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

Immunohistochemical evidence of the presence of metabotropic receptors for γ-aminobutyric acid at the rat neuromuscular junctions

© 2015, Pleiades Publishing, Ltd. in the synapses of the “fast” (m. EDL) and “slow” (m. soleus) skeletal muscles of the rat GABABR1 and GABABR2 subunits of metabotropic receptors for γ-aminobutyric acid (GABA), located primarily on the motor nerve ending membrane were detected by immunohistochemistry and fluorescence microscopy methods

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

Studies of the expression of subunits α2 and β1 of Na⁺/K⁺-ATPase, α1S (L-type) Ca²⁺-channel, and SERCA 1/2/3 of Ca²⁺-ATPase of phasic and postural rat muscles in a model of hypogravity using the method of fluorescent microscopy

© 2016, Pleiades Publishing, Ltd.Using fluorescent microscopy, we found decreased expression of the β1 subunit of Na+/K+-ATPase and subunits of Ca2+-ATPase, increased expression of the α1S subunit of the L-type Ca2+-channel, and no changes in the expression of the α2 subunit of Na+/K+-ATPase in rat postural muscle under the conditions of modeled hypogravity. In the phasic muscle, we observed decreased expression of the β1 subunit, which was similar to that found in the postural muscle, whereas the other studied parameters remained without alterations. However, a decrease in the fluorescence intensity of the β1 subunit was insignificant due to a high variability of data. Thus, hypogravity negatively influenced primarily those skeletal muscles that are responsible for static load

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

Muscarinic cholinoreceptors (M1-, M2-, M3- and M4-type) modulate the acetylcholine secretion in the frog neuromuscular junction

© 2017 Elsevier B.V.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 Ca2+/high Mg2+ 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

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

Semaphorins Are Likely to Be Involved in the Control of Hibernation

© 2016, Springer Science+Business Media New York.Hibernation is unique mammals’ phenotype demonstrated surviving in seasonal adverse environment conditions. During hibernation, all of systems of organs undergo alterations in their metabolic activity and physiology similar to different physiological conditions associated with human diseases and injuries, which represent a proper model for studying new approaches in clinical treatment. In this study, we have analyzed expression of semaphorins in small hibernator edible dormouse (Glis glis). Semaphorins are factors which involved in a key process of axon guidance and cell–cell communication and can act as tumor suppressor. We found that gene coding Semaphorin-3D (SEMA3D), previously reported to be specific for brain and heart of mammals, represents one of the most upregulated transcripts in the muscles of the hibernating dormice. Furthermore, another member of the same family, Semaphorin-5B (SEMA5B), was strongly induced in the spinal cord of hibernating animals. These observations make semaphorin group, recently attracting more attention due to anti-tumor activity, one of the target for in-depth analysis in relation to the molecular mechanisms of hibernation

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

Immunohistochemical research of reaction of motoneurons of lumbar spinal cord of the mice that were in 30-day flight on the BION-M1 biosatellite on a week readaptation to conditions of Earth gravitation

© 2016, Human Stem Cell Institute. All rights reserved.Earlier, by an immunohistochemical method we define that after 30-day space flight in motoneurons of mice lumbar spinal cord immunoexpression of the proteins responsible for synaptic transfer of a nervous impulse and proteins of heat shock proteins decrease. In this research for an assessment of animals recovery process dynamics after space flight we studied an immunoexpression of the proteins participating in synaptic transfer of a nervous impulse (synaptophisyne, and PSD95), neurotrophic factors (a vascular endothelial factor of growth - VEGF and its receptor - Flt-1) and heat shock proteins (Hsp25 and Hsp70) in motoneurons of lumbar spinal cord of a mice after 30-day space flight on the BION-M1 biosatellite and the subsequent week readaptation to conditions of Earth gravitation. In this research by immunohistochemical method determine that after a week of animals staying in the Earth gravitation conditions the immunoexpression of synaptophisyne continued to decrease while the expression of PSD95, Hsp25, Hsp70 and VEGF increased in relation to the animals removed from experiment right after 30-day flight. The obtained data confirm functional plasticity of spinal cord motoneurons in the conditions of gravitation force changing. The fact, which is especially interesting, that "switches on" of neurons protective mechanisms (strengthening of heat shock proteins and neurotrophic factor expression) happens not in response to hypogravitation influence, but only a week after return of animals to conditions of Earth gravitation