Changes in the asynchronism of transmitter release in a neuromuscular synapse and the time course of evoke postsynaptic signals during growth and branching og frog nerve ending

The influence of both growth and branching of a nerve terminal on the asynchronism of transmitter release and the time-course of evoked postsynaptic responses was investigated using a model of a frog neuromuscular synapse in which the nerve terminal represents a population of spatially isolated active zones. It was shown that the appearance of additional branching in proximal parts of the nerve ending leads to decrease in the asynchronism of transmitter release, an increase in quantum content and the amplitude of the postsynaptic signal, and the shortening of its phase of growth. It was found that the asynchronism of transmitter release has a much stronger influence on the time-course of end plate currents compared with end plate potentials. The factors strengthening and weakening the asynchronism of transmitter release in a neuromuscular synapse and the reasons for various length and branching of vertebrate nerve terminals are considered.     

The single nerve ending of the frog sartorius muscle: its ultrastructural characteristics and mediator secretion

Electron microscopy and extracellular recordings were used for the investigation of structural and functional peculiarities of single frog sartorius muscle nerve terminal. It has been found that the diameter, length of the synaptic contact and quantity of synaptic vesicles decreased from proximal to distal parts of the nerve terminal. A number of varicosities, separated from each other by schwann cells, have been revealed along the course of the nerve terminal. This indicates the existence of an interrupted synaptic contact. Both the evoked and spontaneous transmitter release decreased from the initial to the end parts of the nerve terminal. The data obtained suggest that there is a correlation between structural heterogeneity and the differences in the transmitter 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⁺⁺.     

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.     

The generation and spread of excitation in the motor nerve ending of the frog

In experiments on the frog cutaneous-pectoris muscle under the visual control the evoked responses of nerve endings were recorded in proximal, central and distal parts of the terminal. At the fixed position of the recording electrode the local iontophoretic application of TTX to different terminal parts and Ranvier's nodes was performed. It was concluded, that local currents at more proximal terminal parts provide the depolarization of the located parts and conduction of excitation to the nonexcitable parts. Inward currents at behind located and already excited parts are shortened and decreased by local currents from more distal parts during the propagation of excitation. It results in shortening of the action potential and decreasing of the transmitter release in more proximal parts. This effect disappeared along the nerve terminal due to decreasing of inward Na current at the end parts.     

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.     

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.     

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse – II. Proximal–distal measurements

Neurotransmitter release from different parts of frog motor nerve terminals is often non-uniform. There is a decrease in release efficacy from the distal regions of frog motor nerve terminal branches. Since release is thought to occur near the double arrays of large intramembranous particles that constitute the pre-synaptic active zones (AZs), we have examined quantitatively the proximal–distal distribution of AZ structure, using a novel freeze-fracture technique that produces replicas of large fractions of terminals, including the region of nerve entry. This enables us to know the proximal–distal orientation of each branch. From 23 end-plates we have obtained fractures of 72 branches. For 27 of these branches we have obtained continuous fractures both greater than 25 μm in length and with sufficient information to determine their proximal–distal polarity. Only a few of these branches showed a marked distal decrease in AZ length/unit length of terminal, while several junctions had short regions (5–10 μm), either proximally or distally, that exhibited amounts of AZ that were substantially greater or smaller than the mean value for that terminal branch. The terminal area, post-synaptic gutter width and nerve terminal width all exhibit some distal decline concomitant with the distal tapering of nerve terminal branches. AZ length tends to have the least decline compared to the other parameters. Thus, the vast majority of frog motor nerve terminal branches do not display a significant proximal-distal gradient in the amount of AZ structure/μm terminal length. The present data do not provide an obvious ultrastructural correlate for the distal decline in transmitter release that some authors have observed.     

The intracellular calcium and mechanisms of endocytosis of synaptic vesicles at the frog nerve ending

In the experiments on frog motor nerve endings of cutaneous pectoris muscle, made by extracellular recording of synaptic signals, it has been shown that the increase in intracellular calcium ion concentration in the nerve ending (by enhance of extracellular potassium ion concentration, or by addition of caffeine) leads to an increase in the miniature end-plate potential frequency, which is preserved over the whole period (about 10 min) of action of these substrates. The rhythmic stimulation of motor nerve (20 or 100 imp/s) quickly leads to a decrease in the end plate potentials amplitude. It has been shown by fluorescent microscopy with the use of endocytotic marker FM 1-43 that in the course of a short time exposition (5 min) in a high potassium solution (40 mM) or caffeine (5 mM), light spots appeared in the nerve ending. This shows that synaptic vesicles undergo intensive processes of endocytosis. During a longer exposition (30 min) no light spots were revealed, whereas the nerve ending width increased. This data allowed to propose that the process of endocytosis was blocked. In the presence of even lower concentrations of potassium ions and caffeine, and during a long rhythmic stimulation (20 or 100 imp/s) no blocking of endocytosis was revealed. It is concluded that high concentrations of intracellular calcium in the frog motor nerve ending leads to a reversible block of endocytosis, while exocytosis in synaptic vesicles is proceeding.     

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.     

Analysis of living motor nerve ending of a frog by endocytotic fluorescent marker FM 1-43

In our experiments on motor nerve endings of the frog cutaneous pectoris muscle, using fluorescent marker FM 1-43, the intensity and topography of endocytosis were investigated after the initiation of massive exocytosis of synaptic vesicles by increasing the extracellular potassium concentration. Using FM 1-43, fluorescent spots were shown to appear, looking as accumulations of synaptic vesicles in the active zone region. The forms and sizes of luminous spots and the distances between them were analysed. Considerable variations in brightness and total areas of fluorescent spots per a length unit in different regions of the nerve ending were revealed in addition to a proximal-distal gradient of these parameters along the nerve terminal. Peculiarities of topography and intensities of luminescence in the most terminal regions of the nerve ending are described. The obtained data are discussed in terms of the exo- and endocytosis cycle of synaptic vesicles in the active zone region, and from the point of view of the plasticity of the motor nerve ending and active zones. The factors involved in the transmitter release nonuniformity are analysed.     

The mechanisms of short-term forms of synaptic plasticity

In experiments on the frog cutaneous pectoris muscle in cases of different external calcium concentrations, using extracellular recording technique, processes of facilitation and depression of transmitter release during the high-frequency stimulation were investigated. On the ground of experiments using intracellular mobile calcium buffers BAPTA-AM and EGTA-AM, it was proposed that at least two (low- and high-affinity) calcium-binding sites underlie the facilitation. Both the facilitation and the depression were accompanied by such transformations of underlied of nerve ending responses as changes of the third phase amplitude. Application of potassium channel blockers allowed us to reveal the significant contribution of changes of duration of the AP repolarisation phase and, accordingly, the changes of magnitude of calcium influx to development of facilitation and depression of transmitter release. It was also revealed that, during the high-frequency rhythmic stimulation, the increase of asynchrony of transmitter release leading to decrease of facilitation and increase of depression occurred. It was concluded that the forms of short-term synaptic plasticity--facilitation and depression, were caused by various presynaptic mechanisms: the increase of concentration of "local" and accumulation of "residual" calcium, the changes of calcium influx, increase of temporal course of secretion, the impairment of equilibrium between the depletion and restoration of mediator supply. Due to some of these processes and specific conditions of synapse functioning, the facilitation of the depression of transmitter release occurred.     

Adaptation of motor nerve fibers to physical activity

The effects induced by training on the H-reflex of soleus and lateral gastrocnemius muscles have been studied on 19 adult male volunteers; out of these, 10 were non-trained subjects and the remaining 9 were top level athletes engaged in sports requiring very rapid and intense contractions (sprinters and volley-ball players). It has been observed that the latency of the M response is significantly higher in the athletes than in the non-trained subjects. Instead, no significant differences were observed between these two groups, concerning the latency of H response. The increase of M response latency is likely due to a decrease of nerve conduction velocity in the terminal part of motor fibers. The possibility that this conduction speed decrease could be dependent on sprouting and/or terminal branching growth of the motor nerve ending is discussed.     

Influence of the muscle fibre end geometry on the extracellular potentials

Intra- and extracellular action potentials of isolated frog muscle fibres were recorded at different distances to the end of the fibre. The first and second time derivatives of the intracellular action potentials were also recorded. The intracellular action potentials and their first and second time derivatives were almost the same regardless of the place of recording. With the decrease in the axial distance to the end the extracellular action potentials changed gradually in a complicated manner from a shape similar to the second time derivative into a shape similar to the first time derivative. Extracellular potentials, having two negative maxima, were recorded over the terminal taper part of the fibres.These alterations were simulated by a mathematical model. It was shown that the changes in the shape of the extracellular action potentials around the end of the fibres were mainly due to the existence of the fibre end though a better correspondence of the experimentally recorded and the calculated extracellular action potentials was obtained when the morphology of the fibre end was taken into consideration.     

Action of exogenic acetylcholine on the potassium currents of frog motor nerve endings

Using nerve-muscle preparations of the cutaneous pectoris muscle of the frog, and recording extracellular evoked electrical responses from a nerve ending (NE), we have studied the mechanisms by which exogenic acetylcholine (ACh) influences the ion currents of a motor nerve ending. We have established that ACh in concentrations of 0.1–0.6 mmoles/liter causes an increase in the third phase of the NE response, and in concentrations of 0.7–2.0 mmoles/liter, suppresses it. We have found that tubocurarine and atropine do not block the effects of ACh. It has been shown that the ACh-induced increase in the amplitude of the third phase of the NE response is seen against a background of calcium channel blockers, and it can be eliminated by 4-aminopyridine (4-AP) and tetraethylammonium. The inhibitory effect of ACh on the third phase of the NE response is not present in calcium-free solutions, nor when the calcium current is blocked. A discussion is given of the mechanisms of exogenic and endogenic ACh action on the potential-dependent and calcium-activated potassium current of the NE.S. V. Kurashov Medical Institute, Ministry of Health of the Russian Federation, Kazan. Translated from Neirofiziologiya, Vol. 24, No. 6, pp. 678–683, November–December, 1992.     

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.     

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.     

Transmitter release at the active zone of motor nerve endings

Transmitter release sites were located in the motor nerve ending of the frog cutaneous-pectoris muscle using three extracellular electrodes. Transmitter release sites were found to be grouped in a direction cutting across the nerve ending and reflecting transmitter release and active release zones (AZ). Measurements from these groups showed that most transmitter release takes place at the center of the AZ, declining towards the periphery and to either side of this zone. All AZ were found to take place in spontaneous release with a low extracellular concentration of calcium ions present, compared with only a proportion in evoked release. Advantages of the triple as opposed to the dual micro-electrode technique are analyzed. It was found that transmitter release in spatially isolated AZ at the nerve ending leads to a polymodal distribution pattern of the amplitude of uniquantal signals during extracellular recording. The part played by AZ in transmitter release is discussed.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Moscow. V. I. Ul'yanov-Lenin State University, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 318–327, May–June, 1990.     

Role of intracellular Ca2+ in stimulation-induced increases in transmitter release at the frog neuromuscular junction

Under conditions of reduced quantal content, repetitive stimulation of a presynaptic nerve can result in a progressive increase in the amount of transmitter released by that nerve in response to stimulation. At the frog neuromuscular junction, this increase in release has been attributed to four different processes: first and second components of facilitation, augmentation, and potentiation (e.g., Zengel, J. E., and K. L. Magleby. 1982. Journal of General Physiology. 80:583-611). It has been suggested that an increased entry of Ca2+ or an accumulation of intraterminal Ca2+ may be responsible for one or more of these processes. To test this hypothesis, we have examined the role of intracellular Ca2+ in mediating changes in end-plate potential (EPP) amplitude during and after repetitive stimulation at the frog neuromuscular junction. We found that increasing the extracellular Ca2+ concentration or exposing the preparation to carbonyl cyanide m- chlorophenylhydrazone, ionomycin, or cyclopiazonic acid all led to a greater increase in EPP amplitude during conditioning trains of 10-200 impulses applied at a frequency of 20 impulses/s. These experimental manipulations, all of which have been shown to increase intracellular levels of Ca2+, appeared to act by increasing primarily the augmentation component of increased release. The results of this study are consistent with previous suggestions that the different components of increased release represent different mechanisms, and that Ca2+ may be acting at more than one site in the nerve terminal.     

Investigations of thermotropic phase changes in peripheral nerve of frog and rat. A spin label study

The temperature-dependent fluidity of myelin of frog and rat peripheral nerve (Nervus ischiadicus) was studied using the spin label technique. In frog nerve a phase change was detected at 38 °C. In rat nerve no sharp phase change could be established, and the lipid-depleted frog and rat nerve also showed no transition. From the spectral data, it was concluded that in frog and rat nerve the lipid-protein interactions are different, i.e. species dependent. Ca²⁺-depletion of frog nerve caused a loss of transition, while rat nerve remained unaffected. Thus it was indicated that, in frog nerve, Ca²⁺ is involved in the phase change. In the total lipid extract of frog nerve a phase change centered at 32 °C occurred, while the total lipid extract of rat nerve again showed no transition. It is suggested that a connexion exists between our results and investigations on the temperature dependence of an axonal conduction block of nerve.