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Effects of gamma-aminobutyric acid (GABA) and some selective GABAergic ligands on the quantal acetylcholine (ACh) release in the frog neuromuscular contacts were investigated using combination of microelectrode technique with fluorescent and immunohistochemical assays. Significant attenuation of ACh release was observed in the presence of GABA as well as selective GABA A and GABA B receptor agonists. Neither GABA A nor GABA B antagonists abolished to full extent this effect of GABA. Fluorescent assay allowed to detect the GABA-induced opening of K + channels, which was inhibited by XE-991, a selective antagonist of K v 7 type. Electrophysiological recordings of endplate potentials in the presence of XE-991 confirmed the contribution of K v 7 type potassium channels to the effects of GABA on ACh release that was not associated with GABA A and GABA B receptors activation. Note that XE-991 effectively precluded the action of retigabine, neuronal K v 7 channel opener, on ACh release. Immunohistochemical assay revealed that frog mature skeletal muscle fibers contain a significant amount of GABA, and substantial amount of GABA can be released in the extracellular space at the muscle contractions induced by prolonged high-frequency nerve stimulation. Besides, some binding sites for exogenous GABA were detected on the plasma membranes. It is concluded that GABA, in addition to affecting GABA A and GABA B receptors, can directly activate K v 7 channels, thereby negatively modulating the evoked ACh release. Endogenous GABA may serve as a retrograde regulator of neurotransmitter exocytosis., Competing Interests: Declarations Ethics Approval The experimental protocol met the requirements of the EU Directive 2010/63/EU and was approved by the Local Ethical Committee of Kazan Federal Scientific Centre (Protocol #3/February 28, 2023) as well as by the Local Ethical Committee of Kazan State Medical University (Protocol #5/May 27, 2014). The current study was conducted in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Consent for Publication Not applicable. Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All authors consent to publish the article., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Despite the long history of investigations of adrenergic compounds and their biological effects, specific mechanisms of their action in distinct compartments of the motor unit remain obscure. Recent results have suggested that not only skeletal muscles but also the neuromuscular junctions represent important targets for the action of catecholamines. In this paper, we describe the effects of adrenaline and noradrenaline on the frequency of miniature endplate potentials, the quantal content of the evoked endplate potentials and the kinetics of acetylcholine quantal release in the motor nerve endings of the mouse diaphragm. Noradrenaline and adrenaline decreased the frequency of the spontaneous release of acetylcholine quanta. The effect of noradrenaline was prevented by the β adrenoreceptor blocker propranolol, whereas the action of adrenaline was abolished by the α adrenoreceptor antagonist phentolamine. Noradrenaline did not alter the quantal content of endplate potentials, while adrenaline suppressed the evoked release of acetylcholine. Blocking the α adrenoreceptors prevented the decrease in quantal secretion caused by adrenaline. Quantal release became more asynchronous under noradrenaline, as evidenced by a greater dispersion of real synaptic delays; in contrast, adrenaline synchronized the release process. Our data suggest an involvement of α and β adrenoreceptors in the diverse modulation of the frequency of miniature endplate potentials, the quantal content of the evoked endplate potentials and the kinetics of acetylcholine quantal secretion in the mouse neuromuscular junction. Moreover, the adrenoblockers affected both the evoked and spontaneous quantal release of acetylcholine, suggesting the presence of endogenous catecholamines in the vicinity of cholinergic synapses., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Muscarinic cholinoreceptors regulate the neurosecretion process in vertebrate neuromuscular junctions. The diversity of muscarinic effects on acetylcholine (ACh) secretion may be attributed to the different muscarinic subtypes involved in this process. In the present study, the location of five muscarinic receptor subtypes (M1, M2, M3, M4 and M5) on the motor nerve terminals of frog cutaneous pectoris muscle was shown using specific polyclonal antibodies. The modulatory roles of these receptors were investigated via assessment of the effects of muscarine and specific muscarinic antagonists on the quantal content of endplate currents (EPCs) and the time course of secretion, which was estimated from the distribution of "real" synaptic delays of EPCs recorded in a low Ca 2+ /high Mg 2+ solution. The agonist muscarine decreased the EPC quantal content and synchronized the release process. The depressing action of muscarine on the EPC quantal content was abolished only by pretreatment of the preparation with the M3 blockers 4-DAMP (1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide) and J 104129 fumarate ((αR)-α-Cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate). Moreover, antagonists of the M1, M2, M3 and M4 receptors per se diminished the intensity of secretion, which suggests a putative up-regulation of the release by endogenous ACh., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Acetylcholinesterase ( AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine, thereby limiting spillover and duration of action. This study demonstrates the existence of an endogenous mechanism for the regulation of synaptic AChE activity. At the rat extensor digitorum longus neuromuscular junction, activation of N-methyl- d-aspartate ( NMDA) receptors by combined application of glutamate and glycine led to enhancement of nitric oxide ( NO) production, resulting in partial AChE inhibition. Partial AChE inhibition was measured using increases in miniature endplate current amplitude. AChE inhibition by paraoxon, inactivation of NO synthase by Nω-nitro- l-arginine methyl ester, and NMDA receptor blockade by dl-2-amino-5-phosphopentanoic acid prevented the increase in miniature endplate current amplitude caused by amino acids. High-frequency (10 Hz) motor nerve stimulation in a glycine-containing bathing solution also resulted in an increase in the amplitude of miniature endplate currents recorded during the interstimulus intervals. Pretreatment with an NO synthase inhibitor and NMDA receptor blockade fully eliminated this effect. This suggests that endogenous glutamate, released into the synaptic cleft as a co-mediator of acetylcholine, is capable of triggering the NMDA receptor/ NO synthase-mediated pathway that modulates synaptic AChE activity. Therefore, in addition to well-established modes of synaptic plasticity (e.g. changes in the effectiveness of neurotransmitter release and/or the sensitivity of the postsynaptic membrane), another mechanism exists based on the prompt regulation of AChE activity. [ABSTRACT FROM AUTHOR]