Action of botulinum A toxin and tetanus toxin on synaptic transmission

Intracellular recordings of the spontaneous activity from mammalian spinal cord neurons in culture demonstrated different sensitivities of excitatory and inhibitory synaptic transmission for the action of tetanus toxin (Tetx) and botulinum toxin type A (Botx). The effects of Tetx and Botx on spontaneous and nerve-evoked transmitter release were compared under identical experimental conditions in experiments on in vitro poisoned mouse diaphragms. At 37 degrees C completely paralyzed endplates are characterized by a very low frequency of spontaneous miniature endplate potentials (m.e.p.p.s) and by a 100% failure to evoke endplate potentials (e.p.p.s) in response to single nerve stimuli. Striking differences in the action of both toxins have been observed when the very low transmitter release probabilities of paralyzed nerve-muscle preparations were increased by tetanic nerve stimulation and/or application of potent K+-channel blockers and/or by reduction of temperature to 25 degrees C. While Botx did not change the short latency between nerve impulse and postsynaptic response, Tetx produced a temporal dispersion of the quantal release suggesting that the toxins act at different sites in the chain of events that result in transmitter release. To find further evidence to support the different actions of the toxins the spontaneous transmitter release was studied in more detail. Tetx blocked preferentially the release of so-called large mode m.e.p.p.s without affecting the frequency of the small mode ones. In contrast, Botx strongly inhibited both the small and large mode m.e.p.p.s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discrepancies between spontaneous and evoked synaptic potentials at normal, regenerating and botulinum toxin poisoned mammalian neuromuscular junctions

Amplitudes and times to peak of spontaneous miniature endplate potentials (m.e.p.ps) and evoked quantal endplate potentials (e.p.ps) were compared at normal, regenerating and botulinum toxin poisoned neuromuscular junctions of the extensor digitorum longus muscle of the rat. At normal junctions the mean time to peak of m.e.p.ps was longer and more variable than that of similar-sized e.p.ps. At endplates where nerve regeneration was induced by mechanical crushing of the motor nerve the frequency of m.e.p.ps was reduced and their amplitude distribution was broader than normal. The distribution of times to peak of m.e.p.ps was considerably broader than that of quantal e.p.ps recorded at the same endplates. At neuromuscular junctions poisoned with botulinum toxin type A, spontaneous and evoked transmitter release were greatly reduced. The amplitude distribution of m.e.p.ps was wider than that of e.p.ps and the time to peak of e.p.ps was about twice as fast as and less variable than that of m.e.p.ps. To explain the observed differences in time to peak among m.e.p.ps and between m.e.p.ps and quantal e.p.ps we suggest that some m.e.p.ps, but not e.p.ps, originate from transmitter quanta released from sites at a greater distance from postsynaptic receptors or that the release or diffusion process for acetylcholine is more prolonged when producing some of the m.e.p.ps. Such mechanisms produce at normal junctions a small population of m.e.p.ps with prolonged times to peak, at regenerating junctions a greater proportion of such m.e.p.ps and in botulinum toxin poisoning a majority.     

Transmitter release in botulinum-poisoned muscles

Examination of miniature end-plate potentials (m.e.p.ps) in rat skeletal muscle poisoned in vivo by botulinum toxin type A reveals the presence of two populations of potentials. One population which corresponds to m.e.p.ps in unpoisoned muscles and to quantal end-plate potentials. The frequency of these m.e.p.ps is greatly reduced by botulinum toxin. The second population of m.e.p.ps has quite different characteristics. These m.e.p.ps have a more variable, but generally much larger amplitude, and their time to peak is longer than normal m.e.p.ps. The frequency of these m.e.p.ps increases during poisoning and reaches 0.3-1 Hz after 10-14 days. In addition to the variability in amplitude and time-to-peak these m.e.p.ps differ from those at unpoisoned junctions by being unaffected by procedures which alter extra- or intracellular Ca2+ concentrations. The appearance of this Ca2+-insensitive spontaneous quantal secretion of acetylcholine is apparently not a direct effect of the toxin but secondary to blockade of impulse transmission since it also appears at unpoisoned end-plates when transmission is impaired for other reasons. Procedures which increase the intracellular Ca2+ concentration in nerve terminals restore transmitter release from botulinum toxin poisoned nerves. Furthermore, the block caused by the toxin is very temperature-dependent, a reduction in temperature relieving the block. Since presynaptic Ca2+ currents are unaltered by the toxin it is proposed that the block of transmission is due to a reduction in the calcium content of the nerve terminal to a level where the amount of Ca2+, which normally enters, is insufficient to activate transmitter release.     

The mechanism of β-bungarotoxin action. I. Modification of transmitter release at the neuromuscular junction

The protein, β-bungarotoxin, a presynaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, is known to inhibit mitochondrial function. Within 30 min after adding the toxin to a rat diaphragmphrenic nerve preparation, the quantal content increased tenfold and the frequency of miniature endplate potentials increased fourfold. No increase in miniature endplate potential frequency was seen in the absence of extracellular calcium. Since mitochondria may be involved in regulating intracellular calcium levels, the rate at which the transmitter release is turned off was studied by measuring delayed release in the presence and absence of toxin. Delayed release is elevated about eightfold by the toxin. If delayed release is due to residual calcium, as has been hypothesized, these data may be explained if the toxin does not alter the amount of calcium which enters the terminal, but rather the rate at which that calcium is removed. Alternatively, a calcium-dependent modification of the release process itself might be produced. The eventual reduction in transmitter output did not appear to result from depletion of the terminal of releaseable packets of transmitter, but does require extracellular calcium.     

Polymodal distribution of synaptic delays in the frog neuromuscular junction

Synaptic delay of single-quantum response with low mean quantal size (0.05–1) was measured during experiments on preparations of frog neuromuscular junctions using extracellular focal recording of presynaptic action potentials and endplate currents. It was found that distribution of these synaptic delays is of a polymodal nature and mean intermodal interval equaled 0.22±0.01 msec over 13 experiments. An increase in quantal size produced only a redistribution of mode weighting, while mean modal interval remained unchanged. A reduction in temperature induced an increase in the modal interval with the temperature coefficient Q₁₀=2.42±0.14 (n=15). The explanation is suggested that the process of quantal transmitter release is determined by interaction between the calcium-dependent mechanism for raising the likelihood of release on the one hand and the rhythmic operation of the system producing transmitter release on the other. The latter stage in the process depends on temperature, not intracellular Ca²⁺ concentration. The polymodal distribution of synaptic delay reflects the rhythmic operation of the transmitter release zone.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 748–756, November–December, 1986.     

Sexual differentiation and hormonal regulation of the laryngeal synapse in Xenopus laevis

In Xenopus laevis frogs, sex differences in adult laryngeal synapses contribute to sex differences in vocal behavior. This study explores the development of sex differences in types of neuromuscular synapses and the development and hormone regulation of sex differences in transmitter release. Synapses in the juvenile larynx have characteristics not found in adults: juvenile muscle fibers can produce subthreshold or suprathreshold potentials in response to the same strength of nerve stimulation and can also produce multiple spikes to a single nerve stimulus. Juvenile laryngeal muscle also contains the same synapse types (I, II, and III) as are found in adult laryngeal muscle. The distribution of laryngeal synapse types in juveniles is less sexually dimorphic than the distribution in adults. Analysis of quantal content indicates that laryngeal synapses characteristically release low amounts of transmitter prior to sexual differentiation. Quantal content values from male and female juveniles are similar to values for adult males and are lower than values for adult females. When juveniles are gonadectomized and treated with exogenous estrogen, quantal content values increase significantly, suggesting that this hormone may increase transmitter release at laryngeal synapses during development. Gonadectomy alone does not affect quantal content of laryngeal synapses in either sex. Androgen treatment decreases quantal content in juvenile females but not males; the effect is opposite to and smaller than that of estrogen. Thus, muscle fiber responses to nerve stimulation and transmitter release are not sexually dimorphic in juvenile larynges. Transmitter release is strengthened, or feminized, by the administration of estradiol, an ovarian steroid hormone. © 1995 John Wiley & Sons, Inc.     

Presynaptic effects of arachidonic acid and prostaglandin E2 in the frog neuromuscular synapse

Arachidonic acid and prostaglandin E2 decreased the frequency of miniature endplate currents without changing their amplitude-temporary parameters. They also reduced the evoked transmitter release and the amplitude of the 3rd phase of nerve ending response corresponding to the voltage-dependent K(+)-current. Using perineural recording, It was shown that arachidonic acid and prostaglandin E2 decreased the Ca2+ currents of nerve endings. Indometacin: inhibitor of cyclooxygenase, enhanced the evoked transmitter release and decreased the 3rd phase of nerve ending response. Indometacin prevented the effects of arachidonic acid on evoked transmitter release, whereas the effects of arachidonic acid on the 3rd phase was preserved. Prostaglandin E2 seems to mediate the effects of arachidonic acid on spontaneous and evoked transmitter release, Ca(2+)- and Ca(2+)-activated K(+)-currents. Moreover, the arachidonic acid and prostaglandin E2 exerted their own effects upon voltage-dependent potassium current of motor nerve ending.     

Inhibitory synaptic currents in voltage-clamped locust muscle fibres desensitized to their excitatory transmitter

Inhibitory junctional currents (i.j.c.s) have been examined in locust muscle fibres to give properties of GABA-channels activated by the neurally released transmitter. A nerve-muscle preparation is described which has proved suitable for voltage-clamp analysis of inhibitory transmission. I.j.c.s were recorded from fibres in which excitatory synapses had been desensitized with glutamate, to abolish excitatory junctional currents. This procedure had no apparent effect on inhibitory channel properties. The time constant of decay of the i.j.c. was 7.7 +/- 0.3 ms, slightly exceeding the time constant of the membrane noise induced by externally applied GABA. Peak i.j.c. conductance decreased with hyperpolarization. I.j.c.s showed measurable fluctuations permitting an estimate of the mean size of the quantal events composing the i.j.c. Their mean size coincided with the spontaneously occurring miniature inhibitory junctional currents that could be directly recorded in some fibres. The inhibitory nerve-impulse released an average of 35 transmitter packets at sites distributed along the muscle fibre length. Since each m.i.j.c. produced a current of about 0.6 nA (at Vm = -80 mV, ECl = -40 mV) the single quantum of inhibitory transmitter opens 600-1000 postsynaptic chloride channels. This is roughly three to four times the number of channels opened by the excitatory transmitter packet at glutamate synapses in the same fibres.     

Mediator secretion in the nerve-muscle synapse in the frog following long-term effect of calcium-free solutions

The changes of spontaneous and evoked transmitter release in condition of long time (1-4 hours) incubation in Ca-free solution with EGTA adding, were investigated with extracellular recordings in experiments on the nerve-muscular junction of the frog cutaneous-pectoris muscle. Using the method of three extracellular microelectrodes recordings of the monoquantal postsynaptic signals, it was shown that during action of Ca-free solutions the topography of transmitter release changed, the specific spatial organization of points of transmitter release was disrupted. These changes remained after returning to the initial solution. The obtained data suggest that the Ca2+ free solution leads to disruption of active zones of nerve ending. In condition of low initial extracellular Ca2+ concentrations (0.15-0.4 mmol/l), the active zones disorganization led to decreasing of average amplitude of the end-plate currents (EPC) by decreasing their quantal content, increasing their time-course and decreasing the frequency of the miniature end-plate currents (MEPC). The sharp displacement of dependence of quantal contents of EPC in extracellular Ca2+ concentration to a higher Ca2+ concentration without significant changes of slope was revealed. In condition of high (1.8 mmol/l) concentration of Ca2+, the long action of Ca-free solutions leads to decreasing of amplitude of EPC too, but it was less obvious than in condition of initial low Ca2+ concentration. It is supposed that intra- and extracellular Ca concentration provides the support of the typical morpho-functional organization of the mechanisms of transmitter release at the nerve ending of the frog. The disorganization of active zones leads to separation of the elements, which take part at the transmitter release process and reduces the efficiency of secretion.     

Effect of carbacholine on proximal and distal regions of motor nerve terminals in the frog]

Carbacholine depressed postsynaptic currents in the frog m. sartorius leaving intact presynaptic currents in proximal and distal portions of the motor nerve ending. The carbacholine depressing action was followed by an increase in the time gap between the beginning of presynaptic depolarisation and subsequent quantal release. This effect was considerably more obvious in the distal portions of the nerve endings. Effect of extracellular potassium was evident in a diminishing of presynaptic currents due to membrane depolarisation. The data obtained suggest that carbacholine presynaptically depresses synaptic transmission via metabotropic cholinergic receptors controlling the time course of the transmitter release.     

Modulation of the kinetics of evoked quantal release at mouse neuromuscular junctions by calcium and strontium

The effects of calcium and strontium on the quantal content of nerve-evoked endplate currents and on the kinetic parameters of quantal release (minimal synaptic delay, value of main mode of synaptic delay histogram, and variability of synaptic delay) were studied at the mouse neuromuscular synapse. At low calcium ion concentrations (0.2-0.6 mmol/L), evoked signals with long synaptic delays (several times longer than the value of main mode) were observed. Their number decreased substantially when [Ca(2+)](o) was increased (i.e. the release of transmitter became more synchronous). By contrast, the early phase of secretion, characterized by minimal synaptic delay and accounting for the main peak of the synaptic delay histogram, did not change significantly with increasing [Ca(2+)](o). Hence, extracellular calcium affected mainly the late, 'asynchronous', portion of phasic release. The average quantal content grew exponentially from 0.09 +/- 0.01 to 1.04 +/- 0.07 with the increase in [Ca(2+)](o) without reaching saturation. Similar results were obtained when calcium was replaced by strontium, but the asynchronous portion of phasic release was more pronounced and higher strontium concentrations (to 1.2-1.4 mmol/L) were required to abolish responses with long delays. Treatment of preparations with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) (25 micromol/L), but not with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM) (25 micromol/L), abolished the responses with long delays. The dependence of quantal content and synchrony of quantal release on calcium and strontium concentrations have quite different slopes, suggesting that they are governed by different mechanisms.     

The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.     

4-aminoquinoline-induced 'giant' miniature endplate potentials at mammalian neuromuscular junctions

4-Aminoquinoline (4-AQ) in concentrations around 200 micrometers induces, within minutes of its application to isolated mouse or rat neuromuscular junctions, the appearance of a population of miniature endplate potentials (m.e.p.ps) with a larger than normal amplitude, so-called giant m.e.p.ps (g.m.e.p.ps). With amplitudes 2-12 times the modal value of m.e.p.p. amplitude, the population of g.m.e.p.ps varied between 15 and 45% of the total population of m.e.p.ps. There was no increase in the frequency of m.e.p.ps but a positive correlation between the frequency of g.m.e.p.ps and the total frequency of m.e.p.ps. In many instances the rise time and decay time of g.m.e.p.ps were prolonged compared to normal. Elevated extracellular calcium concentrations increased the frequency of m.e.p.ps but had no effect on g.m.e.p.p. frequency. High extracellular potassium concentrations markedly increased m.e.p.p. frequency but failed to influence g.m.e.p.p. frequency. Similar observations were made with ethanol 0.1 M, ouabain 200 micrometers or black widow spider venom. Botulinum toxin type A markedly reduced total m.e.p.p. frequency but 4-AQ still induced g.m.e.p.ps. Nerve stimulation failed to release quanta corresponding to the g.m.e.p.ps. G.m.e.p.ps seemed to originate from quantal acetylcholine release from the nerve terminal since they were abolished by surgical denervation and by the addition of d-tubocurarine to the medium. Blockade of voltage-sensitive calcium or sodium channels by, respectively, manganese ions or tetrodotoxin failed to affect the appearance and the frequency of g.m.e.p.ps. The electrophysiological findings and a statistical analysis of the characteristics of the m.e.p.ps indicate that they belong to two populations. One population is accelerated by the depolarization-release coupling mechanism responsible for evoked transmitter release and is characterized by an amplitude distribution and a process in time that indicate that they correspond to releases occurring at 'active zones' in the nerve terminal. The second population of m.e.p.ps is uninfluenced by nerve terminal depolarization and transmembrane calcium fluxes. This population apparently originates from sites dispersed in the nerve terminal membrane and outside the 'active zones'. 4-AQ increases the frequency of this second m.e.p.p. population without affecting the first population.     

Latency of transmitter release at crayfish motor nerve endings examined by intracellular depolarization

Latency of release of individual quanta of transmitter was studied at neuromuscular junctions of a crayfish (Procambarus clarkii). Postsynaptic quantal currents were recorded at individual motor nerve endings with a macropatch electrode while the subterminal axon branch was depolarized by current passed through an intracellular microelectrode. For depolarizing currents of moderate size, the latency of transmitter release did not change when the duration of the depolarizing current was altered. Previous studies in which a contrary result was obtained may have been compromised by artefacts or by the sampling methods employed. The present results do not support the hypothesis of a depolarization-induced "repressor" of quantal release.     

Critical quantum content for shortening of endplate currents in the frog skeletal muscle.

The decay time of endplate currents was followed during progressive lowering of quantum content of endplate responses by reduced Ca2+. A certain critical value of about 100 quanta was found, when the decay of endplate currents remained constant even though the quantal content was reduced further.     

A method for testing an extended poisson hypothesis of spontaneous quantal transmitter release at neuromuscular junctions

A statistical method for testing the Poisson hypothesis of spontaneous quantal transmitter release at neuromuscular junctions has been proposed. The notion of the Poisson hypothesis is extended so as to allow for nonstationarity in the data, since nonstationarity is commonly seen in the occurrence of spontaneous miniature potentials. Special emphasis has been put on the nonstationary analysis of the quantal release. A time scaling technique has been introduced and is discussed for the analysis. Artificially generated data, which simulate three types of nonstationary spontaneous quantal release, i.e., Poisson, non-Poisson-clustered, and non-Poisson-ordered types, were analyzed to demonstrate the effectiveness of the method. Some sets of miniature endplate potentials, intracellularly recorded at frog sartorius neuromuscular junctions in low Ca++ and high Mg++ solutions showing apparent nonstationarities, were analyzed as illustrative examples. The proposed method will extend the range of applicable data for the statistical analysis of spontaneous quantal transmitter release.     

Estimation of parameters for a model of transmitter release at synapses.

We consider models for the release of transmitter in response to nerve impulses, where it is assumed that quanta of transmitter are released from some of n sites, the probability of release from any site being p. It is assumed that the quantal size is either a constant or is distributed as a normal or a gamma variate. Observations on both spontaneous potentials and evoked potentials are used to obtain moment estimated of n and p. Large sample estimates of the standard errors of these estimates are given.     

Temperature Effect on Proximal to Distal Gradient of Quantal Release of Acetylcholine at Frog Endplate

The conduction velocity of the nerve terminal, mean quantal content, and release latencies of uniquantal endplate currents (EPCs) were recorded in proximal, central, and distal parts of the terminal by extracellular pipettes located 5, 50, and 100 mm from the end of myelinated nerve trunk. The spike conduction velocity, minimal latency, modal value of the latency histograms, and time interval during which 90% of EPCs released (P₉₀) at distal, central, and proximal part of the frog nerve terminal have different temperature dependency between 10° and 28°C. As shown by the size and time-course of reconstructed multiquantal EPCs, the secretion synchronization, which is greatest in distal parts, compensates at least partly for the progressive slowing of spike conduction velocity in the proximodistal direction, in particular at lower temperatures.