Cytoskeletal Protein Septins Participate in the Modulation of the Kinetics of Acetylcholine Quanta Release at Neuromuscular Junction

© 2016, Springer Science+Business Media New York.In the presynaptic nerve terminal, some families of cytoskeletal proteins can participate in the neurosecretion modulation. Septins, GTP-binding cytoskeletal proteins, form hetero-oligomeric complexes both among themselves and with other synaptic proteins. Previously, it was reported that in the cell cultures, septins can interact with SNARE complex, NSF and SNAP-25, suggesting septin involvement in the exocytosis of neuromediator. Here, we describe effects of septin blockade on the time course of acetylcholine quantal release at mice neuromuscular junction under different frequency stimulation of motor nerve. Forchlorfenuron (FCF), a synthetic cytokinin, is the inhibitor of septin polymerization which specifically impairs assembly and disassembly of septin hetero-oligomers without affecting the actin or tubulin polymerization. FCF in the concentrations from 20 to 100 μM decreased the intensity of the spontaneous and evoked release of acetylcholine quanta. Block of septin dynamics resulted in changes in the kinetics of quantal release: the synchronization of quanta secretion was observed at low and high frequencies of nerve stimulation. Thus, septins are important regulators of spontaneous and evoked neurotransmitter secretion, since disruption of their interaction with SNARE protein complex leads to changes in kinetics of neurotransmitter quanta secretion

Effect of Noradrenaline on the Kinetics of Evoked Acetylcholine Secretion in Mouse Neuromuscular Junction

© 2018, Pleiades Publishing, Ltd. Abstract: In contrast to frog neuromuscular synapses, where noradrenaline (norepinephrine) and its analogues caused synchronization of the acetylcholine release process, in mouse diaphragm endplates noradrenaline increased the degree of asynchrony of neurosecretion. The effect of noradrenaline on release timing persisted at different levels of external calcium ions (0.25–2.0 mM) and was abolished in presence of both α- and β‑adrenoblockers phentolamine and propranolol. The computer reconstruction of multiquantal endplate currents accounting for experimentally observed modification of release kinetics under noradrenaline showed that the rise time of postsynaptic response changes to a greater extent than the amplitude and falling phase of the multiquantal responses. We conclude that there exists a principal difference in the action of noradrenaline in the cholinergic neuromuscular synapses of warm-blooded and cold-blooded animals that can be accounted for by the differences in the type of adrenoreceptors involved in the modulation of synaptic transmission and/or in the involvement of distinct intracellular pathways triggered by receptor activation

Contribution of Ryanodine Receptors in Forming Presynaptic Ca²⁺ Level and Cholinergic Modulation in Response to Single Potential in Frog Neuromuscular Junction

© 2016, Springer Science+Business Media New York.Ca2+ entering through voltage-gated Ca2+ channels plays a key role in the initiation of neurotransmitter secretion. However, intracellular Ca2+ storages such as endoplasmic reticulum (ER) can also contribute in the presynaptic Ca2+ level forming. Also ryanodine-dependent Ca2+-induced Ca2+ channels of endoplasmic reticulum may contribute in secretion inhibiting action of cholinomimetics. In this work, we use a photometric method for estimating the relative change in the level of presynaptic Ca2+ ions (Ca2+ transient). We have shown that during low-frequency stimulation Ca2+ release from the ER is involved in forming of presynaptic Ca2+ level. And more likely those effects of the cholinomimetics on Ca2+ transient are not related to Ca2+ release from the endoplasmic reticulum

Calcium Transient and Quantal Release in Mouse Neuromuscular Junction Under Extracellular Calcium Concentration Change

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. In mouse neuromuscular junction, the amplitude of the presynaptic calcium (Ca2+) transient was measured and correlated with mediator release at different extracellular Ca2+ concentrations. Fluorescent calcium-sensitive dye Oregon Green 488 BAPTA 1 hexapotassium salt was used for Ca2+ transient registration. The quantal content of release was assessed by the amplitude of the endplate potentials (EPPs) and was measured using intracellular microelectrodes. The amplitude of the EPPs changed more significantly than the amplitude of the Ca2+ transient when the extracellular calcium concentration was changed. Linear approximation of the dependence of the quantal content on the amplitude of the Ca2+ transient on double logarithmic scale gave a slope showing that the biochemical cooperativity was 2.86. The obtained value is comparable with the data calculated earlier in the neuromuscular junction of the rat and other synapses using electrophysiological measurements. Our data suggest that the change of the Ca2+ transients recorded from the whole volume of the nerve terminal properly reflects the variation of calcium concentration responsible for the neurotransmitter release in active zone. Thus, analysis of the bulk Ca2+ transient can be used to evaluate the calcium entry into the nerve endings and compare it with the number of quanta released under different conditions

Action of ATP on Ca²⁺-Transient in Different Parts of the Frog Motor Nerve Ending

© 2016, Springer Science+Business Media New York.Electrophysiological evidence indicates a difference in neurotransmitter secretion along the motor nerve terminals of the frog neuromuscular junction. This includes a decrease of the minimal synaptic delay value and a reduction in the quantal content of the evoked endplate currents from the proximal to distal portion of the motor nerve ending. Besides, various physiologically active compounds may have different effects on the acetylcholine secretion in the proximal and distal parts of the nerve terminal. Here, we explored the effects of ATP on Ca2+-transient using optical detection methods with high-speed camera in different parts of the frog nerve terminal. There was shown a significant inhibitory effect of ATP on Ca2+-transient in both the proximal and distal regions of nerve terminals. However, in different parts of nerve endings, any significant differences in ATP effects were not found. Thus, ATP decreases the Ca2+-transient along the entire presynaptic terminal

Calcium Transient Registration in Response to Single Stimulation and During Train of Pulses in Mouse Neuromuscular Junction

© 2016, Springer Science+Business Media New York.Calcium (Ca2+) is a key ion involved in transmitter release in chemical synapses. Optical recording of fluorescence changes of Ca2+ indicators is one of the most frequently used methods to measure intracellular Ca2+ dynamics. This technique is based on use of Ca2+-binding fluorescent dyes which change their emission intensity after binding to Ca2+. The most crucial step in this type of experiments is loading of Ca2+ dye. In this paper, we present the method of Ca2+-sensitive dye loading to mammalian nerve endings through the stump of the nerve. We represent Ca2+ transient registered parameters in response to a single motor nerve stimulus. The study of Ca2+ dynamics during high frequency stimulation close to real pattern of synaptic transmission allows us to understand such fundamental process as synaptic plasticity. We describe the results obtained during the registration of Ca2+ transient caused by the rhythmic motor nerve stimulation. Intracellular level of Ca2+ estimated by the amplitude of Ca2+ transient rises with the increase of stimulation frequency. The amplitude of Ca2+ transient decreases after blocking of voltage dependent Ca2+ channels by cadmium. The obtained data showed that detected increase of fluorescence intensity is induced by Ca2+ influx through the voltage-gated Ca2+ channels to the nerve ending during an action potential. This dye-loading method is suitable for registration of presynaptic Ca2+ dynamics under both single nerve stimulus and rhythmic activity

Homocysteine aggravates ROS-induced depression of transmitter release from motor nerve terminals: Potential mechanism of peripheral impairment in motor neuron diseases associated with hyperhomocysteinemia

© 2015 Bukharaeva, Shakirzyanova, Khuzakhmetova, Sitdikova and Giniatullin. Homocysteine (HCY) is a pro-inflammatory sulphur-containing redox active endogenous amino acid, which concentration increases in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). A widely held view suggests that HCY could contribute to neurodegeneration via promotion of oxidative stress. However, the action of HCY on motor nerve terminals has not been investigated so far. We previously reported that oxidative stress inhibited synaptic transmission at the neuromuscular junction, targeting primarily the motor nerve terminals. In the current study, we investigated the effect of HCY on oxidative stress-induced impairment of transmitter release at the mouse diaphragm muscle. The mild oxidant H2O2 decreased the intensity of spontaneous quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl Daspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS

Calcium modulation of the kinetics of evoked quantum secretion in neuromuscular synapses of cold- and warm-blooded animals

© 2015, Pleiades Publishing, Ltd. Calcium entry into the nerve endings through voltage-dependent calcium channels triggers a chain of events leading to exocytosis of neurotransmitter, providing the transmission of excitation through the synapse. In this regard, a significant role of calcium ions and presynaptic calcium channels in the modulation of secretion is evident. However, the question of the contribution of different types of voltage-dependent calcium channels in the calcium regulation parameters of the quantal secretion still remains unclear. The secretion kinetics characterizes a degree of synchrony of the neurotransmitter release. In recent decades it is regarded as one of the important factors maintaining the effectiveness of the synaptic transmission. Since neuromuscular synapses of frogs and mice are classical objects of physiological and pharmacological studies, the results of which are summarized and extrapolated to other synapses, it is interesting to compare changes of the acetylcholine secretion in these synapses under different conditions of calcium entry into the nerve endings. In this review we discuss the data on the neuromuscular synapses of frogs and mice and analyze some aspects of calcium regulation and involvement of different types of voltage-dependent calcium channels in the modulation of the acetylcholine secretion kinetics

Kinetics of neurotransmitter release in neuromuscular synapses of newborn and adult rats

The kinetics of the phasic synchronous and delayed asynchronous release of acetylcholine quanta was studied at the neuromuscular junctions of aging rats from infant to mature animals at various frequencies of rhythmic stimulation of the motor nerve. We found that in infants 6 (P6) and 10 (P10) days after birth a strongly asynchronous phase of quantal release was observed, along with a reduced number of quanta compared to the synapses of adults. The rise time and decay of uni-quantal end-plate currents were significantly longer in infant synapses. The presynaptic immunostaining revealed that the area of the synapses in infants was significantly (up to six times) smaller than in mature junctions. The intensity of delayed asynchronous release in infants increased with the frequency of stimulation more than in adults. A blockade of the ryanodine receptors, which can contribute to the formation of delayed asynchronous release, had no effect on the kinetics of delayed secretion in the infants unlike synapses of adults. Therefore, high degree of asynchrony of quantal release in infants is not associated with the activity of ryanodine receptors and with the liberation of calcium ions from intracellular calcium stores. © 2014 ISDN