Co-operative action a calcium ions in transmitter release at the neuromuscular junction

1. The quantitative dependence of transmitter release on external calcium concentration has been studied at the frog neuromuscular junction, using intracellular recording and taking the amplitude of the end-plate potential (e.p.p.) as an index of the number of packets released.2. The relation between [Ca] and the e.p.p. is highly non-linear. The initial part of this relation on double logarithmic co-ordinates gives a straight line with a slope of nearly four (mean 3.78 +/- 0.2 S.D. in 28 experiments). Addition of a constant amount of Mg reduces the e.p.p. without altering the slope of the log e.p.p./log Ca relation.3. The slope of this logarithmic relation diminishes as [Ca] is raised towards the normal level.4. The results are explained quantitatively on the hypothesis that Ca ions combine with a specific site X on the nerve terminal forming CaX, and that the number of packets of acetylcholine released is proportional to the fourth power of [CaX].5. The analysis suggests that a co-operative action of about four calcium ions is necessary for the release of each quantal packet of transmitter by the nerve impulse.     

The mechanism of presynaptic inhibition at the crayfish neuromuscular junction

Summary After stimulation of the excitatory nerve fiber potential changes (e.n.t.p.s) were recorded extracellularly from the excitatory nerve terminals on crayfish muscle. The influence of neural presynaptic inhibition on these e.n.t.p.s was analysed.Diphasic e.n.t.p.s recorded at some distance from the nerve terminal were increased by inhibition. Diphasic e.n.t.p.s recorded nearer to the terminal could be abolished by inhibition, their negative phase was more affected than the positive one and sometimes they were made monophasic positive. These findings were interpreted to represent a block of conduction or a central shift of a preexisting block of conduction by inhibition of the excitatory nerve terminal.Monophasic positive e.n.t.p.s recorded at the ultimate terminal were much reduced by inhibition. The release of the excitatory transmitter substance is not decreased proportional to the reduction of the positive e.n.t.p.s by presynaptic inhibition.All effects of presynaptic inhibition on shape and amplitude of the e.n.t.p. can be explained by an increased membrane conductance for chloride and/or potassium ions in the terminal.

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Zusammenfassung Am Krebsmuskel wurden mit Hilfe von extracellulären Mikroelektroden nach Reiz der erregenden Nervenfaser von ihren Endigungen Potentialänderungen (e.n.t.p.) abgeleitet. Die Effekte der neuralen präsynaptischen Hemmung auf diese e.n.t.p. wurden analysiert.Diphasische e.n.t.p., die in einiger Entfernung von der Endigung abgeleitet wurden, wurden durch die Hemmung vergrößert. Dagegen konnten diphasische e.n.t.p., die näher der Endigung gemessen wurden, durch die Hemmung sehr stark reduziert werden; ihre negative Phase wurde mehr betroffen als die positive, und diphasische e.n.t.p. konnten so zu monophasisch-positiven werden. Diese Befunde wurden als Anzeichen einer Blockade der Erregungsfortleitung, oder einer zentralen Verschiebung einer schon bestehenden solchen Blockade, durch die Hemmung interpretiert.An der äußersten Nervenendigung wurden monophasisch positive e.n.t.p. registriert. Diese wurden durch die Hemmung sehr stark verkleinert. Die Freisetzung des erregenden Überträgerstoffes durch die Nervenendigung wurde durch die Hemmung nicht proportional zur Amplitude der positiven e.n.t.p. herabgesetzt.Alle durch präsynaptische Hemmung bewirkten Veränderungen der Form und Amplitude der e.n.t.p. können durch eine Erhöhung der Membranleitfähigkeit der Nervenendigung für Chlorid- und/oder Kaliumionen erklärt werden.

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With 8 Figures in the Text

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This investigation was supported by grants from the Deutsche Forschungsgemeinschaft.     

Synaptic vesicle recycling at the neuromuscular junction in the presence of a presynaptic membrane marker

Staining of the presynaptic axonal membrane of the neuromuscular junction with horseradish peroxidase-labeled α-bungarotoxin was utilized as a marker for observing directly the fate of this membrane during the process of synaptic vesicle release and recycling. The neuromuscular junctions of frog sartorius-sciatic nerve preparations were stained with horseradish peroxidase-α-bungarotoxin and stimulated by electrical stimulation of the nerve, high concentration of external potassium ions, and black widow spider venom. Some preparations were stimulated in the presence of exogenous horseradish peroxidase tracer after incubation in the conjugate and were found to contain horseradish peroxidase within many synaptic vesicles, indicating that the conjugate did not affect the process of synaptic vesicle recycling. Stimulation was followed by depletion of synaptic vesicles and appearance of axolemmal infoldings and membranous cisternae. With rest after electrical and potassium stimulation, synaptic vesicles were reconstituted and terminals assumed a more normal appearance. Membrane staining after stimulation occurred in the axolemmal infoldings, some of the intra-axonal cisternae, and in a few coated vesicles. However, all synaptic vesicles were unreactive, in either rested or unrested terminals. Thus, axonal membrane labeled with horseradish peroxidase-α-bungarotoxin did not become incorporated into new synaptic vesicles.These observations support a mechanism of recycling of synaptic vesicles by specific retrieval of vesicle membrane or constituents from the axolemma.     

Quantal independence and uniformity of presynaptic release kinetics at the frog neuromuscular junction

1. Amplitude and latency fluctuations of the end-plate potential at the frog neuromuscular junction were studied simultaneously at low temperatures, using intracellular or focal extracellular recording techniques and average quantal contents between 0.5 and 3.2. At the release rates studied, the evoked release of one quantum has in most cases no significant effect on the probability of subsequent quantal release to the same stimulus, confirming the mutual independence of quantal releases in this preparation.3. An equation derived from Poisson's law was applied to a histogram of the latencies of the first quantum released on each of a series of trials, to predict the average quantal content of end-plate responses originating at various times after nerve stimulation. The shape of the predicted time distribution of quantal contents usually agreed closely with that of the experimentally observed time distribution of end-plate response amplitudes. This agreement demonstrates that both the amplitude and the latency fluctuations of the end-plate response result from one presynaptic stochastic process that is uniform in magnitude and time course after each stimulus.4. Analysis of extracellular records from synaptic regions with a history of extensive activity often suggested the existence of depressive interaction among quantal releases, perhaps caused by depletion of the supply of releasable quanta.     

An analysis of the action of a false transmitter at the neuromuscular junction.

1. The action of monoethylcholine (MECh) on neuromuscular transmission has been studied by electrophysiological methods. 2. End-plate potentials (e.p.p.s.) in curarized rat muscle were unaffected or slightly increased in amplitude by MECh (0-1-1 mM). Stimulation at 3 Hz for about 30 min in the presence of MECh caused a progressive decline in e.p.p. amplitude, and a shortening of the e.p.p. time course. These changes were reversed by addition of choline to the medium. Similar changes in amplitude, but no change in time course, occurred when the preparation was stimulated in the presence of hemicholinium or triethylcholine. 3. Extracellular recordings of miniature end-plate potentials in frog muscle showed that stimulation in the presence of MECh caused the time constant of the exponential decay of the m.e.p.p.s. to decrease by 42%. The amplitude of intracellular m.e.p.p.s. was reduced by 45%. These changes were maximal by the time about 3 X 10(5) quanta had been released. 4. Voltage clamp experiments in rat muscle in which miniature end-plate currents (m.e.p.c.s) were recorded showed that stimulation in the presence of MECh reduced the amplitude (by 33%) and the decay time constant (by 42%). 5. Analysis of end-plate current flucutations produced by local application of acetylcholine (ACh) and acetylmonoethycholine (AMECh) to voltage clamped rat end-plates showed that the amplitude of the elementary current events was the same for both compounds whereas the average channel lifetime was 44% shorter for AMECh than for ACh. 6. The voltage-sensitivity of the channel lifetime (measured from end-plate current fluctuations) was the same for ACh and AMECh. The voltage-sensitivity of the m.e.p.c. decay time constant was the same as that found from noise measurements. The shortened m.e.p.c.s. (false m.e.p.c.s.) occurring after stimulation in the presence of MECh also showed the same voltage-sensitivity. 7. Both normal and false m.e.p.c.s. were prolonged by neostigmine by almost the same factor; false m.e.p.c.s. were thus shorter than normal m.e.p.c.s. even when cholinesterase was inactivated. Experiments with progressive curarization of neostigmine-treated end-plates suggested that the fraction of transmitter molecules bound is smaller for false than for normal m.e.p.c.s. The difference implies that the false transmitter has one quarter of the affinity of ACh for the receptors. 8. It is concluded that stimulation in the presence of MECh gives rise to a false transmitter, presumably AMECh, which has a lower affinity for receptors than ACh, and gives rise to ionic channels with a shorter average lifetime than those activated by ACh.     

Correlation of presynaptic and postsynaptic events during establishment of long-term facilitation at crayfish neuromuscular junction

Repetitive stimulation (10-20 Hz) of the motor axon supplying the opener muscle in the crayfish leg produces long-lasting enhancement of excitatory postsynaptic potentials. This long-term facilitation (LTF) was investigated by recording simultaneously from the presynaptic nerve terminal and from the innervated muscle fiber with intracellular microelectrodes. On cessation of stimulation, the facilitated postsynaptic potential declines in amplitude when monitored with low-frequency test stimuli. A rapid decline (phase I) occurs over the first 30 s and is succeeded by a more gradual decline lasting several minutes (phase II). Finally, a residual potentiation with a very slow decay (phase III) persists for several hours. Simultaneous pre- and postsynaptic recordings were made during induction of LTF with stimuli delivered at 20 Hz for 10 min. During the tetanus, excitatory postsynaptic potentials were enhanced 20-fold, while action potentials in the presynaptic terminal declined in amplitude from 108.6 to 97.2 mV, and the presynaptic membrane became hyperpolarized by 6.4 mV. The Na+ pump inhibitor ouabain (0.5-1.0 mM) abolished the hyperpolarization, indicating that the latter resulted from activation of an electrogenic Na+ pump. The reduction in amplitude of the presynaptic action potential was consistent with a reduced transmembrane concentration gradient for Na+. Thus, it is suggested that a significant accumulation of Na+ occurs during repetitive stimulation of crayfish motor axons. Decay of phase II of LTF, but not of phases I or III, had approximately the same time course as the decay of Na+ accumulation in the terminals, monitored by changes in the presynaptic action potential. Thus it is probable that in crayfish this phase of LTF is linked to an increased intraterminal Na+ concentration. Injection of Na+ from a microelectrode into the presynaptic terminal produced enhancement of the excitatory postsynaptic potential lasting for many minutes, as well as changes in presynaptic membrane potential and action potential similar to those seen during repetitive stimulation. The results provide the first direct measurements of electrical and ionic changes in axonal terminals during prolonged periods of activity leading to LTF, and support the hypothesis that accumulation of intraterminal Na+ is associated with one phase of LTF.     

alpha-latrocrustatoxin increases neurotransmitter release by activating a calcium influx pathway at crayfish neuromuscular junction

alpha-latrocrustatoxin (alpha-LCTX), a component of black widow spider venom (BWSV), produced a 50-fold increase in the frequency of spontaneously occurring miniature excitatory postsynaptic potentials (mEPSPs) at crayfish neuromuscular junctions but did not alter their amplitude distribution. During toxin action, periods of high-frequency mEPSP discharge were punctuated by periods in which mEPSP frequency returned toward control levels. EPSPs were increased in amplitude during periods of enhanced mEPSP discharge. alpha-LCTX had no effect when applied in Ca(2+)-free saline, but subsequent addition of Ca(2+) caused an immediate enhancement of mEPSP frequency even when alpha-LCTX was previously washed out of the bath with Ca(2+)-free saline. Furthermore removal of Ca(2+) from the saline after alpha-LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus alpha-LCTX binding is insensitive to Ca(2+), but toxin action requires extracellular Ca(2+) ions. Preincubation with wheat germ agglutinin prevented the effect of alpha-LCTX but not its binding. These binding characteristics suggest that the toxin may bind to a crustacean homologue of latrophilin/calcium-independent receptor for latrotoxin, a G-protein-coupled receptor for alpha-latrotoxin (alpha-LTX) found in vertebrates. alpha-LCTX caused "prefacilitation" of EPSP amplitudes, i.e., the first EPSP in a train was enhanced in amplitude to a greater degree than subsequent EPSPs. A similar alteration in the pattern of facilitation was observed after application of the Ca(2+) ionophore, A23187, indicating that influx of Ca(2+) may mediate the action of alpha-LCTX. In nerve terminals filled with the Ca(2+) indicator, calcium green 1, alpha-LCTX caused increases in the fluorescence of the indicator that lasted for several minutes before returning to rest. Neither fluorescence changes nor toxin action on mEPSP frequency were affected by the Ca(2+) channel blockers omega-agatoxin IVA or Cd(2+), demonstrating that Ca(2+) influx does not occur via Ca(2+) channels normally coupled to transmitter release in this preparation. The actions of alpha-LCTX could be reduced dramatically by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid. We conclude that induction of extracellular Ca(2+) influx into nerve terminals is sufficient to explain the action of alpha-LCTX on both spontaneous and evoked transmitter release at crayfish neuromuscular junctions.     

The termination of transmitter action at the crustacean excitatory neuromuscular junction.

1. Excitatory junctional currents (e.j.c.s) and miniature excitatory junctional currents (mine.e.j.c.s) have been followed by recording the focal extracellular potential at excitatory neuromuscular junctions of Maia squinado, the Spider Crab. 2. If L-aspartate (concentrations less than or equal to 1 mM) is present in the saline, the average e.j.c. is prolonged by an increase in the duration of its falling phase. 3. No change occurs in the time course of the probablility of release of quanta in the e.j.c. as determined from the histogram of first quantal latencies. 4. The min.e.j.c. is also prolonged by L-aspartate (concentrations less than or equal to 0-5 mM). The rise time increases slightly, the pre-exponential period of the decay phase almost doubles, and the time constant of the final exponential decay increases to a value larger than the average lifetime of the elementary conductance event produced by L-glutamate. 5. Changes in e.j.c.s and min.e.j.c.s produced by aspartate show a striking similarity to the action of neostigmine on the time course of the vertebrate end-plate current. It is proposed that aspartate exerts its action by blocking a process normally reponsible for clearing the synaptic cleft of transmitter.     

Probing the endogenous Ca2+ buffers at the presynaptic terminals of the crayfish neuromuscular junction

Ca2+ indicators of varying affinity and mobility were pressure injected into the presynaptic axon of the inhibitor of the crayfish neuromuscular junction (NMJ). Fluorescence transients recorded at a 2-kHz resolution were used to probe physiological parameters governing the decay of fluorescence transients within 100 ms after an action potential (early decay). Blocking Ca2+ extrusion or Ca2+ sequestration processes did not significantly alter early decay, arguing against a role for either mechanism. Fluorescence transients recorded with low mobility or fixed indicators exhibited early decay similar to that recorded with indicators of comparable affinity but high mobility, suggesting that early decay was not due to the rate of Ca2+-indicator diffusion. The extent of early decay correlated closely with the affinity, but not mobility, of the Ca2+ sensitive dyes tested. These results implicate intrinsic buffers with slow Ca2+ binding kinetics as the most likely determinants of early decay. However, computer simulations showed that intrinsic buffers with a slow binding rate are unlikely to be the only ones present in the system because the slow kinetics would be unable to buffer incoming Ca2+ during an action potential and would result in momentary indicator saturation. In fact, experimental data show that the peak amplitude of an action potential activated Ca+ transient is about 20% of the maximal fluorescence intensity activated by prolonged Ca2+ influx. We conclude that endogenous buffering at the crayfish NMJ includes both fast and slow components, the former being fast enough to compete with fast Ca2+ indicators, and the latter dictating the early decay.     

Activation of transmitter release by strontium and calcium ions at the neuromuscular junction

1. The interaction between Ca and Sr ions on quantal transmitter release at the frog neuromuscular junction was studied, using conventional electrophysiological techniques.

2. While Ca ions always activate transmitter release, the activating action of Sr ions depends on the Ca ion concentration in the medium; at low [Ca], strontium ions enhance the release, but at higher [Ca] they inhibit it. It is postulated that there is a [Ca] at which Sr ions do not affect transmitter liberation.

3. When Sr activates release, its effect and the effect of Ca add in a more than linear fashion.

4. Magnesium ions inhibit the release induced by Sr.

5. The results can be explained by assuming that Ca and Sr act on the same site, at some stage of the process of quantal transmitter release. The affinity of both ions towards the sites is approximately the same, but the effectiveness of Sr is much smaller.

     

An analysis of the role of calcium in facilitation at the frog neuromuscular junction

1. The force-velocity relationship and the stress-strain curve of the so-called series elastic component (s.e.c.) of frog sartorius, semitendinosus and gastrocnemius have been determined during shortening against a given force (isotonic quick-release) and at high speed (controlled release): (a) from a state of isometric contraction and (b) after stretching of the contracted muscle. In both cases the muscle was released from the same length: this was usually slightly greater than the muscle's resting length.2. The muscle released immediately after being stretched is able to shorten against a constant force, P, equal to or even greater than the isometric force, P(0), at the same length. When the force P applied to the muscle is reduced below P(0) the velocity of shortening is greater after stretching, and the force-velocity curve is therefore shifted along the velocity axis: the shift is maximal when P is near to P(0) and it decreases rapidly with decreasing P.3. The extent of shortening of the s.e.c. required to make the force fall from P(0) to zero is 50-100% greater when the muscle is released immediately after stretching than when it is released from a state of isometric contraction. This difference is found by using either the controlled release method or the isotonic quick-release method.4. If a time interval is left between the end of stretching and the onset of shortening of the contracted muscle (controlled release method), the length change of the s.e.c., for a given fall of the force, is reduced and approaches that taking place when the muscle is released from a state of isometric contraction.5. Curare does not affect the results described above, indicating that these do not depend on modification of the neuromuscular transmission.6. It is concluded that stretching a contracted muscle modifies temporarily: (a) its elastic characteristics, as shown by the greater amount of mechanical energy released for a given fall of the force at the muscle's extremities, and (b) its contractile machinery, as it is suggested by the change of the force-velocity relationship.     

The effect of procaine on the action of acetylcholine at the neuromuscular junction.

1. The effect of procaine on acetylcholine-induced membrane fluctuations (ACh noise) was studied by intracellular and focal external recording from end-plates of frog sartorius muscle. 2. With intracellular recording, ACh noise (the ratio of variance to mean depolarization) was substantially reduced by procaine, suggesting a greatly decreased amplitude of the elementary potential change. 3. Spectral analysis of the ACh noise indicated a dual average time course of the underlying "shot effects", similar to the complex shape (brief spike followed by a long tail of low intensity) of the end-plate current in pro-aine-treated muscle. 4. The post-synaptic blocking action of procaine can be largely explained by the drastic shortening of the initial high-intensity phase of the end-plate conductance change. 5. The average time course of the ACh shot effects is discussed in terms of alternative hypotheses, one attributing the complex shape to each elementary event, the other involving sequential reaction steps which produce two unequal populations of ion gates of different "life-times".     

Safety factor at the neuromuscular junction

Reliable transmission of activity from nerve to muscle is necessary for the normal function of the body. The term 'safety factor' refers to the ability of neuromuscular transmission to remain effective under various physiological conditions and stresses. This is a result of the amount of transmitter released per nerve impulse being greater than that required to trigger an action potential in the muscle fibre. The safety factor is a measure of this excess of released transmitter. In this review we discuss the practical difficulties involved in estimating the safety factor in vitro. We then consider the factors that influence the safety factor in vivo. While presynaptic transmitter release may be modulated on a moment to moment basis, the postsynaptic features that determine the effect of released transmitter are not so readily altered to meet changing demands. Different strategies are used by different species to ensure reliable neuromuscular transmission. Some, like frogs, rely on releasing a large amount of transmitter while others, like man, rely on elaborate postsynaptic specialisations to enhance the response to transmitter. In normal adult mammals, the safety factor is generally 3-5. Both pre- and postsynaptic components change during development and may show plasticity in response to injury or disease. Thus, both acquired autoimmune and inherited congenital diseases of the neuromuscular junction (NMJ) can significantly reduce, or even transiently increase, safety factor.