Spontaneous quantal and subquantal transmitter release at the Torpedo nerve-electroplaque junction

Focal electrodes were used to record the spontaneous miniature potentials generated on delimited patches of innervated membrane in the Torpedo electric organ. The main population of miniature potentials followed a bell-shaped amplitude distribution. In addition, we observed a second class of spontaneous events that were smaller and whose amplitude distribution was skewed. These sub-miniatures formed an homogenous population together with the regular miniatures with respect to their time course versus amplitude relationship. They were thus probably generated at the same sites. The proportion of potentials that were subminiature was less than 10% in resting, freshly excised tissue, but it increased markedly:

(i) when the tissue was kept for 24–28 h in vitro after excision;

(ii) in the period following a brief heat challenge or

(iii) stimulation to exhaustion;

(iv) in the presence of dinitrophenol or dinitrofluorobenzene. In all these conditions, we measured the acetylcholine, adenosine 5′-triphosphate and creatine phosphate content of the tissue and found a correlation between the relative number of subminiature potentials and the lack of energy rich molecules.

It is concluded that subminiature potentials are present in the electric organ as in neuromuscular junctions. They are probably produced at the same sites as the regular miniature potentials and their relative occurrence seems to increase greatly when the nerve terminals are in a state of energy deficiency.     

Synaptic potentials in nerve cells of the stellate ganglion of the squid

1. Intracellular recordings were made from nerve cells in the stellate ganglion of the squid.2. Stimulation of the preganglionic nerve evoked excitatory or inhibitory synaptic potentials, or a combination of both. Antidromic stimulation of the stellar nerves also evoked excitatory and inhibitory potentials in the cells. With both types of stimulation the synaptic potentials were built up of contributions from several axons indicating considerable convergence of excitatory and inhibitory inputs on the cells.3. Inhibitory, as well as excitatory, miniature synaptic potentials were recorded from the cells even after impulse activity had been blocked by tetrodotoxin.4. Glutamate applied iontophoretically to some cells produced a depolarization of their membranes. In other cases glutamate evoked a hyperpolarizing potential. Application of glutamate caused a decrease in the amplitude of excitatory synaptic potentials.     

Spontaneous quantal transmitter release: a statistical analysis and some implications

1. Miniature end-plate potentials (m.e.p.p.s) were intra- and extracellularly recorded from neuromuscular junctions in rat phrenic nerve-diaphragm preparations in vitro.2. Statistical analysis of the intervals between m.e.p.p.s showed that when the mean number of events in time t was plotted as a function of the variance of the events in time t there was a significant deviation from the straight line relationship expected for a Poisson process. Computer simulation showed that this deviation is explicable if release was generated by the random phasing of the activity of a number of releasing sites.3. There was no indication that release of one quantum influences the probability of release of remaining quanta (drag, clustering). It is suggested that m.e.p.p.s whose amplitude is larger than the mode result from the release of the contents of vesicles whose volume is also supramodal.4. The effects of depolarization of nerve terminals upon the variance-mean curve suggest an increase in the activity of sites rather than an increase in their number.5. Statistical analysis indicated at least 200 +/- 100 (mean +/- 1 S.E.) releasing sites. This number is of the same order as the number of sites of vesicle aggregation and presynaptic membrane density seen in electron micrographs of nerve terminals of this preparation.     

The effect of prolonged depolarization on synaptic transfer in the stellate ganglion of the squid

1. Depolarization of the giant axon terminal of the squid causes local calcium influx which gives rise to transmitter release and post-synaptic response, and which under certain experimental conditions leads to a regenerative action potential in the presynaptic terminal itself.2. There has been conflicting evidence in the literature on the question whether the calcium permeability change in the terminal is rapidly inactivated, or whether it can persist with little diminution for hundreds of milliseconds during a depolarizing voltage step.3. Results are presented which show that there is little ;calcium inactivation', even when very large depolarizing steps are imposed on the terminal and maintained for periods of 1-2 sec.4. Contrary indications are examined and found to be attributable to an increase of potassium conductance, rather than direct inactivation of calcium conductance.     

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.     

Inhibition of quantal transmitter release by the polycationic ligand cationized ferritin

Summary Cationized ferritin (200 g ml^–1) causes a reduction in both spontaneous and evoked transmitter release at the frog neuromuscular junction. The reduction in EPP amplitude probably reflects reduced Ca²⁺-influx since full recovery can be achieved by elevation of [Ca²⁺]_o. The effect on MEPP frequency is independent of [Ca²⁺]_o and may well reflect a direct effect on exocytosis.     

Control of quantal transmitter release at frog's motor nerve terminals

Motor terminals on the cutaneous pectoris muscle of the frog were depolarized by current pulses through the recording macro-pathch-clamp electrode and the resulting quantal release was measured (excitation blocked with TTX). Above a threshold release increased very steeply with depolarization until saturation was approached. The dependence of release on duration of depolarization was even steeper: doubling pulse duration often produced more than 100-fold release (early facilitation) Distributions of delays of quantal release after the depolarization pulse were determined for wide ranges of depolarizations and pulse durations. The shape of these distributions was little affected by large changes in average release; increasing the temperature from 0°C to 10°C about halved the time scale of the distributions. Lengthening the depolarization from 0.5 to 6 ms produced a latency shift: the distributions of delays were shifted by almost the increase in pulse duration. At 5–6 ms pulse duration a few releases occurred during the final millisecond of the pulse. It is suggested that the time course of the phasic release is not controlled by the time course of changes in intracellular calcium concentration, but by an activator which is produced about proportional to supra-threshold pulse amplitude and duration, and that this activator effects release with a cooperativity of 6–7. An additional depolarization produced repressor is responsible for the minimum delay.Supported by the Deutsche Forschungsgemeinschaft     

Control of quantal transmitter release at frog's motor nerve terminals

Quanta of transmitter were released from motor nerve terminals of the frog by a depolarizing 'releasing pulse'. 'Modulating pulses' were subthreshold for release; pre-pulses were added directly before and post-pulses directly after the releasing pulse. Modulating depolarization pulses enhanced release up to 20-fold, and such hyperpolarizations suppressed release up to 10-fold. Pre- and post-pulses were about equally effective. In a wide range these modulations did not affect the facilitation of a test-EPSC by the preceding releasing pulse; modulation thus is not mediated by changes in Ca-inflow. It is suggested that phasic release is largely controlled by an 'activator' which is generated by depolarization, and that modulating pulses increase this activator when depolarizing, and decrease this activator below its resting level if hyperpolarizing. If an interval was interposed between pre- and releasing pulse, the modulating effect decreased very steeply with increasing interval for the first 2 ms, and much slower for longer intervals. Distributions of delays of quantal releases showed a time course of decay very similar to the decay of modulation with increasing interval. Both decays may reflect the exponential decay of activator. Depolarizing post-pulses increased the minimal synaptic delay and the delay of maximal release, and hyperpolarizing ones had the opposite effects. They are interpreted to modulate the generation and decay of a 'repressor', which is produced by depolarization and is responsible for the minimal synaptic delay and the delayed maxima of release. A speculative scheme of interactions of [Ca]i, activator and repressor is discussed.     

Muscarinic inhibition of quantal transmitter release from the magnesium-paralysed frog sartorius muscle

The existence of presynaptic muscarinic acetylcholine receptors on motor nerve terminals of the isolated frog sartorius muscle was investigated. The modulatory role of these receptors was studied by observing the effects of muscarinic ligands on the frequency of miniature endplate potentials and on the quantal content of endplate potentials. The agonist oxotremorine reduced in concentration-dependent fashion the frequency of spontaneous potentials and the amplitude of evoked potentials. Also, high concentrations of oxotremorine depolarized the postsynaptic membrane and reduced the amplitude of the miniature endplate potentials. The depolarizing action of the drug was blocked byd-tubocurarine. The muscarinic antagonist atropine attenuated agonist-induced reductions in endplate potential amplitude and miniature endplate potential frequency but did not affect the depression in amplitude of the spontaneous potentials evoked by oxotremorine. It is concluded that activation of presynaptic muscarinic receptors inhibits the release of acetylcholine from motor nerve terminals.

Atropine itself had no effect on the quantal content of evoked potentials or on the frequency of spontaneous potentials suggesting that the nerve terminal is not affected by non-quantal acetylcholine.     

A study of the mechanism of quantal transmitter release at a chemical synapse

1. The nerve-muscle preparation of the cutaneous pectoris of the frog has been used to study quantal transmitter release.2. When the osmotic pressure of the external solution is raised 1.5-2 fold, the frequency of miniature end-plate potentials (m.e.p.p.s) rises by 1.5-2 orders of magnitude. This effect is independent of the presence of Ca(2+) ions and of the nature of the substances by which the osmotic pressure has been increased.3. In Ca(2+) free hypertonic solution the nerve impulse still invades the nerve terminals but does not alter the frequency of the m.e.p.p.s.4. The arrival of the impulse in the terminals causes an immediate increase in the rate of quantal release, provided divalent cations are present whose passage through the axon membrane is facilitated by excitation (Ca(2+), Sr(2+), Ba(2+)).5. Divalent cations which penetrate only slightly (Mg(2+), Be(2+)) lower the frequency of m.e.p.p.s and suppress the end-plate potential (e.p.p.) evoked by an impulse, in the presence of Ca(2+) ions. Be(2+) is a more effective inhibitor than Mg(2+).6. In Ca(2+) free solutions, adding Mg(2+) causes an increase in the frequency of m.e.p.p.s evoked by depolarization of the nerve endings or by treatment with ethanol.7. The trivalent cation La(3+) is more effective than divalent cations are in increasing the frequency of m.e.p.p.s. The tetravalent cation Th(4+) also raises the m.e.p.p. frequency.8. The observations summarized in paragraphs 2-7 indicate that the frequency of m.e.p.p.s at a constant temperature depends only on the concentration of uni-, di- and trivalent cations inside the nerve ending. It is suggested that the internal cation concentration influences the adhesion between synaptic vesicles and the membrane of the nerve ending.9. For a model experiment, artificial phospholipid membranes have been used to study the effect of uni-, di-, tri- and tetravalent cations on the adhesion process. At pH 7-7.4, the time required for adhesion to take place decreases with increasing cation concentration in the bath. Ca(2+) ions are 100-1000 times more effective than K(+) ions; La(3+) and Th(4+) ions are still more effective. The ;adhesion time' decreases when the pH is lowered; it increases greatly with lowering of temperature.10. The hypothesis is put forward that the mutual adhesion of artificial vesicles made of phospholipid membranes, and the adhesion between synaptic vesicles and the membrane of the nerve ending arise by a common mechanism. In both cases, the important factor is the influence of cations on the electric double layer at the membrane surface.     

The stochastic properties of spontaneous quantal release of transmitter at the frog neuromuscular junction

1. Earlier results showed that it is unlikely that spontaneous quantal release of transmitter at the frog neuromuscular junction is produced by a Poisson process.

2. Data sets were tested, by using the u statistic, to see whether if they are assumed to be generated by a Poisson process, the mean interval is changing monotonically with time. By this critieria, some of the data sets are stationary, others are not.

3. A variety of mathematical transforms are employed on empirical data sets to characterize the properties of the spontaneous quantal release.

(a) The intensity function, which calculates the frequency distribution of all possible combinations of intervals, shows an excess of short intervals, without any sign of periodicity.

(b) The variance—time curve, which estimates the accumulated variance of the series as a function of time into the series, lies significantly above the Poisson prediction.

(c) The power spectrum, whether calculated on the intervals or on the number of intervals in time bins, deviates significantly from the Poisson prediction at the low frequencies.

(d) The ln-survivor curve has two phases: a concave section for the short intervals, and a roughly linear section for the intervals of greater length.

These transforms indicate that the min.e.p.p.s are clustered.

4. A series of models for spontaneous quantal release were considered.

(a) A Poisson model. Rejected because of consistent failure to fit the data.

(b) A periodic model. Rejected because the intervals should be ordered rather than clustered.

(c) A time-dependent model, in which quantal release is governed by a Poisson process with a mean interval that is oscillating in time. This model will generate clustering; by the transforms the model can be shown to closely fit the data. However, an autocorrelation of min.e.p.p. amplitudes shows that there is a relationship between the amplitudes and their position in the series. This is not predicted by the time-dependent oscillating model.

(d) A branching Poisson model, in which a primary release, generated by a Poisson process, is likely to be followed by one or more subsidiary releases from the same site. The parameters of the branching model can be determined from ln-survivor curves. Theoretical curves, created with these parameters, give power spectra, variance—time curves, and ln-survivor curves that strongly resemble those calculated from the data. The model also predicts a significant autocorrelation of amplitudes.

5. Min.e.p.p.s recorded with an extracellular electrode also fit well to a branching Poisson model.

6. The effects of raised [Ca²⁺]_o on the intervals between min.e.p.p.s were studied. In our experiments the change in extracellular solution did not produce any notable change in release statistics.

7. The effects of elevated [K⁺]_o on the intervals between spontaneous releases were studied. Depolarization of the nerve terminal increases the frequency of primary releases and decreases the chance of having subsidiary releases.

8. Possible physical mechanisms by which quantal release of transmitter from a nerve terminal would fit a branching Poisson model are described.

     

Polyphasic synaptic potentials in the ganglion of the mollusc, Navanax.

1.Included in the ensemble of synaptic input received by identified neurones in the ganglion of the marine mollusc Navanax are biphasic synaptic potentials, consisting of a depolarization followed by a hyperpolarization. 2. Both phases are chemically mediated as judged by their susceptibility to a high magnesium medium and neither exhibits depression with repetition. 3. The hyperpolarizing phase has a reversal potential of about -50mV, which varies only with changes in the external chloride concentration. This phase is unaffected by cholinolytics. 4. The depolarizing phase reverses at a more positive potential, is probably the result of a change in sodium conductance and is blocked by hexamethonium and high concentrations of eserine. 5. The biphasic synaptic potentials are therefore similar in many respects to the biphasic response evoked by iontophoretic application of acetylcholine on to these cells, suggesting that the two types of cholinergic receptors previously characterized on these neurones are both functional.