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.

     

Interaction between sodium and calcium ions in the process of transmitter release at the neuromuscular junction

1. The interaction between Na and Ca ions on quantal transmitter release at the frog neuromuscular junction has been studied, using intracellular recording and averaging of responses.2. At low calcium concentrations, partial withdrawal of Na ions increases end-plate potential (e.p.p.) amplitudes and quantal content (m) and decreases the amplitude of the miniature e.p.p.s (m.e.p.p.s). Under these conditions the relation between [Ca] and m is highly non-linear. When plotted on double logarithmic co-ordinates withdrawal of [Na] causes a nearly parallel shift of this relation.3. Mutual interaction occurs between Ca, Na and Mg in transmitter release. With a constant low [Ca] in the medium, withdrawal of [Na] produces a smaller increase in m when [Mg] is high, than when [Mg] is low.4. In the presence of normal [Ca] (1.8 mM), [Na] withdrawal decreases the amplitude of the e.p.p. and produces a small decrease in m.5. The results can be explained by assuming that [Na] reduction has two mutually opposing effects on transmitter release: it makes more sites available for the action of Ca, and it lowers the amplitude of the action potential in the nerve terminals. The former effect dominates at low, the latter at high, calcium concentrations.     

Lithium ions and the release of transmitter at the frog neuromuscular junction.

1. Transmitter release has been examined at the frog neuromuscular junction when all or part of the Na of the Ringer is replaced by Li ions. 2. No immediate change occurs in either the mean quantal content of the end-plate potential or the miniature end-plate potential frequency on changing to Li Ringer but over the following hour both these quantities increase by more than two orders of magnitude. 3. During thefirst 30-40 min of an exposure to Li-Ringer the m.e.p.p. frequency rises exponentially with a time constant of 10 min, and the mean quantal content of the e.p.p. grows by addition of extra evoked quanta, the increment rising exponentially with a time constant the same as that of the m.e.p.p. frequency. 4. Following this initial period in Li-Ringer the m. e.p.p. frequency accelerates to a peak of several hundred quanta per second and then declines slowly over the next few hours. Just before the m.e.p.p.frequency peak the conduction velocity of the presynaptic action potential declines and shortly afterwards synaptic transmission fails as the action potential no longer conducts into the terminals. 5. The rise in the m.e.p.p. frequency during the first 30-40 min is independent of the [Ca-2+]o. At subsequent times before the peak external Cabecomes progressively more effective in accelerating the m.e.p.p. frequency and in the presence of 1mM-EGTA spontaneous release stabilizes at 60-80 quanta/sec. 6. The [Li-+]o strongly influences the rate of increases in both evoked and spontaneous release but not their extent; replacing only half the Na of the Ringer by Li increases the time constants of the increases to about 30 min. 7. Rises in the m.e.p.p. frequency can be irreversibly accelerated by tetanizing the nerve in a Li-Ringer in which the Ca has been chelated by EGTA. The extent of the increases in the m.e.p.p. frequency is dependent on the number of pulses in the tetanus and is little affected by the frequency of stimulation. Accumulation of Li ions inside the presynaptic terminals probably underlies the changes in spontaneous release. 8. When only 10 percent of the Na of the Ringer is replaced by Li-+ ions the magnitude of post-tetanic potentiation of the e.p.p. and of the post-tetanic rise in the m.e.p.p. frequency is increased. Under these conditions changes in facilitation of the e.p.p. are small. 9. Various mechanisms by which Li could alter transmitter release are discussed and it is suggested that intracellular Ca sequestering mechanisms of the presynaptic terminals are affected when an end-plate is exposed to Li-Ringer.     

Delayed release of transmitter at the frog neuromuscular junction

1. After the end-plate potential (e.p.p.) there is an increase in the frequency of the miniature e.p.p.s. This delayed release of transmitter was studied at the frog neuromuscular junction, using conventional intracellular and extracellular recording techniques. E.p.p. amplitude was kept subthreshold by subnormal concentrations of activating divalent ions.

2. The ratio delayed release: initial release had values between 2 and 140%, depending on the experimental conditions; it decreased with an increase in Ca concentration and quantal content.

3. Delayed release is larger at low temperature than at room temperature.

4. Delayed release is statistically independent of the amplitude of the preceding e.p.p.

5. The time course of the decay of the delayed release is monotonic when strontium is the activating divalent ion; it shows a `dip' in more than 50% of the cases when Ca activated release.

6. The results were discussed in terms of the residual Ca ion hypothesis.

     

Competition between sodium and calcium ions in transmitter release at mammalian neuromuscular junctions

1. Frequencies of miniature end-plate potentials (m.e.p.p.s) were recorded at neuromuscular junctions in rat diaphragm-phrenic nerve preparations in vitro.2. In the presence of raised [K] (15-20 mM) lowering [Na] caused a rapid increase in m.e.p.p. frequency whether [Ca] was low or normal. Raising [Na] towards the normal concentration (162 mM) caused a slow fall in frequency and raising [Ca] in the range 0.32-2 mM caused a slow increase in frequency. These effects were less in the normal [K] (5 mM).3. Mean m.e.p.p. frequencies were determined for solutions containing 15 mM-K and combinations of [Ca] and [Na]. M.e.p.p. frequency varied inversely with [Na] when [Ca] was constant. In each of the three Na concentrations used (162, 113 and 65 mM) raising [Ca] in the range 0.32-2 mM increased m.e.p.p. frequency but when raised above 2-3 mM, Ca depressed frequency.4. A model was proposed in which Ca affected transmitter release by changing the concentration in the presynaptic membrane of a complex CaX to which the rate of transmitter release was directly proportional. Higher concentrations of Ca depressed transmitter release by inactivating CaX. Sodium ions competitively depressed release either by competing with calcium ions for association with X or by reducing the affinity of X for Ca.5. When [Na] was lowered in solutions containing raised [Mg] and [Ca], the increase of mean m.e.p.p. frequency was greater than that observed in raised [Ca] and normal [Mg] and was of the same order as the increases seen in low [Ca]. The result was interpreted to indicate either that Na and Mg do not compete with Ca at the same site or that Mg affects the affinity of X for Ca and Na.6. The effect of lowering [Na] on m.e.p.p. frequency was a specific effect of Na ions. When LiCl was substituted for NaCl, the increase of m.e.p.p. frequency persisted. Changes in [Cl] had no effect on m.e.p.p. frequency.7. There was a linear relation between the mean logarithm of m.e.p.p. frequencies and [K], the slope of the relation increasing as [Na] was lowered. Conversely, lowering [Na] caused a greater increase in m.e.p.p. frequency as [K] was raised.8. The variation of m.e.p.p. frequencies in a diaphragm was roughly proportional to a second or higher power of [Na] and inversely proportion to [Ca]. It was thought that this could be due to differences in chelation of Ca which were more apparent at low Ca concentrations.9. The similarities between the effects of Na, Ca and K on m.e.p.p. frequency and the effects of these ions on Ca-influx in heart muscle led to the suggestion that transmitter release is proportional to the concentration of a negatively charged complex of a carrier X with one calcium ion (CaX) at the internal surface of the membrane and that changes in membrane potential affect transmitter release by changing the distribution or location of CaX in the membrane.     

On facilitation of transmitter release at the toad neuromuscular junction

1. The time dependence of the increase in amplitude (facilitation) of a second end-plate potential (e.p.p.) elicited within 10-100 msec of a preceding e.p.p. was examined at neuromuscular junctions in sartorius muscles of toads. Facilitation was defined by two characteristics, initial facilitation and the time constant of its exponential decay.2. The time constant of decay of facilitation was longer at lower temperatures and the Q(10) was 4.3 in the range 10-25 degrees C. There was no significant effect of temperature on initial facilitation.3. Ouabain (10(-4)-10(-3)M), lithium substitution for sodium, sodium azide (5 mM) and N-ethylmaleimide (NEM, 0.1 mM) initially had no effect on initial facilitation or the decay of facilitation. After some time, they all caused a longer time constant of decay of facilitation and a depression of initial facilitation.4. It was concluded that the decay of facilitation is not directly dependent on active transport of sodium ions, calcium efflux, ATP-dependent movements of calcium or mitochondrial uptake of calcium following an action potential.5. Ouabain, lithium, sodium azide, and NEM all caused an increase in transmitter release. This effect, and the late effects on facilitation, were thought to be due to an increase in intracellular calcium concentration in nerve terminals.6. No relationship was found between the quantal content of e.p.p.s (range, 0.8-100) and initial facilitation, or the time constant of decay of facilitation.7. Substitution of strontium for calcium ions caused a marked prolongation of the time constant of decay of facilitation, and a depression of initial facilitation.8. The results were consistent with the hypothesis that the time constant of decay of facilitation is related to the rate of disappearance of an ;active' complex of calcium (CaA) which, of itself, is not sufficient for transmitter release. It is suggested that an action potential produces CaA which decays with the time constant of facilitation and CaS, a short-life complex of calcium which decays with the time constant of the phasic release of transmitter. The release of transmitter is proportional to some function of [CaA] and [CaS].     

The kinetics of transmitter release at the frog neuromuscular junction

1. Fluctuations in the latency of focally recorded end-plate currents were analysed to determine the time course of the probabilistic presynaptic process underlying quantal release evoked after single nerve stimuli at the frog neuromuscular junction.

2. The early falling phase of the presynaptic probability function can be fitted by a single exponential over two orders of magnitude of quantal release rate. The time constant of the early falling phase is about 0·5 msec at 11° C, and increases with decreasing temperature with a Q₁₀ of at least 4 over the range 1-12° C.

3. After this early exponential fall, quantal release probability returns to control levels with a much slower time course.

4. Conditioning nerve stimuli increase the magnitude and slightly prolong the early time course of release evoked by a test stimulus. When facilitation is calculated for matched time intervals following the conditioning and testing stimuli, it is found that the magnitude of the small, late residual tail of release is facilitated by a greater percentage than the magnitude of larger, early portions of release.

5. These results are discussed in terms of the hypothesis (Katz & Miledi, 1968) that evoked release and facilitation are mediated by a common presynaptic factor which activates release in a non-linear manner.

     

Strontium and quantal release of transmitter at the neuromuscular junction

1. Previous work has shown that in calcium-free solutions nerve impulses invade the motor nerve terminals at the neuromuscular junction, but fail to release transmitter. In these conditions, strontium ions applied iontophoretically to a minute part of a junction, or to the whole muscle by bath application, restore to the nerve impulse its ability to release transmitter.2. As with calcium, the transmitter released in the presence of strontium is in the form of packages (quanta) whose release can be predicted from Poisson's Theorem.3. The mean number of quanta released by a nerve impulse increases with the concentration of strontium. Strontium is much less effective than calcium in equimolar concentrations.4. Transmitter quanta released in the presence of strontium evoke larger unit potentials than quanta released in the presence of calcium. The larger size of the Sr-unit potentials is caused by a prolongation of transmitter action, presumably due to a post-synaptic effect of strontium.5. Neuromuscular transmission was blocked in some fibres when the concentration of strontium was raised beyond 10 mM. This junctional block was presumably due to a failure in the propagation of nerve impulses.6. The post-stimulation increase in the frequency of miniature end-plate potentials, which is normally seen in calcium solutions, is also observed when calcium is substituted by strontium. The post-stimulation effect increases with the concentration of strontium.7. It is concluded that strontium can substitute for calcium in the process of quantal release of transmitter. The physico-chemical mechanism of this substitution remains unknown.     

A new type of transmitter release at the neuromuscular junction

Examination of spontaneous miniature endplate potentials (MEPPs) in murine skeletal muscle has revealed that in conditions such as botulinum poisoning, during nerve terminal regeneration or in the presence of the drug 4-aminoquinoline, two types of acetylcholine release are responsible for the MEPPs. In addition to the MEPPs which correspond to the quantal component of a nerve impulse-evoked endplate potential a second type of acetylcholine release occurs. The latter type of transmitter release gives rise to MEPPs with a more prolonged time-to-peak and frequently a larger than normal amplitude. It is unaffected by nerve terminal depolarization and transmembrane Ca2+ fluxes. The relationship between MEPP frequency and temperature has a Q10 of about 12 compared to 2-3 for normal MEPPs. In botulinum-poisoned muscles this secretory type of transmitter release dominates, being exclusively present in muscles where nerve stimulation fails to release transmitter. In normal muscle such a release is induced by 4-aminoquinoline which may cause up to 45% of all the spontaneous MEPPs to be of that kind. It is suggested that the described spontaneous secretion of acetylcholine serves in inductory and neurotrophic function.     

Depression of transmitter release at the neuromuscular junction of the frog

1. Depression of transmitter release produced by preceding conditioning stimulation was studied at the frog's neuromuscular junction.2. Depression occurred when transmitter release was restricted to a short length of nerve terminal, or when release was initiated by electrotonic depolarization of the terminals after action potentials were abolished by tetrodotoxin.3. Quantitative studies revealed a non-linear relationship between the estimated magnitude of ;zero-time' depression and the amount of transmitter released by conditioning stimulation.4. Two stimuli separated by 20-200 msec were given and the ratio of the end-plate potential amplitudes (V(2)/V(1)) was measured. This amplitude ratio increased during depression produced by stimuli preceding the test pair.5. These observations may be explained by assuming that depression is associated with a reduction in release probability as well as a depletion of transmitter available for release.     

Presynaptic membrane potential and transmitter release at the crayfish neuromuscular junction

Release of transmitter was evoked at neuromuscular junctions of the crayfish opener muscle by passage of current through an intracellular electrode impaling a branch of the motor axon close to a muscle fiber. Membrane-potential changes in the presynaptic axon branch were monitored, together with postsynaptic potentials. Depolarization of impaled secondary axonal branches by more than 10 mV led to an increase in asynchronous transmitter release. The release was facilitated by prolonged (50-500 ms) depolarizations and it decayed rapidly when depolarization was terminated. Ca2+ was essential for facilitated release; however, no indication of a Ca spike was found at the recording site. Input-output curves for the synapse were obtained by applying depolarizing pulses of varying amplitude to the axon branch. Transmitter output was strongly influenced by both amplitude and duration of the applied depolarization. During normal synaptic transmission, propagated Na+-dependent action potentials were recorded in the secondary axonal branches but there was no evidence for a calcium-dependent component for these action potentials. Evoked release was dependent on Ca2+ and was steeply dependent on the amplitude of the action potential, which could be made variable in size by application of tetrodotoxin (TTX). Prolonged depolarization of axonal branches resulted in enhancement of transmitter release evoked by an action potential. The enhancement occurred in spite of a simultaneous reduction of the amplitude of the action potential. Morphological features of the terminals were investigated after injection of lucifer yellow into the axon. An electrical model incorporating the morphological features suggests that membrane-potential changes set up in the main axon reach the nearest terminals with 30-40% attenuation, while events originating in the terminals would be severely attenuated in the main axon. Comparison of the crayfish synapse with other frequently studied synapses shows both similarities and differences, suggesting that it is not possible to apply findings made in one synapse to all others.     

Estimates of probability of transmitter release at the mammalian neuromuscular junction

1. Intracellular records of end-plate potentials (e.p.p.s) were obtained from curarized neuromuscular junctions in the rat diaphragm.2. Statistical estimates of the quantal release probability (p) were made at individual junctions from measurements of the means and variances of e.p.p. amplitudes at two different levels of Ca concentration. It was assumed that the release process was binomial and that the number of quanta available for release (n) was independent of external Ca.3. These estimates of p were compared with those obtained by measuring depression of e.p.p. amplitudes after single conditioning shocks and assuming that the depression was due to depletion of n. The statistical estimates were consistently smaller.4. This disparity, plus the additional observation that depression was not linearly related to the number of quanta released by one or more conditioning shocks, suggested that depression was not due entirely to depletion and that the depletion assumption led to an over-estimate of p.5. A third method of estimating p from measurements of the decline of e.p.p. amplitudes during rapid stimulation also appeared to result in an over-estimate.     

The binomial nature of transmitter release at the crayfish neuromuscular junction

1. Transmitter release at excitatory junctions on the opener muscle of the crayfish dactyl was studied by recording junctional potentials with extracellular micro-electrodes.2. At low temperatures, evoked release was dispersed sufficiently in time for potentials produced by individual quanta to be counted, and the mean (m) and variance (sigma(2)) of the quantum content distribution for a series of trials measured directly. These values were used to calculate the average probability of quantal release (p), assuming a binomial distribution.3. For all values of m and p, the observed release pattern (number of 0, 1, 2, 3,... quantal releases during a series of trials) was approximated closely by the corresponding binomial distribution. However, Poisson predictions differed significantly from the observed quantal distribution for values of p > 0.2.     

Stochastic properties of spontaneous transmitter release at the crayfish neuromuscular junction

1. Miniature excitatory junctional potentials (min.e.j.p.s) were recorded with an intracellular electrode from the adductor muscle of the dactyl of the first or second walking leg of the crayfish, Orconectes virilis.

2. The intervals between the min.e.j.p.s were compared to the exponential prediction by five goodness of fit tests. The results indicate that the intervals are not exponentially distributed.

3. The autocorrelogram of intervals shows that the intervals are unlikely to be independent.

4. A stochastic analysis that includes the power spectrum of intervals, the variance—time curve, and the ln-survivor curve suggest that there is a clustering of min.e.j.p.s. The results are similar to those on the frog neuromuscular junction.

5. An autocorrelogram of the min.e.j.p. amplitudes suggests that sizes are not independently distributed.

6. These results, which are similar to those previously reported from the frog neuromuscular junction, support the use of the branching Poisson process as a theoretical model for the stochastic properties of spontaneous quantal release of transmitter.

     

Tonic release of transmitter at the neuromuscular junction of the crab.

1. Synaptic transmission was studied at the neuromuscular junction of the crab Ocypoda cursor, using conventional electrophysiological technique. 2. It was found that fibres of the extensor muscle and those composing the internal layer of the closer muscle have only post-synaptic inhibition (S fibres) while the fibres at the external layer of the closer muscle have in addition presynaptic inhibition (R fibres). 3. In S fibres, addition of GABA reduces input membrane resistance (Rm) and e.p.s.p. amplitude approximately to the same degree. The effect shows desensitization. In R type fibres, GABA reduces the e.p.s.p. much more than expected from changes in Rm. The post-synaptic effect of GABA on Rm shows desensitization, while the presynaptic effect does not show desensitization. 4. In about 50 percent of the cases, after desensitization occurred, Rm increased by about 10-30 percent above the control. Similar increase in Rm occurred after application of picrotoxin. These results suggest that initially the membrane resistance was lower due to tonic release of inhibitory transmitter. 5. The Q10 of Rm was found to vary between 2 and 3. In Ca2+ free media, Cl- free media, or in picrotoxin the Q10 is about 1-3. 6. In R fibres, addition of picrotoxin increased the amplitude of the e.p.s.p. by 30-60 percent above the expected increase due to changes in Rm. 7. In S fibres the mean slope of log e.p.s.p. vs. log [Ca2+] was found to be 1-63, while in R fibres the slope was 0-93. These results suggest the presence of tonic release of the inhibitory transmitter which acts both post-synaptically and presynaptically.     

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.     

Zinc competitively inhibits calcium-dependent release of transmitter at the mouse neuromuscular junction

The effect of zinc on the release of transmitter was investigated in preparations of mouse diaphragm by conventional microelectrode techniques. The frequency (FP of miniature end-plate potentials (MEPPs), elevated by Ca²⁺ in high K⁺ medium, was reduced by zinc in a concentration-dependent fashion. When the extracellular concentration of Ca²⁺ ([Ca²⁺]_o) was varied in the absence of zinc, a linear relationship between log(F) and log([Ca²⁺]_o) was obtained. When the effect of zinc was depicted graphically, it was found that zinc shifted the relationship between log(F) and log([Ca²⁺]_o) to the right, with respect to the control in the absence of zinc, without altering the slope. Zinc also reduced the quantal content (m) of end-plate potentials (EPPs). As [Ca²⁺]_o was varied in the absence of zinc, a linear relationship between ln(m) and ln([Ca²⁺]_o) was observed. Zinc shifted this linear relationship between ln(m) and ln([Ca²⁺]_o) to the right, with respect to the control, without altering the slope. Thus, zinc reduced both the asynchronous and the phasic release of transmitter. These results suggest that zinc competes with Ca²⁺, and this conclusion is confirmed by examination of a modified Lineweaver-Burk plot of the data. Zinc probably inhibits the entry of Ca²⁺ into the nerve terminals, thereby inhibiting transmitter release.