Location and functioning of transmitter release sites in frog neuromuscular junction

End-plate potentials (EPP) and miniature EPP (MEPP) were recorded in a single neuromuscular synapse of the frog sartorius muscle by means of two microelectrodes with a resistance of 0.5–2.0 M. Groups of signals (fields), reflecting transmitter secretion in spatially distinct release sites were identified by extracellular recording on MEPP amplitude scatter diagrams. Release sites in the nerve ending were found to be unevenly distributed, to be grouped in certain areas, and to differ in their probability of secretion of a quantum of transmitter. Comparison of fields on MEPP and uniquantal EPP amplitude scatter diagrams in solution with low Ca⁺⁺ concentration (0.2–0.4 mM) showed that ability to induce evoked and spontaneous transmitter release at the release site differs, and that sometimes a release site does not participate in evoked secretion. The results of simultaneous recording of synaptic potentials using extra- and intracellular electrodes indicate that transmitter secretion in spatially distinct groups of release sites leads to the appearance of polymodality in the distribution of amplitudes of intracellularly recorded MEPP and uniquantal EPP.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 152–160, March–April, 1985.     

Effect of substances blocking calcium channels (verapamil,D-600, manganese ions) on transmitter release from motor nerve endings in frog muscle

Experiments on isolated frog nerve-muscle preparations showed that manganese ions (0.4–5.0 mM) inhibit evoked transmitter release by reducing the quantum composition of the end-plate potentials, and they intensify spontaneous transmitter release to a certain extent by increasing the frequency of miniature potentials. Verapamil (1 · 10^–6–5·10^–5 g/ml) and D-600 (2.5·10^–5 g/ml), by contrast with manganese ions, do not inhibit evoked release, but also intensify spontaneous release of the transmitter. All the agents tested prevent the potentiating effect of imidazole (3 mM). During repetitive stimulation, verapamil disturbs action potential generation in the motor nerve. Manganese ions had no such action. It is concluded that between the calcium channels of motor nerve endings and the calcium channels of heart muscle or the neuron soma there are molecular differences, expressed as sensitivity to the blocking action of verapamil and D-600.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 415–422, July–August, 1977.     

Effects of ethimizol on frog neuromuscular junction

The effects were studied of ethimizol, a substance activating memory processes, on features of synaptic transmission during experiments on frog cutaneous pectoris muscle. It was found that the presynaptic action of ethimizol consists of raising the frequency of miniature potentials, when used at a concentration of 0.5–10 mM, and modulating quantal content of synaptic transmission due to changes in binomial quantal release parameters p and n when 0.5–2 mM ethimizol was used. This substance facilitated transmission at synapses with a low initial level of transmitter release. This substance facilitated transmission at synapses with a low initial level of transmitter release. Ethimizol was also found to have a postsynaptic action, consisting of reducing amplitude at a concentration of 5–10 mM and prolonging synaptic currents and potentials when concentrations of 0.5–10 mM were used. The latter effect produced a considerable increase in the time integral of endplate potentials. The postsynaptic action of ethimizol is perhaps seen in its effects on features of postsynaptic ionic channels. The effects of ethimizol are discussed with a view to how it may act within the central nervous system as a nonspecific modulator.A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 757–763, November–December, 1985.     

Action of thiamine on neuromuscular transmission in the frog

The action of thiamine on neuromuscular transmission in the frog sartorius muscle was investigated. It was found that thiamine at a concentration of 1×10^–14 to 1×10^–4 M increases transmitter secretion at the nerve endings. This is demonstrated by the increased frequency, amplitude, and quantal content of miniature endplate potentials, and is due to the enhanced likelihood of transmitter release. The role of thiamine in regulating synaptic transmission and the mechanism of its interaction with thiamine-sensitive receptors are examined.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 794–800, November–December, 1985.     

Inhibitory Miniature Potentials in the Stretch Receptor Neurons of Crayfish

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K₂SO₄ electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K₂SO₄), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl^- into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and γ-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl^- permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.     

Effect of thiamine on the processes responsible for acetylcholine secretion in the frog neuromuscular synapses

The effect of vitamin B₁ (thiamine, 10^–10–10^–3 M) on direct (transmitter secretion) and recurrent (resynthesis of the transmitter and its storage in synaptic vesicles) processes of acetylcholine (ACh) secretion was studied in the frog neuromuscular junction. In Ca²⁺-containing extracellular medium, the facilitatory effects of thiamine and -latrotoxin (an increase in the frequency of miniature end-plate potentials, MEPP) were additive, regardless of the sequence of their application. After partial exhaustion of the synaptic vesicle stores caused by -latrotoxin (2 nM) in Ca²⁺-free extracellular medium, thiamine accelerated the Ca²⁺-induced recovery of the ACh secretion. In the presence of thiamine, there were two phases in the dependence of quantum content of an end-plate potential (EPP) on stimulation frequency, which are typical of the effects of Sr²⁺ and Ba²⁺ on the ACh secretion. Under conditions of depression and postdepression recovery, the effect of thiamine on the time course of the changes in EPP amplitude was similar to that produced by Ba²⁺. Possible mechanisms of the effects of vitamin B₁ on the processes responsible for the ACh secretion and the dependence of the MEPP frequency on the concentrations of thiamine and thiamine diphosphate are discussed in light of the above results.Neirofiziologiya/Neurophysiology, Vol. 26, No. 4, pp. 291–298, July–August, 1994.     

Temperature dependence of spontaneous quantal and nonquantal transmitter release from motor nerve endings in the mouse

Spontaneous quantal and nonquantal acetylcholine release were investigated at an ambient temperature range of 10–35°C in a preparation of white mouse hemidiaphragm. Quantal transmitter release was assessed by the frequency of miniature endplate potentials and nonquantal secretion by the level of H-effect. Spontaneous quantal release rose exponentially in step with increasing temperature. Two relative maxima, one of 20°C and the other of 35°C were noted in the temperature dependence of nonquantal transmitter release. Nonquantal release of acetylcholine did not take place at a temperature of 10°C. The effective energy of activation of quantal release was calculated as 57.0 kJ/mole over the temperature range investigated; that of the nonquantal release process at intervals of 15–20°C and 25–35°C measured 45.5 and 38.2 kJ/mole respectively. It is suggested that an active transport system process rather than simple diffusion of acetylcholine molecules is responsible for nonquantal release of this neurotransmitter.S. V. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 18, No. 3, pp. 361–367, May–June, 1986.     

Effects of cholinergic compounds on spontaneous quantal transmitter release at the frog neuromuscular junction

The effects of nicotinic and muscarinic mimetics and lytics on spontaneous quantal transmitter secretion from the motor nerve endings were investigated during experiments on theRana temporaria sartorius muscle. Acetylcholine and carbachol reduced the frequency of miniature endplate potentials both in a normal ionic medium and in one with potassium ion concentration raised to 10 mM. Similar effects were produced by nicotinic agonists, namely nicotine, tetramethylammonium, and suberyldicholine, whereas muscarinic mimetics — methylfurmetide, oxotremorine, and F-2268 (L- and D-stereoisomers) — did not affect transmitter release. Neither d-tubocurarine, benzohexonium, nor atropine abolished the presynaptic effects of carbachol and acetylcholine. It is concluded that nicotinic cholinoreceptors are present at the frog motor nerve endings which modify spontaneous transmitter release and differ in their pharmacological properties from recognized N-cholinoreceptors of the motor and autonomic systems of the higher vertebrates.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 586–593, September–October, 1986.     

Effect of imidazole,guanidine, and theophylline on transmitter release in the frog neuromuscular synapse in relation to temperature and calcium ion concentration in the medium

The effects of imidazole, guanidine, and theophylline on spontaneous (frequency of miniature end-plate potentials) and evoked (quantum composition of end-plate potentials) transmitter release were compared in isolated sartorius muscles ofRana temporaria at different temperatures and during changes in the calcium concentration in the external solution. All three substances increased the quantum composition of the end-plate potentials and the frequency of the miniature end-plate potentials at 20°C and in 0.5 mM calcium. As regards their effect on the quantum composition the substances could be arranged in the following order: imidazole guanidine theophylline; as regards their effect on frequency: theophylline imidazole guanidine. Theophylline increased spontaneous release, whereas imidazole and guanidine increased evoked transmitter release more than the rest. Comparison of the effect of these substances at 20 and 7°C showed that only the action of theophylline on spontaneous release depends on temperature. The effect of imidazole and theophylline on frequency was independent of the calcium concentration in the medium. Differences in the mechanism of action of these compounds on spontaneous and evoked acetylcholine release are discussed.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 430–436, July–August, 1977.     

Functional heterogeneity at the frog sensory-motor synapse

This article compares four models of amplitude fluctuations in postsynaptic potentials. The convolution of two binomial distributions and the beta model proved the best fit with experimentally obtained data (as compared with the binomial model). The beta model is based on the assumption that the probability of quantal transmitter release is a random variable with a beta distribution. Numbers of quantal generators as estimated by the beta model were found to resemble numbers of morphological identifiable synaptic boutons. Findings from research using this model showed that the binomial parameter n may be interpreted as the number of transmitter release sites functioning with a probability in excess of 0.2. The findings obtained confirm the postulated functional diversity of release sites at interneuronal synapses.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 780–788, November–December, 1989.     

Biochemical characteristics of a muscle perfusate and its effects on mediator release in the neuromuscular synapse

Histidine-containing compounds (HCC) were found, using diazoreaction and reaction with diethylpyrocarbonate, a specific histidine reagent, in the perfusate of the frog hindlimb vasculature. After gel filtration on sephadex G-25 about 80% of HCC were eluted with a fraction with molecular mass less than 5000 kD. The studies of the perfusate influence on the characteristics of quantum secretion of transmitter in the preparation of frog cutaneopectoral muscle have shown that the perfusate increased the quantum content of end-plate potentials (EPP) due to increasing binominal parameter and decreasing frequency of miniature EPP. The characteristics of presynaptic action of the perfusate were similar to those of exogenous histidine.     

A model of mediator secretion in the neuromuscular synapse based on spatial heterogeneity of acetylcholine quantum release probability

Spatial heterogeneity of the probability of release of a quantum of mediator (p_i) along the course of a nerve ending was found in experiments with extracellular recording of end-plate potentials of the frog sartorius muscle. The intensity of the effect of Ca⁺⁺ was shown to depend on the initial p_i. Intracellular recording showed that an increase in the extracellular concentration of these ions between 0.2 and 1.8 mM is accompanied by an increase in the binomial parameters n and p. On the basis of the results a model of mediator liberation in the neuromuscular synapse was constructed, and by means of it coefficients of distribution of p_i can be determined along the course of the nerve ending and the number of functioning release points established. To assess these parameters, values of n and p must be calculated for two closely similar values of the extracellular Ca⁺⁺ concentration.S. V. Kurashov Kazan' Medical Institute, Ministry of Health of the RSFSR. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 233–240, May–June, 1982.     

Electrophysiological aspects of synaptic transmission at the electromotor junction of Torpedo marmorata

Miniature and stimulus-evoked electroplaque potentials were recorded in Torpedo electrocytes intracellularly and extracellularly and analysed quantitatively. Tetrodotoxin reversibly blocked stimulus evoked potentials but hardly affected spontaneous miniature potentials in amplitude and frequency. The quantum content of stimulus-evoked potentials varied between 150 and 400 in normal saline and decreased in low Ca2+ high Mg2+ solution. Quantal release conformed to binomial statistics and allowed determination of the release parameters p and n. Analysis of the time constant of decay of spontaneous miniature electroplaque currents showed variation around a mean of 0.75 +/- 0.16 msec (SD) which was greatly prolonged by application of neostigmine.     

Effect of carbachol on spontaneous transmitter release from rat motor nerve endings depending on extracellular potassium concentration

The effect of carbachol (10 µM) on the frequency of miniature end-plate potentials (MEPP) was studied in experiments on the Wistar rat soleus muscle during a change in extracellular potassium concentration from 2 to 15 mM. Between the range of potassium concentrations from 2 to 7.5 mM the cholinomimetic had no effect on spontaneous transmitter release. In higher potassium concentrations carbachol caused an increase in the frequency of MEPP. This facilitatory effect increased in strength with an increase in potassium concentration; at 15 mM the frequency of MEPP was increased up to 160%. The results confirmed the previous hypothesis that the action of the mimetic on spontaneous transmitter release, relaized through presynaptic acetylcholine receptors, depends on the initial level of polarization of nerve endings.S. V. Kurashov Kazan' State Medical Institute. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 470–475, July–August, 1984.     

Miniature End-plate Potentials at Mammalian Neuromuscular Junctions Poisoned by Botulinum Toxin

IT is generally accepted that botulinum toxin entirely blocks transmitter release from motor nerve terminals without affecting nerve conduction or the sensitivity of the muscle membrane to acetylcholine. In particular, it has been reported that with both acute and chronic intoxication with type A botulinum, miniature end-plate potentials (m.e.p.p.s.) disappear completely from a muscle at about the time that transmission is blocked^1,2. The action of botulinum toxin has been reinvestigated following acute application of toxin to the rat diaphragm in vitro and chronic paralysis of rat soleus muscle following a single intramuscular injection of toxin; miniature potentials have been observed to persist following blockade of neuromuscular transmission.     

Chemically Mediated Transmission at a Giant Fiber Synapse in the Central Nervous System of a Vertebrate

The hatchetfish, Gasteropelecus, possesses large pectoral fin adductor muscles whose simultaneous contraction enables the fish to dart upwards at the approach of a predator. These muscles can be excited by either Mauthner fiber. In the medulla, each Mauthner fiber forms axo-axonic synapses on four "giant fibers," two on each side of the midline. Each pair of giant fibers innervates ipsilateral motoneurons controlling the pectoral fin adductor muscles. Mauthner fibers and giant fibers can be penetrated simultaneously by microelectrodes close to the synapses between them. Electrophysiological evidence indicates that transmission from Mauthner to giant fiber is chemically mediated. Under some conditions miniature postsynaptic potentials (PSP's) are observed, suggesting quantal release of transmitter. However, relatively high frequency stimulation reduces PSP amplitude below that of the miniature potentials, but causes no complete failures of PSP's. Thus quantum size is reduced or postsynaptic membrane is desensitized. Ramp currents in Mauthner fibers that rise too slowly to initiate spikes can evoke responses in giant fibers that appear to be asynchronous PSP's. Probably both spikes and ramp currents act on the same secretory mechanism. A single Mauthner fiber spike is followed by prolonged depression of transmission; also PSP amplitude is little affected by current pulses that markedly alter presynaptic spike height. These findings suggest that even a small spike releases most of an immediately available store of transmitter. If so, the probability of release by a single spike is high for any quantum of transmitter within this store.     

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.     

Effect of dimedrol on the function of the neuromuscular synapse and the generation of action potentials by motor nerve fibers

Microelectrode registration of synaptic potentials in the frog cutaneous-pectoris muscle has shown dimedrol (7.9 X 10(-5) M) to act on synaptic transmission decreasing the quantal content, estimated by mean EPP amplitude to mean miniature EPP amplitude ratio, the quantal content calculated by variation coefficient of EPP amplitude being unaffected. The data suggest possible transmitter release and depletion of mediator stock. The experiments on isolated motor nerve fibers have demonstrated dimedrol to cause the increase in transmitter release probability by widening the action potentials in the terminals and thus enhancing Ca2+ influx.     

Statistical analysis of the structure of a train of miniature endplate potentials following different treatments of the process of transmitter secretion

The process of transmitter release has been statistically analysed with the use of a rat phrenic nerve-diaphragm preparation in which spontaneous transmitter secretion had been changed by ouabain, 4-aminopyridine and tetanus toxin. In all cases significant deviations of the statistics of miniature end-plate potentials (MEPP) impulse flows from Poisson process and amplitude distributions of MEPP from normal have been obtained. By the statistical characteristics two groups of processes have been distinguished: 1) normal and ouabain where certain consistency of the processes suggests the organization of transmitter release sites and 2) 4-aminopyridine and tetanus toxin where the temporary characteristics of the process in conjunction with the appropriate transformation of MEPP amplitude distribution apparently suggests breakdown of the mechanism of spontaneous synchronization of transmitter quanta release.     

Transmitter release in proximal and distal parts of a nerve terminal of the frog sartorius muscle

Evoked synaptic potential were recorded extracellularly in experiments on a nervemuscle preparation of the frog sartorius muscle. A decrease in evoked transmitter release was found from the proximal to the distal parts of the nerve ending, due to a decrease in the probability of transmitter quantum release. The terminal portions of the synapse are less sensitive than the proximal parts to changes in Ca⁺⁺ concentration, they show less marked facilitation of transmitter release during paired and repetitive stimulation, and exhibit deeper and more rapidly developing depression. It is concluded that differences in transmitter release in the terminal parts of the synapse are due to the low reserves of transmitter and the lower premeability of the presynaptic membrane to Ca⁺⁺.