (line 412) and dystrophic (line 413) chickens during reinnervation after nerve crush. Control (nondenervated) dystrophic muscle fibers had a significantly greater RMP (−77.2 vs. −74.1 mV), extrajunctional ACh sensitivity (0.34 vs. 0.04 mV/nC), and incidence of repetitive firing of directly elicited action potentials than did normals. Miniature end-plate potential (MEPP) amplitude in dystrophic fibers was significantly reduced (0.26 vs. 0.38 mV). Six to 7 days after nerve crush, muscle fibers from both lines of chickens showed a significant reduction in RMP and an increase in extrajunctional ACh sensitivity. During this time spontaneous MEPPs were absent and the incidence of repetitive firing was decreased. No significant difference was noted between chicken lines in any of the properties studied. The return of normal properties associated with reinnervation occurred primarily between days 9 and 40. Repolarization of the RMP was clearly evident by day 9 in both lines, but dystrophic fibers showed a slightly earlier and greater degree of repolarization. Similarly, initial decreases in extrajunctional ACh sensitivity and the reappearance of MEPPs were observed on day 9 with dystrophic and day 12 with normal fibers. Neurally elicited action potentials were first recorded on day 11 for dystrophic and day 12 for normal fibers. Finally, multiple firing of directly elicited action potentials associated with reinnervation attained the same incidence (20 to 21% of fibers) on day 12 in dystrophic and day 14 in normal fibers. The results suggest that dystrophic chicken muscle has an enhanced capacity for reinnervation following nerve crush.

Endplate blocking actions of lophotoxin.

Effects of lophotoxin (LTX), a neurotoxin isolated from Pacific sea whips Lophogorgia rigida and Lophogorgia chelensis, on neuromuscular transmission were assessed in the rat phrenic nerve-hemidiaphragm preparation using conventional microelectrode recording techniques, and in the frog cutaneous pectoris preparation using two microelectrode voltage clamp techniques. LTX (2-25 microM) produced a progressive, irreversible block of miniature endplate potential (m.e.p.p.) and endplate potential (e.p.p.) amplitude. M.e.p.p. amplitude histograms were shifted markedly in the direction of lower amplitude by LTX. These effects occurred following a latency of 25-40 min. The latency to onset of block was decreased with increasing LTX concentrations. In some preparations, LTX produced a transient increase in m.e.p.p. frequency during the first 5 min of application; however, m.e.p.p. frequency then declined to complete block. The depressant effect of LTX on m.e.p.p. and e.p.p. amplitude progressed to complete block irrespective of the LTX concentration. LTX also blocked the endplate depolarization produced by iontophoretic application of acetylcholine (ACh). The resting membrane potential of skeletal muscle fibres was unaffected by LTX. In voltage clamp experiments, LTX (15 microM) depressed the peak amplitude of the endplate current (e.p.c.) nearly uniformly at potentials between -120 and +60 mV. LTX did not affect the e.p.c. reversal potential or the kinetics of e.p.c. decay suggesting that LTX does not block open ACh channels. E.p.c. block by LTX was also progressive and irreversible. The results indicate that LTX blocks neuromuscular transmission by a postjunctional action. The binding site of LTX may be different from that of ACh.