Modern Concepts of Cholinergic Neurotransmission at the Motor Synapse

© 2018, Pleiades Publishing, Ltd. Cholinergic synaptic contact between motor neuron and skeletal muscle fiber is perhaps one of the core objects for investigations of molecular mechanisms underlying the communication between neurons and innervated cells. In the studies conducted on this object in the past few decades, a large amount of experimental data was obtained that substantially complemented a traditional view on synaptic transmission. In particular, it was established that (i) acetylcholine is released from the nerve ending in both quantal and nonquantal ways; (ii) molecular mechanisms of the processes of the quantal acetylcholine release—spontaneous and evoked by electrical stimuli—have unique features and can be regulated independently; (iii) acetylcholine release from the nerve ending is accompanied by a release of a number of synaptically active molecules modulating the processes of secretion or reception of the main mediator; (iv) signal molecules affecting the process of cholinergic neurotransmission can be released not only from the nerve ending but also from glial cells and muscle fiber; (v) molecular mechanisms of the regulation of synaptic transmission are highly diverse and go beyond the alteration of the number of the released acetylcholine quanta. Thus, the neuromuscular junction shall be deemed currently as complicated and adaptive synapse characterized by a wide range of multiloop intercellular signaling pathways between presynaptic motor neuron ending, muscle fiber, and glial cells ensuring a high safety factor of synaptic transmission and the possibility of its fine tuning

Adrenoceptors at the Frog Neuromuscular Junction: an Immunohistochemical Study

© 2016, Springer Science+Business Media New York.Previously, it was shown that both adrenaline and noradrenaline potentiate neuromuscular transmission, but which one of the receptors mediates the facilitating effect of catecholamines is still unclear. In this study, we have investigated the presence of different adrenoceptors at isolated preparations of frog cutaneous pectoris muscle by using methods of immunohistochemistry. The immunopositive reaction was observed while using polyclonal antibodies to α1- (α1B and α1D), α2- (α2A, α2B, and α2C), and β-adrenoceptors (β1, β2 and β3). In all the cases, the immunohistochemical staining of the mentioned proteins was localized in the area of the synaptic contact. Thus, at the neuromuscular junction, a wide range of α1-, α2- and β-adrenoceptors was found. It expands our understanding of the endogenous mechanisms of cholinergic neurotransmission regulation and elucidates the aspects of the mechanisms of action of adrenergic agonists, which are still intensively studied or already used for treatment of neuromuscular disorders with a primary neuro- or myopathology, and neuromuscular diseases characterized by a neuromuscular junction pathology

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

© 2016 Wiley Periodicals, Inc.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. Quisqualate's 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. © 2016 Wiley Periodicals, Inc

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction

© 2015 International Society for Neurochemistry. Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABABR1 and GABABR2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release

Regulation of acetylcholinesterase activity by nitric oxide in rat neuromuscular junction via N-methyl-d-aspartate receptor activation

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. NO molecules depress AChE activity in the neuromuscular junction thereby enhancing endplate current amplitude. 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. In addition to well-established modes of synaptic plasticity another mechanism exists based on the prompt regulation of AChE activity. © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd

GABA in developing rat skeletal muscle and motor neurons

© 2020, Springer-Verlag GmbH Austria, part of Springer Nature. In recent years, considerable evidence is accumulated pointing to participation of gamma-aminobutyric acid (GABA) in intercellular signaling in the peripheral nervous system, including, in particular, neuromuscular transmission. However, where in the neuromuscular synapse GABA is synthesized remains not quite clear. We used histochemical methods to detect GABA and l-glutamate decarboxylase (GAD) in developing skeletal muscle fibers and in cultured motor neurons. We found that GABA can be detected already in myocytes, but with further muscle maturation, GABA synthesis gradually attenuates and completely ceases in early postnatal development. We found also that formation of GABA in muscle tissue does not depend on activity of GAD, but presumably proceeds through some other, alternative pathways. In motor neurons, GABA and GAD can be detected at the early stage of development (prior to synapse formation). Our data support the hypothesis that GABA and GAD, which are detectable in adult neuromuscular junctions, have neuronal origin. The mechanism of GABA production and its role in developing muscle tissue need further clarification

Modern Concepts of Cholinergic Neurotransmission at the Motor Synapse

© 2018, Pleiades Publishing, Ltd. Cholinergic synaptic contact between motor neuron and skeletal muscle fiber is perhaps one of the core objects for investigations of molecular mechanisms underlying the communication between neurons and innervated cells. In the studies conducted on this object in the past few decades, a large amount of experimental data was obtained that substantially complemented a traditional view on synaptic transmission. In particular, it was established that (i) acetylcholine is released from the nerve ending in both quantal and nonquantal ways; (ii) molecular mechanisms of the processes of the quantal acetylcholine release—spontaneous and evoked by electrical stimuli—have unique features and can be regulated independently; (iii) acetylcholine release from the nerve ending is accompanied by a release of a number of synaptically active molecules modulating the processes of secretion or reception of the main mediator; (iv) signal molecules affecting the process of cholinergic neurotransmission can be released not only from the nerve ending but also from glial cells and muscle fiber; (v) molecular mechanisms of the regulation of synaptic transmission are highly diverse and go beyond the alteration of the number of the released acetylcholine quanta. Thus, the neuromuscular junction shall be deemed currently as complicated and adaptive synapse characterized by a wide range of multiloop intercellular signaling pathways between presynaptic motor neuron ending, muscle fiber, and glial cells ensuring a high safety factor of synaptic transmission and the possibility of its fine tuning

Adrenoceptors at the Frog Neuromuscular Junction: an Immunohistochemical Study

© 2016, Springer Science+Business Media New York.Previously, it was shown that both adrenaline and noradrenaline potentiate neuromuscular transmission, but which one of the receptors mediates the facilitating effect of catecholamines is still unclear. In this study, we have investigated the presence of different adrenoceptors at isolated preparations of frog cutaneous pectoris muscle by using methods of immunohistochemistry. The immunopositive reaction was observed while using polyclonal antibodies to α1- (α1B and α1D), α2- (α2A, α2B, and α2C), and β-adrenoceptors (β1, β2 and β3). In all the cases, the immunohistochemical staining of the mentioned proteins was localized in the area of the synaptic contact. Thus, at the neuromuscular junction, a wide range of α1-, α2- and β-adrenoceptors was found. It expands our understanding of the endogenous mechanisms of cholinergic neurotransmission regulation and elucidates the aspects of the mechanisms of action of adrenergic agonists, which are still intensively studied or already used for treatment of neuromuscular disorders with a primary neuro- or myopathology, and neuromuscular diseases characterized by a neuromuscular junction pathology

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction

© 2015 International Society for Neurochemistry. Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABABR1 and GABABR2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release