Identification of transmitter release sites in the motor nerve terminal

A model was produced of generation of postsynaptic current following release of a quantum of neurotransmitter from the nerve ending, whereby the law of current density attenuation is defined as j=I/r^b (A), where I is current density at the generation site and j stands at distance r from that site. Coefficient b was shown experimentally to be close to 1 using extracellular techniques of signal recording. Assuming that sites of signal generation and transmitter release are spatially identical, a new technique for determining the coordinates of the transmitter release site in the motor nerve terminal is suggested. This consists of measuring uniquantal signal amplitude by means of three extracellular microelectrodes spaced 5–10 µm apart. We were able to establish, by producing "spatial pictures" of transmitter release based on analysis of several hundred signals in the frog cutaneous pectoris muscle, that release sites are arranged in groups running diagonally to the nerve ending. These groups are thought to reflect transmitter release in active zones of the nerve ending. Advantages, disadvantages, and inaccuracies of the method are identified.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Moscow. V. I. Ulyanov-Lenin University, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 309–318, May–June, 1990.     

Changes in the topography of transmitter release in a neuromuscular synapse reflect plastic alterations occurring in active zones

The topography of transmitter release along the motor nerve terminals (NT) was studied on the frogcutaneous pectoris muscle under normal conditions and following denervation. Coordinates of release sites (RS) of transmitter quanta were determined by extracellular recording of postsynaptic signals using three microelectrodes. It was shown that RS form groupings that reflect transmitter release in individual active zones (AZ). The topography of transmitter release in the distal parts of the NT under normal conditions was shown to differ from that observed in the proximal parts. The difference consists in a lower probability of transmitter release in AZ and a higher probability of this process between AZ, as well as in a change of release profile in individual AZ. Similar differences were found following denervation. It is suggested that these properties may reflect plastic reorganization occurring in AZ in the course of remodelling of neuromuscular synapse and its degeneration.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 253–260, July–August, 1995.     

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.     

Differences between Spontaneous and Evoked Monoquantum Signals in the Frog Neuromuscular Junction

In experiments on the frog cutaneous-pectoris muscle, the amplitude-temporal parameters of monoquantum end-plate currents (EPC) and miniature EPC (mEPC) were investigated using extracellular recording. A significant dependence of the risetime of the signals on their amplitude was found after analyzing mEPC; at the same time, such dependence was absent for EPC. Approaches leading to disorganization of the active zones (AZ) of the nerve ending (NE), prolonged action of a Ca-free solution, and denervation resulted in an increased dependence of the risetime of the monoquantum signals on their amplitude; moreover, these dependences were similar for both mEPC and monoquantum EPC. Mathematical simulation showed that the obtained data could be explained by the spatial heterogeneity of the sites of spontaneous and evoked transmitter release within the regions of the AZ. A new hypothesis interpreting spontaneous and evoked transmitter release is proposed.     

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.     

Analysis of transmission at interneuronal synapses using a convolution of binomial distributions

Analysis of discrete distributions of basic EPSP amplitudes at sensorimotor, proprio- and reticulo-motoneuronal synapses in the frog by means of the convolution of two binomial distributions revealed that the number of transmitter release sites operating was greater than that calculated by means of binomial distribution. Probability levels of transmitter release sites responding in unison to nerve impulses are dissimilar (at not more than 0.1–0.3) and remained constant. Interference with the release mechanism induced blockade of release sites. Impairment of the process triggering transmitter release produced a sharp decline in the probability of release sites operating in unison.A. A. Zhdanov State University, Leningrad; I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from neirofiziologiya, Vol. 20, No. 4, pp. 487–494, July–August, 1988.     

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.     

Pre-synaptic and post-synaptic factors influencing the synchronism in the transmitter secretion and the amplitude and temporal parameters of a postsynaptic response in the neuromuscular junction: a model study

Using a model of the frog neuromuscular junction, we studied the influence of pre-synaptic and post-synaptic factors on the amplitude and temporal parameters of end-plate currents (EPC). A nerve terminal (NT) was supposed to include linearly distributed active zones (AZ) that are able to release a transmitter quantum with a definite temporal distribution of the release probability (AZ DRP) after successive activation of these zones by a spreading action potential (AP). An increase in the length of a terminal, distance between AZ, and time constant of the DRP decline, or a decrease in the AP conduction velocity along the NT determines a decrease in the EPS amplitude and prolongation of its rising phase. These effects result from an increased asynchronism in the transmitter release. An expansion of the temporal parameters of minature EPC leads to an increase in the EPC amplitude, i.e., provides minimization of its loss. Various EPC models are compared, and contributions of the examined pre-synaptic and post-synaptic factors in modifications of the amplitude and temporal EPC parameters are evaluated.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 163–179, May–June, 1995.     

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.     

Ionic currents in nerve terminals of the frog sartorius muscle

Nerve terminal responses produced by stimulating the motor nerve were recorded extracellularly from the nerve endings of the frog sartorius muscle. A triphasic response occurred in the proximal areas of the nerve ending, beginning with a positive phase. Ionotophoretic application of tetrodotoxin, tetraethylammonium, and 4-aminopyridine indicated that the negative phase reflected inward sodium current and the third (positive) phase indicated outward potassium current. A late slow negative component was recorded using CaCl₂-filled electrodes during perfusion of erve-muscle preparations with a calcium-free solution containing tubocurarine. This component was dependent on the Ca²⁺ concentration present in the electrode, increasing when tetraethylammonium and 4-aminopyridine were added and disappearing under the effects of Co²⁺. Similar components were recorded using microelectrodes containing Sr²⁺ and Ba²⁺. It was deduced that the slow components in the response indicate currents passing through voltage-dependent calcium channels in the presynaptic membrane of the nerve ending. The time course of the calcium current is compared with that of transmitter release at the synapse.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. V. I. Ulanov-Lenin Kazan' State University. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 770–779, November–December, 1985.     

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.     

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.     

Effects of high potassium solutions and caffeine on the processes of exo-endocytosis of synaptic vesicles at the frog motor nerve ending

In experiments on the frog motor nerve endings of cutaneous pectoris muscle using fluorescent microscopy it has been shown that initiation of massive transmitter release of synaptic vesicles by high potassium solutions in using endocytotic marker FM 1-43 at the nerve terminals light spots occurred only at some of the nerve terminals or at the some parts of nerve terminal. It has been revealed that application of caffeine increased the number of light terminals. Using extracellular microelectrode recording, we showed that both high potassium solutions and caffeine increased frequency of miniature end-plate potentials in a dose-dependent manner. However, high potassium solutions always increased the frequency of spontaneous transmitter release while caffeine increased it only in some experiments. It was concluded that processes of exo- and endocytosis can be caused both by entry of Ca ions at the nerve ending during depolarization (high potassium solutions) and by Ca release from endoplasmic reticulum (caffeine). Possible spatial localization of endoplasmic reticulum at the motor nerve ending is discussed. The hypothesis of its role at the remodeling of synapse was proposed.     

Presynaptic mechanisms of zinc effects on the neuromuscular transmission]

The effect of zinc ions on presynaptic currents and transmitter release was studied at the neuromuscular junction of the frog cutaneous pectoris muscle preparation with using an extracellular microelectrode. It has been shown that zinc (100 mkM) amplified MEPP frequency at first, but suppressed it later. Zinc affected the presynaptic spike waveform and transmitter release in a concentration-dependent manner. Depending on concentration and time of exposure zinc increased or suppressed transmitter release. Increase of transmitter release was shown to be resulted by blockade voltage gated and calcium activated potassium channels in nerve ending, leading to broad of both presynaptic spike and action potential. Strong change of presynaptic spike waveform after high concentration zinc treatment supposed that under this condition zinc depressed voltage gated calcium and sodium channel leading to decrease of transmitter release. It was concluded that the final and irreversible depression of acetylcholine release by zinc was due to alteration of whole ion conductances in nerve ending and to change of configuration of proteins included in structure of ion channels. It is discussed possible mechanisms of various effects of zinc ions at the neuromuscular synapse.     

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.     

Analysis of factors restricting the use of binomial statistics for studying transmitter release in neuromuscular junctions

Binomial parameters of transmitter secretion were calculated on the basis of analysis of synaptic potentials in the frog sartorius muscle. Negative values of the parameter p were found in some synapses. This happened most often in low Ca²⁺ concentrations and with low amplitude of miniature end-plate potentials. The results were interpreted in terms of a model assuming spatial heterogeneity of probability of transmitter quantum release at different release points. Simulation of transmitter secretion by computer showed that the appearance of negative values of the parameter p and incorrect estimates of n experimentally are connected with the form of distribution of probability of transmitter quantum release in the synapse and with the amplitude of miniature potentials.S. V. Kurashov Kazan' Medical Institute, Ministry of Health of the RSFSR. Translated from Neirofiziologiya, Vol. 16, No. 2, pp. 182–189, March–April, 1984.     

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse – I. Naturally occurring variability in active zone structure

The orderly arrays of intramembranous particles (IMPs) found in the p-face of freeze-fracture replicas of the frog neuromuscular junction (‘active zones’) are believed to be involved in transmitter release. Some or all of the particles represent voltage-dependent Ca2+ channels. Since there is a great heterogeneity in the amount of transmitter released by different frog motor nerve terminals we sought to determine whether active zone (AZ) structure displayed a similar heterogeneity by using a novel freeze-fracture procedure providing large, intact replicas containing significant portions of motor nerve terminals from the cutaneous pectoris muscle of the frog, Rana pipiens. Using only junctions in which more than 50 AZs or more than 50 μm of nerve terminal were included in the fractures, we measured AZ length, AZ intramembranous particle density, terminal width at each AZ, space between AZs, the angle of AZ orientation with respect to the longitudinal axis of the nerve terminal, exposed pre-synaptic nerve terminal surface area and a calculated value for mean AZ length per unit terminal length. The analysis led to the following conclusions. There is an approximate 5-fold range in mean AZ length/micrometre terminal length. Terminal width is a good predictor of AZ length. Particle density does not vary significantly within a given AZ, nor between AZs from the same or different junctions. The distance between AZs is not related to AZ length, i.e. shorter AZs are no more or less likely to be closer to the adjacent AZ. The probability of release from any AZ on action potential invasion is small. If most of the IMPs are Ca2+ channels, either the probability of channel opening or the efficacy of triggering release is very low or both. That the variability in release efficacy in different terminals is much greater than ultrastructural variability in terminals suggests that regulation of release is dominated by physiological processes that do not have obvious ultrastructural correlates. On the other hand, the apparent excess of AZ relative to the number of vesicles released indicates that the amount and variability in amount of AZ is important in ways that need to be elucidated.     

The molecular mechanisms of quantum mediator secretion in the synapse

The modern condition of knowledge about the molecular mechanisms underlying the quantal transmitter release in the central and the peripheric synapses is analysed. The data about the synaptic vesicles types, their forming, transporting to the sites of release at the nerve endings, exo- and endocytosis processes are presented. Ultrastructural and molecular organization of active zone of nerve ending and transmitter release morphofunctional unit--secretosome, which includes synaptic vesicle, exocytosis protein complex and calcium channels, are described. The basic proteins involved in the exo- and endocytosis and their interactions during transmitter release are examined. The role of the intracellular buffer systems, calcium micro- and macrodomains in the quantal transmitter secretion are considered. The reasons of the active zones functional non-uniformity and plasticity and factors reduced transmitter release in the active zone to the single quantum are analysed.     

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.     

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⁺⁺.