Dysfunction of Neuromuscular Synapses in the Genetic Model of Alzheimer's Disease.

The function of synaptic transmission and presynaptic vesicular cycle in the neuromuscular synapses of the diaphragm was studied in transgenic APP/PS1 mice (Alzheimer's disease model). The decrease in the quantal content of end-plate potential, intense depression of the amplitude of terminal plate potentials under conditions of lasting high frequency stimulation (50 Hz), a drastic prolongation of the synaptic vesicle recycling time in APP/PS1 mice in comparison with wild type mice were detected. Manifest dysfunction of the neuromuscular synapses, caused by disordered neurosecretion and recycling of the synaptic vesicles in the presynaptic nerve endings, was detected in the Alzheimer's disease model on transgenic APP/PS1 mice. The study supplemented the notions on the pathogenesis of Alzheimer's disease as a systemic disease, while the detected phenomena could just partially explain the development of motor disorders in this disease.

Sensitivity of intracellular calcium-binding sites for exo- and endocytosis of synaptic vesicles to Sr, Ba, and Mg ions.

Experiments on frog cutaneous-thoracic muscle preparations using electrophysiological (intra- and extracellular recording of postsynaptic signals) and optical (confocal microscopy with the fluorescent endocytic stain FM 1-43) methods were performed to study neurotransmitter secretion and the processes of exo- and endocytosis of synaptic vesicles in motor nerve endings on substitution of extracellular Ca ions with other alkaline earth metals (Sr, Ba, or Mg). Massive asynchronous exocytosis was induced by high-potassium solution, while synchronous exocytosis was induced by prolonged high-frequency stimulation of the motor nerve. The calcium-binding site for asynchronous exocytosis was found to be sensitive to Sr, Ba, and Mg ions, while the site for synchronous exocytosis was only sensitive to Sr ions. During stimulation of both asynchronous and synchronous exocytosis, the calcium-binding site for endocytosis was sensitive to Sr and Ba ions and had the lowest affinity for Sr ions. These experiments led to the conclusion that different intracellular calcium-binding sites exist for the exocytosis and endocytosis of synaptic vesicles and that they have different sensitivities for alkaline earth metals.

[Sensitivity of intracellular calcium-binding sites of exo- and endocytosis of synaptic vesicles to Sr, Ba and Mg ions].

In the experiments on frog motor nerve endings made with electrophysiological (intra- and extracellular recording ofpostsynaptic sygnals) and optical (confocal microscopy with usage of fluorescent marker FM 1-43) approaches, transmitter release and exo-endocytosis of synaptic vesicles after replacement ofextracellular Ca ions by other alkaline earth metal ions (Sr, Ba, Mg) were studied. Massive asynchronous exocytosis with high potassium solution and evoked exocytosis with high-frequency rhythmic stimulation of motor nerve were initiated. It has been shown that the site of asynchronous exocytosis was sensitive to Ca, Sr, Ba and Mg ions whereas the site of evoked (synchronous) exocytosis--to Ca and Sr ions only. After stimulation either asynchronous or synchronous exocytosis site of endosytosis was found to be sensitive to Ca, Sr and Ba ions and possess the lowest affinity to Sr ions. It is concluded that there exist different intracellular calcium-binding sites for exocytosis and endocytosis that possess distinct sensitivity to alkaline earth metal ions.

Topography and affinity of calcium sensors of exo- and endocytosis in motor nerve terminals.

Measurements with extracellular microelectrode technique showed that depolarization of motor nerve terminals in frog cutaneous pectoris muscle with high-potassium solution (40 mM K(+)) increased frequency of miniature end-plate currents. Both fast intracellular calcium chelator BAPTA-AM and slow chelator EGTA-AM equally moderated the increase in the frequency of miniature end-plate currents. Intravital fluorescent microscopy with FM 1-43 dye showed that under conditions of stimulation of neurotransmitter exocytosis and secretion with high-potassium solution, internalization of the dye into newly-formed endocytotic synaptic vesicles proceeded both in the control and in the presence of EGTA-AM. In contrast, internalization of the dye was not observed in the presence of BAPTA-AM. It was concluded that asynchronous exocytosis of synaptic vesicles goes on in the active zones enriched with Ca-channels due to activation of high-affinity Ca-site in Ca-macrodomain. Endocytosis of vesicles is probably initiated by Ca-microdomain during activation of low-affinity Ca-site in the immediate proximity to the Ca channel.

Effects of high-potassium solutions and caffeine on synaptic vesicle exoendocytosis processes in the frog neuromuscular junction.

Studies on frog skin-pectoris muscle preparations using vital fluorescent microscopy showed that stimulation of transmitter secretion using high-potassium solutions with the endocytosis marker FM 1-43 induced bright spots in all motor nerve terminals, these representing accumulations of vesicles undergoing the exoendocytic cycle in the active zones of nerve endings. Stimulation of transmitter secretion with caffeine evoked bright spots only in some nerve terminals and only in some parts of the terminals. In summer, the number of bright spots on stimulation of transmitter secretion by caffeine increased sharply. Extracellular recording of spontaneous synaptic signals showed that high-potassium solutions, like caffeine, produced dose-dependent increases in the frequency of miniature endplate currents. However, while high-potassium solutions always increased the frequency, this occurred with caffeine in only a proportion of experiments. This leads to the conclusion that exoendocytosis processes can occur both because of the influx of Ca(2+) ions into nerve endings as a result of depolarization (high-potassium solutions) and because of the release of Ca(2+) ions from the endoplasmic reticulum (caffeine). The possible spatial localization of the endoplasmic reticulum in nerve endings is discussed. The endoplasmic reticulum is suggested to have a role in synapse remodeling processes.

[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.

[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.

Kiss-and-run quantal secretion in frog nerve-muscle synapse.

Electrophysiological and optical methods were used to study exo- and endocytosis of synaptic vesicles underlying secretion of the neurotransmitter from motor nerve terminals in frog sternocutaneous muscle. Increase in extracellular concentration of K+ or sucrose produced similar increase in the frequency of miniature endplate currents recorded by extracellular microelectrode. Fluorescent microscopy revealed bright spots in nerve terminal during stimulation of secretion with high-potassium solutions in the presence of endocytosis marker FM1-43. These spots corresponded to clusters of synaptic vesicles that passed through the cycles of exo- and endocytosis. Subsequent high-potassium stimulation of exocytosis in normal Ringer solution led to disappearance of marker spots, while in hyperosmotic saline the spots were preserved. No spots were seen after stimulation of neurotransmitter secretion with sucrose in the presence of FM1-43. It is concluded that quantal secretion of the neurotransmitter in frog motor nerve endings can be realized via both complete exocytosis of synaptic vesicles with subsequent endocytosis and kiss-and-run mechanism with the formation of a temporary pore.

Transmitter secretion in the frog neuromuscular synapse after prolonged exposure to calcium-free solutions.

Experiments on neuromuscular synapses from frog skin/chest muscle preparations in conditions of extracellular recording addressed changes in the spontaneous and evoked transmitter secretion after long-term (1.5-6 h) maintenance of preparations in calcium-free solution containing EGTA. Use of three microelectrodes for recording of single-quantum postsynaptic signals showed that calcium-free solution altered the characteristic topography of transmitter secretion in nerve terminals, with widening and fusion of groups of transmitter release. These changes persisted after preparations were returned to the initial solution. These data suggest that calcium-free solutions lead to disorganization of the active zones of nerve endings, At initially low extracellular Ca ion concentrations (0.15-0.4 mM), disorganization of active zones induced by prolonged maintenance of preparations in calcium-free solutions led to decreases in the mean amplitude of endplate currents (EPC) because of decreases in their quantum composition, increases in the time course of transmitter secretion, and decreases in the frequency of miniature endplate currents. The relationship between quantum composition of EPC and the extracellular Ca ion concentration showed a sharp displacement towards higher concentrations, without significant changes in the slope of the relationship. At high initial Ca concentrations (1.8 mM), long-term exposure to calcium-free solutions led to a less marked decrease in EPC amplitude. It is suggested that the extra- and intracellular Ca ion concentrations support the maintenance of the characteristic morphofunctional organization of the apparatus responsible for transmitter secretion in frog nerve endings. Disorganization of the active zones leads to disruption of elements involved in transmitter secretion and decreases in the efficiency of secretion.

[Analysis of living motor nerve ending of a frog by endocytotic fluorescent marker FM 1-43]. / Prizhiznennoe fluorestsentnoe issledovanie dvigatel'nogo nervnogo okonchaniia liagushki s ispol'zovaniem éndotsitoznogo markera 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.

[Mediator secretion in the nerve-muscle synapse in the frog following long-term effect of calcium-free solutions]. / Sekretsiia mediatora v nervno-myshechnom sinapse u liagushki posle dlitel'nogo vozdeistviia beskal'tsievykh rastvorov.

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.