A. N. Tsentsevitsky

Opposite modulation of time course of quantal release in two parts of the same synapse by reactive oxygen species.

Reactive oxygen species (ROS) are potent regulators of transmitter release in chemical synapses, but the mechanism of this action remains almost unknown. Presynaptic modulation can change either the release probability or the time course of quantal release, which was recently recognized as an efficient mechanism determining synaptic efficiency. The nonuniform structure and a big size of the frog neuromuscular junction make it a useful model to study the action of ROS in compartments different in release probability and in time course of transmitter release. The time course (or kinetics) of quantal release could be estimated by measuring the dispersion of the synaptic delays for evoked uniquantal endplate currents (EPCs) under low release probability. Using two-electrode recording technique, the action of ROS on kinetics and release probabilities were studied at the proximal and distal parts within the same neuromuscular junction. The stable ROS hydrogen peroxide (H2O2) increased the dispersion of synaptic delays of EPCs (i.e. desynchronized quantal release) within the distal part but decreased delay dispersion (synchronized quantal release) within the proximal part of the same synapse. Unlike the opposite modulation of kinetics, H2O2 reduced release probability in both distal and proximal parts. Since ATP is released from motor nerve terminals together with acetylcholine and can be involved in ROS signaling, we tested the presynaptic action of ATP. In the presence of the pro-oxidant Fe2+, extracellular ATP, similarly to H2O2, induced significant desynchronization of release in the distal regions. The antioxidant N-acetyl-cysteine attenuated the inhibitory action of ATP on release probability and abolished the action of H2O2 and ATP in the presence of Fe2+, on release kinetics. Our data suggest that ROS induced during muscle activity could change the time course of transmitter release along the motor nerve terminal to provide fine tuning of synaptic efficacy.

Kinetics of acetylcholine quanta release at the neuromuscular junction during high-frequency nerve stimulation

The effects of high‐frequency nerve stimulation (10–100 Hz) on the kinetics of evoked acetylcholine quanta secretion from frog motor nerve endings were studied. The amplitude and temporal parameters of uni‐ and multiquantal endplate currents were analysed to estimate the possible changes in the degree of synchrony of quantal release. The frog neuromuscular synapse is unusually long and we have placed special emphasis on evaluating the velocity of propagation of excitation along the nonmyelinated nerve ending as this might influence the synchrony of release from the whole terminal and hence affect the time course of postsynaptic currents. The data show that high‐frequency firing leads to the desynchronization of acetylcholine release from motor nerve endings governed by at least two independent factors, namely a reduction of nerve pulse propagation velocity in the nonmyelinated parts of the axon and a change of secretion kinetics at single active zones. A computer reconstruction of the multiquantal synaptic response was performed to estimate any contribution of each of the above factors to the total rate of release and amplitude and time characteristics of the endplate currents. The results indicate that modification of the kinetics of neurotransmitter quanta release during high‐frequency firing should be taken into account when mechanisms underlying the plasticity of chemical synapses are under investigation.

Identification of the muscarinic receptor subtypes involved in autoregulation of acetylcholine quantal release from frog motor nerve endings.

5 We have earlier demonstrated that in the course of highhfrequency stimulation of the motor nerve changes in amplitude of the endplate successive potentials (currents) are not only related to variation of the quantal content of synaptic responses, but also result from asynchronous release of acetylcholine (ACh) in individual active zones [1]. Since highhfree quency activity promotes accumulation of endogee nous ACh in a synaptic cleft, it has been hypothesized that changes in a release of neurotransmitter are caused by activation of presynaptic cholinergic recepp tors. This suggestion is supported by previous data on the ability of exogenous ACh and its analogues to reduce the quantal content of the endplate currents (EPCs) and to desynchronize release of individual quanta forming a multiiquantal response [2, 3]. The motor nerve ending is known to have choline receptors of NN and MMsubtypes [2, 4]. The purpose of this study was to elucidate the role of various subtypes of muscarinic choline receptors in autoregulation of the neurotransmitter release in response to rhythmic nerve stimulation at low and high frequencies (0.5 and 100 Hz, respectively) under the condition of physioo logical concentration of calcium ions. In our experiments, we used neuromuscular prepaa rations of m. sartorius isolated from the frog Rana ridd ibunda. The muscle with a nerve fragment was placed in an experimental 33mL chamber with flowing Ringers solution (pH 7.3–7.4) of the following comm The EPCs were recorded using the method of twooelectrode fixation of the membrane potential (twoelectrode voltage clamp). The musclee fiber membrane potential within the area of synapse was at the level of –60 mV. The recording device conn sisting of an analoggtoodigital converter and a comm puter station enabled us to perform highhprecision measurements of synaptic signals and their amplitudee time characteristics. From 100 to 200 of the evoked and miniature EPCs (mEPCs) were recorded for each muscle fiber. The EPC quantal content was evaluated as a ratio of the mean EPC amplitudes of the evoked multii quantal response and mEPC amplitudes [5]. Degree of secretion synchronity was evaluated as a ratio of the rise time durations (20–80% of the amplitude) in multii and oneequantal EPCs [6]. To cause blocking of M11subtype muscarinic receptors, pirenzepine and the new selective blocker VU0255035 [7] were used at a dose of 100 nmol/L. Selective antagonists methoctramine (10 nmol/L) and 44DAMP (1 µmol/L) were used for blocking of M2/M4 and M3 choline receptors, …

Metabotropic and ionotropic glutamate receptors mediate the modulation of acetylcholine release at the frog neuromuscular junction

There is some evidence that glutamate (Glu) acts as a signaling molecule at vertebrate neuromuscular junctions where acetylcholine (ACh) serves as a neurotransmitter. In this study, performed on the cutaneous pectoris muscle of the frog Rana ridibunda, Glu receptor mechanisms that modulate ACh release processes were analyzed. Electrophysiological experiments showed that Glu reduces both spontaneous and evoked quantal secretion of ACh and synchronizes its release in response to electrical stimulation. Quisqualate, an agonist of ionotropic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic receptors and metabotropic Group I mGlu receptors, also exerted Glu‐like inhibitory effects on the secretion of ACh but had no effect on the kinetics of quantal release. Quisqualates inhibitory effect did not occur when a blocker of Group I mGlu receptors (LY 367385) or an inhibitor of phospholipase C (U73122) was present. An increase in the degree of synchrony of ACh quantal release, such as that produced by Glu, was obtained after application of N‐methyl‐D‐aspartic acid (NMDA). The presence of Group I mGlu and NMDA receptors in the neuromuscular synapse was confirmed by immunocytochemistry. Thus, the data suggest that both metabotropic Group I mGlu receptors and ionotropic NMDA receptors are present at the neuromuscular synapse of amphibians, and that the activation of these receptors initiates different mechanisms for the regulation of ACh release from motor nerve terminals.

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

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.

Presynaptic receptors regulating the time course of neurotransmitter release from vertebrate nerve endings

A number of different types of presynaptic receptors was revealed in central and peripheral chemical synapses activated both by main mediator and co-mediators released simultaneously. Physiological significance and mechanisms of functioning of these receptors are not clear yet. They are assumed to provide negative or positive feedback decreasing or increasing the number of neurotransmitter quanta released in response to nerve impulse and thus regulating synaptic transmission. At the same time, there is one more way of secretion process modulation associated with the changes of timing of transmitter release. This mechanism was shown to contribute to the efficiency of synaptic transmission. The role of presynaptic receptors in regulation of the kinetics of quanta release is one of the interesting questions of modern neurophysiology. This paper overviews the results obtained by the authors that demonstrate the contribution of presynaptic receptors of different types into the regulation of temporal parameters of quantal secretion at the vertebrates neuromuscular junction. It was shown that activation of the cholinergic nicotinic receptors leads to a decrease of the amplitude of postsynaptic response not only due to reduction of the quantity of released quanta but also due to increased the level of asynchronous release. On the contrary, the facilitating effect of catecholamines on the neuromuscular synapse is the result of activation of presynaptic β1-adrenoreceptors which leads to greater synchronization of release process and, consequently, to the increase of the amplitude of the postsynaptic response. Presynaptic purine receptors, involved in the modulation the intensity of secretion, are also capable of alteration of the time course of secretion. Activation of ryanodine receptors results in the increase of the number of quanta released with prolonged latencies leading to appearance of the phase of delayed asynchronous neurotransmitter release.

Presynaptic nicotinic cholinoreceptors modulate velocity of the action potential propagation along the motor nerve endings at a high-frequency synaptic activity

Experiments on frog neuromuscular junctions have demonstrated that asynchrony of the acetylcholine quantal release forming the multi-quantal evoked response at high-frequency synaptic activity is caused, in particular, by a decrease in velocity of the action potential propagation along the non-myelinated nerve endings, which is mediated by activation of the α7 and α4β4 nicotinic cholinoreceptors.

Revealing of T-type low-voltage activated calcium channels (CaV3) in frog neuromuscular junctions.

73 Increase in the intracellular level of Ca 2+ concenn tration leads to the initiation of a wide range of molee cular processes, including the activation of Ca 2+ dependent enzymes, gene expression, neurotransmitt ter release, etc. [1]. In spite of the variety of channels and pumps involved in the regulation of the intracelluu lar Ca 2+ metabolism, the main role in Ca 2+ signaling is attributed to voltageeactivated Ca 2+ channels [2]. According to the modern classification based on the structural features of the poreeforming subunit α 1 , all voltageeactivated Ca 2+ channels (Ca V) are divided into three families. The first family It was established that in synapses of the central nervous system Ca 2+ channels of different types [3] may be involved in the process of neurosecretion, whereas in synapses of peripheral nervous system this process is mediated mainly by one type of channels. For example, in mammalian neuromuscular juncc tions, these are Ca V 2.1 (P/QQtype) channels, and in amphibian synapses, these are Ca V 2.2 (NNtype). Howw ever, it was shown that, in addition to the main type of channels, blocking of which leads to the blockage of evoked quantal mediator release, other types of highh voltage activated Ca 2+ channels are also found in motor endplates, and their role in neurotransmission is not entirely clear yet [4–9]. At the same time, the existence and functioning of lowwvoltage activated Ca 2+ channels (of the T type) at neuromuscular juncc tion remains an open question, which was the reason for our study. The purpose of the study was to detect lowwvoltage activated Ca 2+ channels (Ca V 3) in neuromuscular synn apses and to reveal their potential role in neurotranss mission to example of the frog neuromuscular juncc tions. Experiments were carried out on neuromuscular preparations of m. cutaneous pectoris of the frog Rana ridibunda with the use of immunocytochemical and electrophysiological methods. For the immunocytochemical detection and locaa tion of Ca 2+ channels of the Ca V 3 type, the neuromuss cular preparations were fixed in 3% formaldehyde (Sigma, USA), sequentially incubated in 0.3% Triton XX100 (Sigma) for 30 min; in a solution containing 5% of goat serum (Sigma), 1% of bovine serum albumin (Sigma) and 0.01% of Triton XX100 (15 min). Then, the neuromuscular preparations were incubated durr ing 15 h at a temperature of 4°C with rabbit polyclonal antibodies against α 1G …

Presynaptic voltage-dependent calcium channels and regulation of the transmitter release in the peripheral nervous system

Calcium ions entering into the nerve terminal through voltage-dependent calcium channels during action potential initiate neurotransmitter release and thereby play an important role in the transmission of excitation. In addition to the amount of released neurotransmitter quanta (the quantal content of the endplate potential), another important characteristic of exocytosis is the kinetics of release of individual quanta that form total multiquantal response. Previously, we showed the dependence of the temporal parameters of neurotransmitter quantum release on the extracellular calcium ion concentration, but it remained unclear which calcium channel types are involved in the modulation of the quantal content and release kinetics. It is commonly accepted that, in contrast to the central nervous system, wherein the secretion take part various types of voltage-dependent calcium channels, exocytosis in the peripheral nervous system of adult animals mainly occurs owing to one type of calcium channels. This is the P/Q type (Ca V 2.1 in the latest classification) blocked with FTX toxin and ω -agatoxin IVA in mammalian neuromuscular junction. In frog synapses, this is the N type channels (Ca V 2.2), which are blocked by the ω -conotoxin GVIA. At the same time, the ability of FTX, a specifc P/Q channel blocker, to decrease the quantal content in frog synapses and effects of dihydropyridines that block L type (Ca V 1.2) channels in the developing Xenopus synapses, and L type (Ca V 1.2) channels activator effects in frog synapses, taken together, indicate the possibility of participation of other types of calcium channels in the exocytosis regulation in peripheral synapses. The possible role of changes in the activity of not only P/Q channels, but also other calcium channel types, in modulation of the temporal parameters of neurotransmitter release was shown in peripheral synapses of mouse and crayfish. In order to identify the type of voltage-dependent calcium channels modulating of the quanta secretion in frog and mouse synapses, the effects of specific blockers of various channel types on the endplate potentials quantal content and transmitter release kinetics were analyzed. On isolated Rana ridibunda neuromuscular preparation of m. cutaneus pectoris and on the mouse diaphragm the evoked uniquantal endplate currents were registered by the extracellular electrodes and the fluctuations of the real synaptic latency were estimated. Blocking of the P/Q-, N-, and L-channels in nerve-muscle junction of the frog by the appropriate specific blockers led to a decrease in the amount of released quanta, as well as in the synaptic latency variance, that indicate the synchronization of the process of synaptic vesicles exocytosis. Obtained results indicate that in low-calcium solution the kinetics of quanta secretion in the frog synapse may be regulated by N-, P/Q-, and L-calcium channels. In the mouse nerve muscle junctions at low calcium concentration and infrequent stimulation, when asynchrony of the secretion was more pronounced, the block of the P/Q-channels led to a decrease in the amount of released quanta without changing the kinetics of their secretion, while blocking the L-type channels only decreased variance of synaptic latency. The fact that the number of released quanta and the time course of their secretion may change independently when various types of voltage-dependent calcium channels are blocked witnesses that the role of N-, P/Q-, and L-channels in modulation of the process of synaptic vesicles exocytosis is different.

Participation of different types of voltage-dependent calcium channels in evoked quantal transmitter release in frog neuromuscular junctions

389 Calcium ions entering the nerve ending through voltage-dependent calcium channels during action potential initiate neurotransmitter release [1] and thereby play an important role in the synaptic transmission of excitation. It is commonly accepted that, in contrast to the central nervous system, where neurosecretion occurs through various types of voltage-dependent calcium channels [2], exocytosis in the peripheral nervous system of adult animals mainly occurs through one type of calcium channels. This is the P/Q type ( Ca V 2.1 in the current classification) blocked with FTX toxin and ω -agatoxin IVA in neuromuscular junction of homoiothermal animals; in frog synapses, this is the N type of channels ( Ca V 2.2 ), which is blocked by the ω -conotoxin GVIA [5, 6]. At the same time, the ability of FTX, a specific P/Q channels blocker, to decrease the amount of released neurotransmitter in frog synapses [6], dihydropyridine effects of blocking L ( Ca V 1.2 ) channels in the developing Xenopus synapses [7], and L ( Ca V 1.2 ) channels activator effects in frog synapses [8], taken together, indicate a possible participation of other types of calcium channels in the exocytosis regulation in peripheral synapses of poikilothermal animals. In addition to the amount of released neurotransmitter quanta (the quantal content of the endplate potential), another important characteristic of exocytosis is the kinetics of release of individual quanta that form the total multiquantum response [9]. Previously, we showed the dependence of the time course of nerotransmitter quantal release on the extracellular calcium ion concentration [10], but it remained unclear which calcium channel type is involved in the modulation of the quantal content and release kinetics. Recently, data were reported on the possible role of changes in the activity of not only P/Q channels, but also other calcium channel types, in modulation of the time course of neurotransmitter release in peripheral synapses of the mouse and crayfish [11, 12]. The goal of this study was to identify the type of voltage-dependent calcium channels participating in the regulation of both the amount of neurotransmitter quanta released in response to a nerve impulse and time course of its secretion. We analyzed the influence of specific blockers of various channel types on the endplate potentials quantal content and neurotransmitter release kinetics in a frog neuromuscular junction.