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.     

Energy Metabolism and Quantal Acetylcholine Release: Effects of Botulinum Toxin, l-Fluoro-2,4-Dinitrobenzene, and Diamide in the Torpedo Electric Organ

In the Torpedo electric organ, a modified nerve-muscle system, type A botulinum toxin blocked the release of acetylcholine (ACh) quanta, both neurally evoked and spontaneous. At the same time, the toxin increased the release of a class of small miniature potentials (the subminiature potentials), reduced the ATP and more the creatine phosphate content of the tissue, and impaired the activity of creatine kinase (CK). Thus, we compared this pattern of changes with those provoked by 1-fluoro-2,4-dinitrobenzene (FDNB), an efficient inhibitor of CK. As expected, FDNB rapidly inactivated CK, which resulted in a profound depletion of ATP whereas the stores of creatine phosphate were preserved. In addition, FDNB caused conspicuous morphological alterations of nerve endings and ACh depletion. This agent also suppressed evoked and spontaneous quantal release whereas the occurrence of subminature potentials was markedly increased. Diamide, a penetrating thiol oxidizing substance, provoked first a transient rise in quantal ACh release and then blockade of transmission with, again, production of a large number of subminiature potentials. Creatine phosphate was depleted in the tissue by diamide, the ATP content reduced, and CK activity partly inhibited. The morphology of nerve terminals did not show obvious changes with either diamide or botulinum toxin at the stage of transmission failure. Although the three poisons acted by different mechanisms, this resulted in a rather similar pattern of physiological changes: failure of quantal release and enhancement of subquantal release. These results and experiments on synaptosomes indicated that CK inhibition was probably a crucial mechanism for FDNB but not for diamide or botulinum intoxication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunits in quantal transmission at the mouse neuromuscular junction: tests of peak intervals in amplitude distributions

The regular spacing of peaks throughout the amplitude distribution of miniature end-plate potentials, quantal evoked end-plate potentials and quantal currents was demonstrated using autocorrelations and power density spectra calculated from the number of events in the successive bins of the histograms built by Matteson et al. (1979), Kriebel & Florey (1983) and Erxleben & Kriebel (1984). At the same mouse neuromuscular junction, the calculated interpeak was constant for evoked and spontaneous quantal releases, throughout sequential sampling and after change of bin size. The presence of regular peak intervals supports the hypothesis that quantal potentials are composed of potential subunits the size of the smallest subminiature potential. Challenging the hypothesis of an acetylcholine quantum composed of acetylcholine subunits, a postsynaptic origin of the subunit is proposed on the basis of the spatial arrangement in rows of the ACh receptors. The ACh-saturating patch evoked by a quantum release (Land et al., 1980, 1981) activates 10-20 rows of receptors, which is roughly the number of subunits composing a quantal event. Therefore the position of the ACh patch or the continuous variations in its size might cause stepwise variations in the total number of ACh receptors activated by an ACh quantum.     

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.     

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.     

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.     

A comment on Martin's relation.

In the limit of slowly varying synaptic conductance changes, a quantity proportional to the quantal content of a synaptic potential is provided by Martin's formula: 1/(VO/Vp-1), where Vp is the peak amplitude of the synaptic potential and Vo is the synaptic equilibrium potential. If the synaptic conductance change is not slowly varying on the time scale of the postsynaptic cell's membrane time constant, Martin's formula overestimates the effects of nonlinear summation and provides an upper limit for the actual quantal content. In the limit of rapidly varying synaptic conductance changes the quantity ln(1/[1-Vp/Vo]) is proportional to the quantal content. This formula underestimates the effects of nonlinear summation and gives a lower limit for the quantal content for cases in which the synaptic conductance is not rapidly varying. These two formulas used together provide upper and lower bounds to the correction for nonlinear summation of postsynaptic potentials.     

Dehydration affects synaptic transmission at flexor muscle in acute lead-treated mice.

The effect of 24 hrs. water deprivation on spontaneous and evoked transmitter release was studied at flexor nerve terminals of control and lead-treated male C57BL mice. Miniature endplate potentials (MEPPs) and endplate potentials (EPPs) were recorded intracellularly from urethane-anesthetized (2 mg/g, i.p.) control and lead exposed mice in both hydrated and dehydrated conditions. Exposure to lead was made by i.p. injection of lead acetate (1.0 mg/kg) dissolved in a 5% glucose solution 24 hrs. prior to the experiment. Unimodal and bimodal MEPP frequencies decreased with dehydration, while small mode MEPPs remained unchanged and large mode MEPPs increased in frequency. EPP amplitude and quantal content were unchanged by dehydration. Lead treatment by itself reduced the frequency of unimodal and bimodal MEPPs but had no effect on the amplitude of EPPs or of quantal content. However a combination of dehydration and acute lead treatment reduced the frequency of unimodal, bimodal and large mode MEPPs and significantly reduced both EPP amplitude and quantal content. Dehydration apparently reveals an underlying neurotoxic action of lead at the neuromuscular junction. This raises a health concern that people subjected to both lead pollution and dehydration are at greater risk to lead poisoning of the neuromuscular junction.     

Some Models of Neuronal Variability

The pattern of nerve action potentials produced by unit permeability changes (quantal inputs) occurring at random is considered analytically and by computer simulation methods. The important parameters of a quantal input are size and duration. Varying both the mean and the probability density function of these parameters has calculable effects on the distribution of interspike intervals. Particular attention is paid to the relation between the mean rate of excitatory inputs and the mean frequency of nerve action potentials (input-output curve) and the relation between the coefficient of variation for the interval distribution and the mean interval (variability curve). In the absence of action potentials one can determine the parameters of the voltage distribution including the autocorrelation function and the power spectrum. These parameters can sometimes be used to approximate the variability of interspike intervals as a function of the threshold voltage. Different neuronal models are considered including one containing the Hodgkin-Huxley membrane equations. The negative feedback inherent in the Hodgkin-Huxley equations tends to produce a small negative serial correlation between successive intervals. The results are discussed in relation to the interpretation of experimental results.     

Down-regulation of quantal size at frog neuromuscular junctions: possible roles for elevated intracellular calcium and for protein kinase C

Previous work showed that quantal size can be at least doubled at the frog neuromuscular junction by pretreatment with hormones or hypertonic solutions, primarily by the release of more acetylcholine (ACh) per quantum. Once increased, quantal size slowly declined over hours. Quantal size was measured from miniature end-plate potentials (MEPPs) or currents (MEPCs). In the present experiments, preparations in which quantal size had been increased were exposed to 17-25 mM [K+], quantal size decreased within minutes. Release of comparable numbers of quanta by nerve stimulation did not decrease size. K(+)-solutions did not decrease size if Ca2+ was omitted or replaced with Sr2+. The phosphokinase C (PKC) activators phorbol 12,13-diacetate (PDA) and 1-oleoyl-2-acetyl-rac-glycerol (OAG) also decreased quantal size within minutes when applied in a hypertonic solution that increased the rate of spontaneous release. Phorbol 12,13-dideconate, which does not activate PKC, did not decrease quantal size. The size decrease triggered by K(+)-solutions or PKC activators was blocked by 100 microM 1-(5-isoquinolinyl-sulfonyl)-2-methyl-piperazine (H7), a protein kinase inhibitor. Apparently, increasing [K+] elevated intracellular [Ca2+], which activates PKC, and which leads to the down-regulation of quantal size. During the period in which size is decreasing, there appears to be large and normal subpopulations of MEPP sizes, with normal gradually replacing large. This suggests that large quanta are formed by adding additional ACh to preformed quanta shortly before they are available for release.     

Quantal current fields around individual boutons in sympathetic ganglia.

The release of a quantum of neurotransmitter from an active zone of a bouton is accompanied by the flow of extracellular current that creates a potential field about the site of transmitter action beneath the bouton. It is shown theoretically that the density of the field at the peak of the quantal current gives rise to an extracellular potential that declines to values of less than 5 microV at 1.3 microm distance in the circumferential direction around the neuron and equally rapidly in the radial direction away from the neuron. A loose-patch electrode placed over a bouton distorts the quantal field about the bouton and calculations show that under current-clamp conditions, potentials of over 40 microV can be recorded with an electrode of tip diameter 2 microm, provided the separation between the tip and the neuron's surface is about 0.1 microm. Quantal release recorded from visualized boutons on rat monopolar pelvic ganglion cells with loose-patch electrodes is in agreement with the properties of the quantal potential field given in the theoretical analysis.     

Minis: whence and wherefore?

In this issue of Neuron, Sara et al. find that spontaneously released miniature synaptic potentials arise from a pool of vesicles distinct from those released by neural activity. This modification of a basic tenet of the quantal hypothesis has important implications for the analysis of changes in synaptic transmission.     

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.     

A computer system for real-time data acquisition and analysis of biopotentials and quantal content at the neuromuscular junction

A computerized data acquisition system for on-line analysis of the parameters of neuromuscular transmission is described. Both hardware usage and software methodologies are discussed with regard to sampling in real-time and analyzing miniature end-plate potentials (MEPPs), end-plate potentials (EPPs) and quantal content of the evoked transmitter release. Significant features of the program include: (1) automatic threshold determination for MEPP detection; (2) the use of a circular buffer to give pre-trigger information; (3) real-time noise spike rejection; (4) an automatic procedure for EPP failure detection; (5) rapid quantal content determinations by several methods as well as complete MEPP and EPP waveform analysis. The system has proven both accurate and reliable during more than two years of use. Advantages of the system over conventional methods include: (1) increased accuracy and efficiency in data analysis; (2) immediate availability of results; (3) conventional data storage; (4) flexibility to meet changing requirements.     

Statistical methods of quantal analysis in computerized compared with physiological experiments

Amplitude distributions of postsynaptic potentials subject to binomial distribution were simulated in computer-based experiments. Effects of sample size (N) and standard deviation of noise (S_n) on accuracy of determining mean quantal content (m) and quantal value (v) were investigated using four quantal analysis techniques (histograms, variation coefficient, failure and combined techniques). It was found that m and v may be determined fairly accurately (to within 10%) at S_n<2v using the last three techniques mentioned and at S_nv (where N=500–1000). It is possible to obtain similar results for N=50–200 if the experiment is repeated ten times. The possibility of applying such techniques to actual physiological results was confirmed by analyzing an extensive trace (N=1333) of inhibitory postsynaptic potentials in sensorimotor cortex units of unanesthetized rabbits.Brain Research Institute, National Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 206–215, March–April, 1990.     

Interaction of glutamate- and adenosine-induced decrease of acetylcholine quantal release at frog neuromuscular junction

In a frog neuromuscular preparation of m. sartorius, glutamate had a reversible dose-dependent inhibitory effect on both spontaneous miniature endplate potentials (MEPP) and nerve stimulation-evoked endplate potentials (EPP). The effect of glutamate on MEPP and EPP is caused by the activation of metabotropic glutamate receptors, as it was eliminated by MCPG, an inhibitor of group I metabotropic glutamate receptors. The depression of evoked EPP, but not MEPP frequency was removed by inhibiting the NO production in the muscle by L-NAME and by ODQ that inhibits the soluble NO-sensitive guanylyl cyclase. The glutamate-induced depression of the frequency of spontaneous MEPP is apparently not caused by the stimulation of the NO cascade. The particular glutamate-stimulated NO cascade affecting the evoked EPP can be down-regulated also by adenosine receptors, as the glutamate and adenosine actions are not additive and application of adenosine partially prevents the further decrease of quantal content by glutamate. On the other hand, there is no obvious interaction between the glutamate-mediated inhibition of EPP and inhibitory pathways triggered by carbacholine and ATP. The effect of glutamate on the evoked EPP release might be due to NO-mediated modulation (phosphorylation) of the voltage-dependent Ca2+ channels at the presynaptic release zone that are necessary for evoked quantal release and open during EPP production.     

Inhibition of packing of acetylcholine into quanta by ammonium

Soaking frog motor nerve terminals in a hypertonic solution produces an increase in the size of miniature end plate potentials (mepp's) and miniature end plate currents (mepc's) after the preparations are returned to normal Ringer's solution. There is substantial evidence that the size increase occurs because additional acetylcholine (ACh+) is incorporated into the quanta. It has been proposed that ACh+ loading into synaptic vesicles requires a proton gradient. As a step in testing this hypothesis the effects of millimolar concentrations of NH4+, methylamine+, or trimethylamine+ in the extracellular solution on the increase in quantal size were measured. These substances would be expected to accumulate in acid intracellular compartments, which would diminish the acidity. The increase in quantal size was blocked by these substances, in agreement with the idea that the proton gradient is involved in ACh+ accumulation. Tetanic stimulation in solutions containing 5 mM NH4Cl also produces a decrease in quantal size, not seen in controls in NH4+-free solution. The inhibition of transmitter packaging by ammonia may play a role in the neural sequelae of hepatic failure.     

硝苯吡啶对腹腔神经节突触传递的阻断作用

本文应用细胞内记录技术,观察了钙通道阻滞剂硝苯吡啶（nifedipine)对离体豚鼠腹腔神经节突触传递的影响,硝苯吡啶（0.1-10umol/L)不影响所检细胞的静息膜电位,膜电阻及细胞内刺激引起的动作电位,但能显著阻断N-型胆碱能的突触传递,并且这种作用可被低钙模拟、高钙拮抗,硝苯吡啶（10umol/L)也不影响突触后膜对乙酰胆碱（ACh)的敏感性;但在高钾克氏液中,能减少微小兴奋性突触后电位（mEPSPs)的频率;在低钙和高镁克氏液中,能减少量子含量,而对量子大小无影响。结果表明,治疗量的硝苯吡啶(0.1umol/L)通过阻滞突触前膜钙内流及ACh的量子性释放,产生突触阻断作用。这可能是硝苯吡啶降压机理的一个组成部分。     

alpha-Tocopherol modifies lead induced functional changes at murine neuromuscular junction

Lead impacts neuromuscular junction and might induce skeletal muscle weakness. Antioxidants may prevent toxic actions of lead on muscle. In this study, resting membrane potentials, endplate potentials, miniature endplate potentials (MEPPs) and isometric twitch tensions were recorded to investigate effects of alpha-tocopherol (Vitamin E) on lead induced changes at murine dorsiflexor muscle. Moreover, levels of endplate nicotinic receptors were measured by receptor autoradiography. Forty rats were divided into four groups (lead alone, alpha-tocopherol, lead plus alpha-tocopherol and saline). Lead (1 mg/kg, i.p.), was administered daily for 2 weeks and alpha-tocopherol (100 mg/kg, i.p.) was given daily for 3 weeks. Lead treatment significantly reduced twitch tension (from 4.4+/-0.4 to 2.2+/-0.3 g) and delayed half time of decay. MEPP frequencies and quantal content were also significantly reduced after lead treatment. Pretreatment with alpha-tocopherol reversed twitch tension reduction (4.1+/-0.3 g) and modified lead induced delay in half time of decay. Similarly, alpha-tocopherol modified the negative actions of lead exposure on MEPP frequencies and quantal content. Receptor autoradiographic studies revealed significant increase of nicotinic receptor levels at the endplate region of flexor muscle in lead treated mice. However, animals treated with lead plus alpha-tocopherol showed significantly decreased levels of nicotinic receptors. alpha-Tocopherol appears to protect against lead induced neuromuscular dysfunction. These effects of alpha-tocopherol are possibly mediated via a free radical mechanism or modification of calcium homeostasis.     

Morphological and electrophysiological properties of the electromotoneurones of the electric ray Torpedo marmorata in vivo and in in vitro brain slices

The morphology of the adult electromotoneurones of the electric ray Torpedo marmorata has been investigated by intracellular injection of horseradish peroxidase. The results show particularly small dendritic fields, poor dendritic branching, and the existence of two distinct types of dendrites. The electrophysiological data demonstrate that the electromotoneurones behave similarly in vivo and in in vitro brain slices. Observations on spontaneous and stimulus-evoked postsynaptic potentials suggest quantal release of transmitter in the Torpedo central nervous system.