The effects of depolarization of motor nerve terminals upon the release of transmitter by nerve impulses

1. Depolarizing currents were applied to motor nerve terminals in the rat phrenic nerve-diaphragm muscle preparation in vitro.2. During the passage of depolarizing currents the amplitude of the presynaptic nerve action potentials and of end-plate potentials (e.p.p.s) was reduced in proportion to the current strength.3. The reduction in e.p.p. amplitudes was shown to be due to a reduction in the number of quanta released.4. An excess of Mg ions or the previous application of a hyperpolarizing current could prevent the reduction of e.p.p. amplitudes and quantal contents by depolarizing current.5. Depolarizing current application prevented later hyperpolarizing currents affecting e.p.p. amplitudes.     

Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog

1. End-plate potentials were recorded intracellularly at the frog neuromuscular junction bathed in a solution containing a low concentration of calcium and a high concentration of magnesium.2. The muscle was subsequently subjected to ;cholinesterase staining', and the area of the individual end-plates, studied with intracellular electrodes, was measured.3. A positive correlation was found between the end-plate area and the diameter of muscle fibres.4. The mean quantum content (m) showed a positive correlation with the size of end-plates.5. The frequency of spontaneous miniature end-plate potentials was positively correlated with m as well as with end-plate area.6. It is concluded that the amount of transmitter released following nerve stimulation is related to the size of nerve endings.     

Mechanisms underlying short-term modulation of transmitter release by presynaptic depolarization

Presynaptic terminal depolarization modulates the efficacy of transmitter release. Residual Ca²⁺ remaining after presynaptic depolarization is thought to play a critical role in facilitation of transmitter release, but its downstream mechanism remains unclear. By making simultaneous pre- and postsynaptic recordings at the rodent calyx of Held synapse, we have investigated mechanisms involved in the facilitation and depression of postsynaptic currents induced by presynaptic depolarization. In voltage-clamp experiments, cancellation of the Ca²⁺-dependent presynaptic Ca²⁺ current ( I _pCa) facilitation revealed that this mechanism can account for 50% of postsynaptic current facilitation, irrespective of intraterminal EGTA concentrations. Intraterminal EGTA, loaded at 10 m m , failed to block postsynaptic current facilitation, but additional BAPTA at 1 m m abolished it. Potassium-induced sustained depolarization of non-dialysed presynaptic terminals caused a facilitation of postsynaptic currents, superimposed on a depression, with the latter resulting from reductions in presynaptic action potential amplitude and number of releasable vesicles. We conclude that presynaptic depolarization bidirectionally modulates transmitter release, and that the residual Ca²⁺ mechanism for synaptic facilitation operates in the immediate vicinity of voltage-gated Ca²⁺ channels in the nerve terminal.     

Statistics of transmitter release at nerve terminals

This review presents an historical account of the developments of the statistical analysis of quantal transmission over the past half century and of the progress made in using this approach to reveal new properties of nerve terminals. In the early 1950s, Katz and his colleagues showed that evoked transmitter release occurred in quanta at the neuromuscular junction, opening up the study of transmitter release at nerve terminals to statistical analysis. In the subsequent two decades attempts were made to see if evoked quantal release could be described by binomial or compound binomial statistics, as originally suggested by Katz, and to relate the parameters of the statistic to various structures of the nerve terminal. During this period two hypotheses were enunciated, namely the 'vesicle hypothesis', which states that quanta arise as a consequence of the packaging of transmitter in vesicles; and the 'active zone hypothesis', which states that vesicles undergo exocytosis at discrete sites on the nerve terminal. Unsuccessful attempts were made to relate the binomial parameter n to the elements in these hypotheses, that is to the number of active zones possessed by the terminal or the number of vesicles available for release at these zones. This difficulty was part resolved in the late 1970s with the application of non-uniform binomial statistics to transmitter release from nerve terminals, in which n is the number of active zones each with their individual probabilities, p(j). Autocorrelation functions were subsequently introduced to detect if transmitter release is quantised at a particular nerve terminal. Statistical methods which would allow discrimination between different models of transmitter release over the active zones of a terminal were then developed. The introduction of maximum likelihood estimation procedures then allowed estimates to be made of the parameters in the statistical models of quantal release. The application of these procedures to experimental data from a variety of nerve terminals provided evidence for the concept that each synapse, taken as possessing a single active zone, possesses its own individual probability of secretion of a quantum by the exocytosis of a vesicle. In the late 1960s Stevens introduced the first stochastic approach to the analysis of the kinetics of the release of a quantum of transmitter at the neuromuscular junction following an impulse. In the subsequent decades this was developed into an explicit theory for the interaction of proteins involved in regulated exocytosis of a vesicle at an active zone. The parameters were the number of transition steps in the release process (k), each occurring at the same rate (alpha), with the possibility of each of these steps becoming blocked at the same rate (gamma). Maximum likelihood estimation procedures could then be used to obtain these parameter values. The discovery was made in the 1990s of the core proteins of the SNARE complex that govern regulated exocytosis. This offers the possibility in the near future of identifying the kinetic interaction of these proteins with the parameters of the stochastic process of exocytosis which confer a particular probability on individual synapses.     

Some effects of nerve stimulation and hemicholinium on quantal transmitter release at the mammalian neuromuscular junction

1. Rat phrenic nerve-diaphragm preparations have been used to assess some effects of prolonged nerve stimulation on transmitter release.2. The amplitude of the end-plate potentials evoked by prolonged repetitive nerve stimulation fell gradually during stimulation. Most of this fall was due to a reduction in the number of transmitter quanta released by each nerve impulse; however there was also a small reduction in the muscle cell depolarization produced by each quantum of transmitter.3. Repetitive nerve stimulation also produced a small reduction in the amplitude of the miniature end-plate potentials. Recovery of amplitude occurred within about 7-8 min of ceasing stimulation.4. A much greater reduction in miniature end-plate potential amplitude accompanied prolonged nerve stimulation if hemicholinium was present in the bathing solution.5. Estimates of the ;readily available transmitter' (Elmqvist & Quastel, 1965b) were made at intervals following prolonged nerve stimulation. Readily available transmitter was reduced, and recovered over approximately 15 min.6. The relationship of these changes to the changes in nerve terminal synaptic vesicle numbers and volumes induced by similar prolonged nerve stimulation (Jones & Kwanbunbumpen, 1970) is discussed.     

Release of adrenergic transmitter from terminal nerve plexus in artery

Studies of the release of the adrenergic transmitter in the rabbit pulmonary artery have been carried out in vitro. At a stimulation frequency of 10 per second, the release was 52.9 pg per gram wet weight per pulse. The mean number of nodes or varicosities per gram wet weight of artery was 9.3×10⁷. If 80% of the released transmitter is taken up by tissues in the artery wall, these values correspond to the average release of approximately 400 molecules ofl-norepinephrine per node per pulse. The transmitter concentrations in the region of the muscle in the outermost lamina of the vessel during sympathetic activity are discussed in relation to the spatial dimensions of the neuroeffector cleft. The release of the total contents of a vesicle on the average every 7 or 8 pulses from each node is consistent with these requirements.     

Actions of lead on transmitter release at mouse motor nerve terminals

The actions of lead (Pb²⁺) on transmitter release were studied at neuromuscular junctions in mouse diaphragm in vitro. The quantal content of end-plate potentials (EPPs) was reduced by Pb²⁺ in a dose-related manner consistent with inhibition of Ca²⁺ entry into nerve terminals, with a half-maximal effect at 1.4 M (in 0.5 mM Ca²⁺ and 2 mM Mg²⁺). Pb²⁺ also inhibited the increased frequency of MEPPs (f _MEPP where MEPPs denotes miniature EPPs) produced by Ba²⁺ in the presence of raised K⁺, blocking the calculated Ba²⁺ entry half-maximally at 170 M. However, at concentrations of 50–200 nM, Pb²⁺ often increased f _MEPP in 20 mM K⁺ in the presence of Ca²⁺ and acted to promote the irreversible effect of lanthanum (La³⁺) to raise f _MEPP. In nominally Ca²⁺-free solution with 20 mM K⁺, brief (1 min) application of Pb²⁺ (20–320 M) caused rapid dose-dependent reversible rises in f _MEPP. With prolonged exposure to Pb²⁺,f _MEPP rose and then slowly declined; after removal of Pb²⁺, once f _MEPP had fallen to low levels, f _MEPP responded nearly normally to Ca²⁺ or ethanol, but not to Pb²⁺ itself. In 5 mM K⁺, 0 mM Ca²⁺ and varied [Pb²⁺] (where [ ] denotes concentration), nerve stimulation caused no EPPs, but prolonged tetanic stimulation produced increases in f _MEPP graded with [Pb²⁺] that persisted as a tail; results were consistent with growth f _MEPP with the 4th power of intracellular Pb²⁺ and removal of intracellular Pb²⁺ with a time constant of about 30 s. These results suggest that Pb²⁺ acts to block the entry of Ca²⁺ and Ba²⁺ into the terminal via voltage-gated Ca²⁺ channels through which Pb²⁺, at higher concentrations, also penetrates and then acts as an agonist at intracellular sites that govern transmitter release.     

Effects of hexamethonium on transmitter release from the rat phrenic nerve.

Hexamethonium (HEX) was applied to isolated transected diaphragm-phrenic nerve preparations of the rat in order to further elucidate the functional role of the presynaptic nicotinic autoreceptors. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the neuromuscular junctions in the presence and absence of HEX to determine the relative effect of this nicotinic antagonist on end-plate sensitivity and evoked release. In this study we show that HEX enhances transmitter release for the first few stimuli, but this action is not maintained during a train-of-six stimulation. While these results support the hypothesis that transmitter released from the nerve terminal normally has a negative feedback effect by depressing transmitter release it is proposed that HEX also has secondary actions on the neuromuscular junction that are unrelated to autoreceptor blockage. The results with HEX suggests that the presynaptic receptors may differ pharmacologically from the postsynaptic receptors.     

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.     

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     

Do some nerve cells release more than one transmitter?

The concept that each nerve cell makes and releases only one nerve transmitter (widely known as Dale's Principle) has been re-examined. Experiments suggesting that some nerve cells store and release more than one transmitter have been reviewed. Developmental and evolutionary factors are considered. Conceptual and experimental difficulties in investigating this problem are discussed. It is suggested that the term 'transmitter' should be applied to any substance that is synthesised and stored in nerve cells, is released during nerve activity and whose interaction with specific receptors on the postsynaptic membrane leads to changes in postsynaptic activity. Expressed in this way, it seems likely that while many nerves do have only one transmitter, others in some species, during development or during hormone-dependent cycles, employ multiple transmitters.     

Ion channels in presynaptic nerve terminals and control of transmitter release.

The primary function of the presynaptic nerve terminal is to release transmitter quanta and thus activate the postsynaptic target cell. In almost every step leading to the release of transmitter quanta, there is a substantial involvement of ion channels. In this review, the multitude of ion channels in the presynaptic terminal are surveyed. There are at least 12 different major categories of ion channels representing several tens of different ion channel types; the number of different ion channel molecules at presynaptic nerve terminals is many hundreds. We describe the different ion channel molecules at the surface membrane and inside the nerve terminal in the context of their possible role in the process of transmitter release. Frequently, a number of different ion channel molecules, with the same basic function, are present at the same nerve terminal. This is especially evident in the cases of calcium channels and potassium channels. This abundance of ion channels allows for a physiological and pharmacological fine tuning of the process of transmitter release and thus of synaptic transmission.     

An examination of the effects of osmotic pressure changes upon transmitter release from mammalian motor nerve terminals

1. When the frequency of miniature end-plate potentials (m.e.p.p.s) was measured at neuromuscular junctions in rat diaphragm nerve preparations in vitro bathed in solutions having osmolarities between 200 and 700 m-osmoles/l. it was found that m.e.p.p. frequency was transiently increased by exposure to osmotic gradients exceeding 75 m-osmoles/l., and then declined, within 1 hr, to a steady level slightly higher than the control level of frequency. Smaller osmotic gradients caused a maintained increase in m.e.p.p. frequency. E.p.p. quantal content was initially increased and later profoundly decreased upon exposure of preparations to solutions with an osmotic pressure of 500 or 600 m-osmoles/l. but was unaffected by less hypertonic solutions.2. Variation of the Ca or Mg content of the bathing solutions did not alter these effects of osmotic pressure on the early transient increase in m.e.p.p. frequency or e.p.p. quantal content but affected the late steady increase in m.e.p.p. frequency.3. The value of the transient increase in m.e.p.p. frequency was exponentially related to the osmotic gradient in the range 0-300 m-osmoles/l. with a Q(10) of 1.95 (range 11-34 degrees C). Greater osmotic gradients did not further increase m.e.p.p. frequency. Variation of the ionic strength of the bathing medium did not influence osmotic effects upon frequency.4. The discrepancy between the effects of osmotic gradients upon spontaneous and nerve-impulse induced transmitter release was explained by an occlusion of the osmotic effects by depolarization of nerve terminals. Time-course studies showed that in the presence of 20 mM-KCl the m.e.p.p. frequency increase in response to an increase in osmotic pressure was small and was followed by a reduction in frequency to below control levels while osmotic pressure changes had no immediate effect upon m.e.p.p. frequency in solutions containing 30 mM-KCl.5. It was concluded that increased osmotic gradients could release transmitter by a mechanism independent of Ca and of nerve terminal depolarization.6. It is suggested that the initial transient effects of changes of osmotic gradient upon transmitter release are related to flow of water through the nerve terminal membrane, while the later effects are related to nerve terminal volume changes.     

The relation between transmitter release and Ca2+ entry at the mouse motor nerve terminal: role of stochastic factors causing heterogeneity.

The relation between quantal transmitter release and presynaptic Ca2+/Ba2+ entry at the mouse neuromuscular junction was studied, making use of the finding that in the presence of Ba2+ trains of nerve stimuli or brief nerve terminal depolarizations elicit "tails" of raised miniature end-plate potential frequency (fm) that reflect entry of Ba2+ per pulse, and hence effectiveness of pulses in opening Ca2+/Ba2+ channels; at the same time these pulses elicit end-plate potentials. With nerve stimulation in the presence of Ba2+ and Ca2+ and modulation of release by raised Mg2+ or bekanamycin, slopes of log quantal content (m) vs log apparent Ba2+ entry per pulse were close to 4, which is the same as the Hill coefficient for Ba2+ cooperativity derived from other data. With depolarizing pulses of varied intensity, however, similar plots gave slopes close to 2, with Ba2+ alone or in a mixture of Ca2+ and Ba2+. Thus, the relation between transmitter release and Ca2+ (or Ba2+) entry apparently depends upon how entry is varied; varying the numbers of channels opened is not the same as varying ion entry per channel. A mathematical model was developed to examine the consequences of heterogeneity of local Ca2+ (or Ba2+) between release sites, arising because of stochastic variation of number and time course of Ca2+ channels opened per site; the experimental results were consistent with this model. It was therefore concluded that release is normally governed by intracellular Ca2+ close to points of Ca2+ entry through channels; stochastic factors give rise to more release than if Ca2+ were homogeneously distributed. If Ca2+ channels are uniformly close to release sites the average number of channels opened per site per action potential may be as low as 4.     

Reducing transmitter release from nerve terminals influences motoneuron survival in developing rats

Motoneurons in neonatal rats die following injury to the peripheral nerve. However, this vulnerability to nerve injury declines rapidly so that nerve injury at five days of age results in little if any motoneuron death. We have proposed that the role of the target during this critical period of development is to up-regulate the release of transmitter from developing motor nerve terminals. Here we show that reducing the release of acetylcholine from nerve terminals in neonatal rats can affect motoneuron maturation and survival. The soleus muscle in neonatal rats was treated with either magnesium or hemicholinium, and the number of motoneurons that survived was established 10 weeks later by retrograde labelling. Following treatment with magnesium, only 58.1% (+/-10.4 S.E.M., n=5) of the motoneurons in the soleus motor pool survived, although hemicholinium had no effect on motoneuron survival. However, those motoneurons that survived following treatment with either magnesium or hemicholinium did not develop normally since they remained susceptible to axotomy-induced cell death for longer than normal. In adult animals in which the sciatic nerve was crushed at five days of age following prior treatment with either magnesium or hemicholinium, only 27.6% (+/-6.2 S.E.M., n=5) and 44% (+/-6.1 S.E.M., n=4) of motoneurons in the sciatic motor pool survived, respectively, although no motoneurons died following injury alone or when injury was preceded by treatment with control implants containing NaCl. These results indicate that the release of acetylcholine from motor nerve terminals plays an important role in the development and survival of motoneurons.     

The effect of polarizing current on action potential and transmitter release in crayfish motor nerve terminals

Summary Potential changes were recorded from the terminal regions of crayfish motor nerve fibres by means of extracellular microelectrodes. Near to the recording site, a nerve branch was cut and sucked into a close fitting glass capillary electrode. Nerve action potentials were recorded through this electrode; in addition, current could be passed which de- or hyperpolarized the nerve and the terminal.Hyperpolarization of the terminal led to an increased average amplitude of the EPSP (up to 400% of control), which was due to an increased probability of release of quanta of transmitter. The average amplitude of the EPSP rose steeply with increasing current strength up to –6 A, larger hyperpolarizing current had little more effect. Part of this facilitatory effect of hyperpolarization was instantaneous, the full effect, however, developed only within 10 min after hyperpolarization. Similarly, the EPSP returned to the control amplitude only 10 min after the hyperpolarizing current was switched off. During hyperpolarization, the action potentials recorded from the nerve and from the terminal were prolonged by about 100% and increased in amplitude. The frequency of spontaneous synaptic potentials was reduced (down to 12% of control) during hyperpolarization.Depolarization of the terminal had little effect on the EPSP and on terminal potential changes, probably because small current strengths led to block of conduction proximal to the terminal. The frequency of spontaneous synaptic potentials was increased during depolarization of the terminal.The effects of polarizing current on the crayfish motor nerve terminal are very similar to the respective effects on cholinergic vertebrate terminals indicating a very similar mechanism of release of transmitter. Part of the facilitatory effect of hyperpolarization should be due to the prolongation of the action potential, in addition a mobilization of transmitter must be assumed. Possible relations between synaptic facilitation due to repetitive stimulation and hyperpolarization of the terminal are discussed.