The Mechanism of α2 adrenoreceptor-dependent Modulation of Neurotransmitter Release at the Neuromuscular Junctions.

α2-Adrenoreceptors (ARs) are main Gi-protein coupled autoreceptors in sympathetic nerve terminals and targets for dexmedetomidine (DEX), a widely used sedative. We hypothesize that α2-ARs are also potent regulators of neuromuscular transmission via G protein-gated inwardly rectifying potassium (GIRK) channels. Using extracellular microelectrode recording of postsynaptic potentials, we found DEX-induced inhibition of spontaneous and evoked neurotransmitter release as well as desynchronization of evoked exocytotic events in the mouse diaphragm neuromuscular junction. These effects were suppressed by SKF-86,466, a selective α2-AR antagonist. An activator of GIRK channels ML297 had the same effects on neurotransmitter release as DEX. By contrast, inhibition of GIRK channels with tertiapin-Q prevented the action of DEX on evoked neurotransmitter release, but not on spontaneous exocytosis. The synaptic vesicle exocytosis is strongly dependent on Ca2+ influx through voltage-gated Ca2+ channels (VGCCs), which can be negatively regulated via α2-AR - GIRK channel axis. Indeed, inhibition of P/Q-, L-, N- or R-type VGCCs prevented the inhibitory action of DEX on evoked neurotransmitter release; antagonists of P/Q- and N-type channels also suppressed the DEX-mediated desynchronization of evoked exocytotic events. Furthermore, inhibition of P/Q-, L- or N-type VGCCs precluded the frequency decrease of spontaneous exocytosis upon DEX application. Thus, α2-ARs acting via GIRK channels and VGCCs (mainly, P/Q- and N-types) exert inhibitory effect on the neuromuscular communication by attenuating and desynchronizing evoked exocytosis. In addition, α2-ARs can suppress spontaneous exocytosis through GIRK channel-independent, but VGCC-dependent pathway.

Early Alterations in Structural and Functional Properties in the Neuromuscular Junctions of Mutant FUS Mice.

Amyotrophic lateral sclerosis (ALS) is manifested as skeletal muscle denervation, loss of motor neurons and finally severe respiratory failure. Mutations of RNA-binding protein FUS are one of the common genetic reasons of ALS accompanied by a 'dying back' type of degeneration. Using fluorescent approaches and microelectrode recordings, the early structural and functional alterations in diaphragm neuromuscular junctions (NMJs) were studied in mutant FUS mice at the pre-onset stage. Lipid peroxidation and decreased staining with a lipid raft marker were found in the mutant mice. Despite the preservation of the end-plate structure, immunolabeling revealed an increase in levels of presynaptic proteins, SNAP-25 and synapsin 1. The latter can restrain Ca2+-dependent synaptic vesicle mobilization. Indeed, neurotransmitter release upon intense nerve stimulation and its recovery after tetanus and compensatory synaptic vesicle endocytosis were markedly depressed in FUS mice. There was a trend to attenuation of axonal [Ca2+]in increase upon nerve stimulation at 20 Hz. However, no changes in neurotransmitter release and the intraterminal Ca2+ transient in response to low frequency stimulation or in quantal content and the synchrony of neurotransmitter release at low levels of external Ca2+ were detected. At a later stage, shrinking and fragmentation of end plates together with a decrease in presynaptic protein expression and disturbance of the neurotransmitter release timing occurred. Overall, suppression of synaptic vesicle exo-endocytosis upon intense activity probably due to alterations in membrane properties, synapsin 1 levels and Ca2+ kinetics could be an early sign of nascent NMJ pathology, which leads to neuromuscular contact disorganization.

Sympathetic Innervation and Endogenous Catecholamines in Neuromuscular Preparations of Muscles with Different Functional Profiles.

Influence of the sympathetic nervous system on the work of skeletal muscles contractile apparatus is now beyond doubt. However, until recently there was no evidence that the endings of sympathetic nerves can be located in close proximity to the neuromuscular synapses, and there is also no reliable data on how much endogenous adrenaline and noradrenaline can be contained near the synaptic contact in skeletal muscles. In this research, using fluorescent analysis, immunohistochemical and enzyme immunoassays the isolated neuromuscular preparations of three skeletal muscles of different functional profiles and containing different types of muscle fibers were examined. Close contact between the sympathetic and motor cholinergic nerve endings and the presence of tyrosine hydroxylase in this area were demonstrated. Concentrations of endogenous adrenaline and noradrenaline in the solution perfusing the neuromuscular preparation were determined under different modes of its functioning. The effects of α and ß adrenoreceptor blockers on the processes of acetylcholine quantal secretion from the motor nerve endings were compared. The data obtained provide evidence for the presence of endogenous catecholamines in the neuromuscular junction region and their role in modulation of the synaptic function.

Catecholamine-dependent hyperpolarization of the junctional membrane via ß2- adrenoreceptor/Gi-protein/α2-Na-K-ATPase pathway.

We investigated the effects of catecholamines, adrenaline and noradrenaline, as well as ß-adrenoceptor (AR) modulators on a resting membrane potential at the junctional and extrajunctional regions of mouse fast-twitch Levator auris longus muscle. The aim of the study was to find which AR subtypes, signaling molecules and Na,K-ATPase isoforms are involved in the hyperpolarizing action of catecholamines and whether this action could be accompanied by changes in the pump abundance on the sarcolemma. Adrenaline, noradrenaline and specific ß2-AR agonist induced hyperpolarization of both junctional and extrajunctional membrane, but the underlying mechanisms were different. In the junctional membrane the hyperpolarization depended on α2 isoform of the Na,K-ATPase and Gi-protein, whereas in the extrajunctional regions the hyperpolarization mainly relied on α1 isoform of Na,K-ATPase and adenylyl cyclase activities. In both junctional and extrajunctional regions, AR activation caused an increase in Na,K-ATPase abundance in the plasmalemma in a protein kinase A-dependent manner. Thus, the compartment-specific mechanisms are responsible for catecholamine-mediated hyperpolarization in the skeletal muscle.

Adrenoceptors Modulate Cholinergic Synaptic Transmission at the Neuromuscular Junction.

Adrenoceptor activators and blockers are widely used clinically for the treatment of cardiovascular and pulmonary disorders. More recently, adrenergic agents have also been used to treat neurodegenerative diseases. Recent studies indicate a location of sympathetic varicosities in close proximity to neuromuscular junctions. The pressing question is whether there could be any effects of endo- or exogenous catecholamines on cholinergic neuromuscular transmission. It was shown that the pharmacological stimulation of adrenoceptors, as well as sympathectomy, can affect both acetylcholine release from motor nerve terminals and the functioning of postsynaptic acetylcholine receptors. In this review, we discuss the recent data regarding the effects of adrenergic drugs on neurotransmission at the neuromuscular junction. The elucidation of the molecular mechanisms by which the clinically relevant adrenomimetics and adrenoblockers regulate quantal acetylcholine release from the presynaptic nerve terminals and postsynaptic sensitivity may help in the design of highly effective and well-tolerated sympathomimetics for treating a number of neurodegenerative diseases accompanied by synaptic defects.

Adrenaline Facilitates Synaptic Transmission by Synchronizing Release of Acetylcholine Quanta from Motor Nerve Endings.

The long history of studies on the effect of catecholamines on synaptic transmission does not answer the main question about the mechanism of their action on quantal release in the neuromuscular junction. Currently, interest in catecholamines has increased not only because of their widespread use in the clinic for the treatment of cardiovascular and pulmonary diseases but also because of recent data on their possible use for the treatment of certain neurodegenerative diseases, muscle weakness and amyotrophic sclerosis. Nevertheless, the effects and mechanisms of catecholamines on acetylcholine release remain unclear. We investigated the action of noradrenaline and adrenaline on the spontaneous and evoked quantal secretion of acetylcholine in the neuromuscular junction of the rat soleus muscle. Noradrenaline (10 µM) did not change the spontaneous acetylcholine quantal release, the number of released quanta after nerve stimulation, or the timing of the quantal secretion. However, adrenaline at the same concentration increased spontaneous secretion by 40%, increased evoked acetylcholine quantal release by 62%, and synchronized secretion. These effects differ from those previously described by us in the synapses of the frog cutaneous pectoris muscle and mouse diaphragm. This indicates specificity in catecholamine action that depends on the functional type of muscle and the need to take the targeted type of muscle into account in clinical practice.

Prenatal hyperhomocysteinemia induces oxidative stress and accelerates 'aging' of mammalian neuromuscular synapses.

Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H2O2) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM 1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM 1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H2O2 and lipid peroxidation products in the diaphragm muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine.

Reorganization of Septins Modulates Synaptic Transmission at Neuromuscular Junctions.

Septins (Sept) are highly conserved Guanosine-5'-triphosphate (GTP)-binding cytoskeletal proteins involved in neuronal signaling in the central nervous system but their involvement in signal transmission in peripheral synapses remains unclear. Sept5 and Sept9 proteins were detected in mouse peripheral neuromuscular junctions by immunofluorescence with a greater degree of co-localization with presynaptic than postsynaptic membranes. Preincubation of neuromuscular junction preparations with the inhibitor of Sept dynamics, forchlorfenuron (FCF), decreased co-localization of Sept with presynaptic membranes. FCF introduced ex vivo or in vivo had no effect on the amplitude of the spontaneous endplate currents (EPCs), indicating the absence of postsynaptic effects of FCF. However, FCF decreased acetylcholine (ACh) quantal release in response to nerve stimulation, reduced the amplitude of evoked quantal currents and decreased the number of quanta with long synaptic delays, demonstrating the presynaptic action of FCF. Nevertheless, FCF had no effect on the amplitude of calcium transient in nerve terminals, as detected by calcium-sensitive dye, and slightly decreased the ratio of the second response amplitude to the first one in paired-pulse experiments. These results suggest that FCF-induced decrease in ACh quantal secretion is not due to a decrease in Ca2+ influx but is likely related to the impairment of later stages occurring after Ca2+ entry, such as trafficking, docking or membrane fusion of synaptic vesicles. Therefore, Sept9 and Sept5 are abundantly expressed in presynaptic membranes, and disruption of Sept dynamics suppresses the evoked synchronous and delayed asynchronous quantal release of ACh, strongly suggesting an important role of Sept in the regulation of neurotransmission in peripheral synapses.

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

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 D-aspartate (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.

Bayesian analysis of the kinetics of quantal transmitter secretion at the neuromuscular junction.

The timing of transmitter release from nerve endings is considered nowadays as one of the factors determining the plasticity and efficacy of synaptic transmission. In the neuromuscular junction, the moments of release of individual acetylcholine quanta are related to the synaptic delays of uniquantal endplate currents recorded under conditions of lowered extracellular calcium. Using Bayesian modelling, we performed a statistical analysis of synaptic delays in mouse neuromuscular junction with different patterns of rhythmic nerve stimulation and when the entry of calcium ions into the nerve terminal was modified. We have obtained a statistical model of the release timing which is represented as the summation of two independent statistical distributions. The first of these is the exponentially modified Gaussian distribution. The mixture of normal and exponential components in this distribution can be interpreted as a two-stage mechanism of early and late periods of phasic synchronous secretion. The parameters of this distribution depend on both the stimulation frequency of the motor nerve and the calcium ions' entry conditions. The second distribution was modelled as quasi-uniform, with parameters independent of nerve stimulation frequency and calcium entry. Two different probability density functions for the distribution of synaptic delays suggest at least two independent processes controlling the time course of secretion, one of them potentially involving two stages. The relative contribution of these processes to the total number of mediator quanta released depends differently on the motor nerve stimulation pattern and on calcium ion entry into nerve endings.

Septin dynamics are essential for exocytosis.

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.

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.