Amplitude Of Endplate Potentials Research Articles

Magnesium Sulfate and Its Versatility in Anesthesia: A Comprehensive Review.

In the field of general anesthesia, magnesium sulfate (MgSO4) has become a valuable adjunct because it provides a range of benefits that enhance and optimize conventional aesthetic procedures. This review highlights the various intra-anesthetic benefits of MgSO4 while examining its complex function in the treatment using anesthesia. Magnesium inhibits the release of acetylcholine at the motor endplate and blocks calcium channels at presynaptic nerve terminals. This reduces the amplitude of endplate potential and the excitability of muscle fibers, which increases the potency of a neuromuscular blockade by nondepolarizing neuromuscular blockers. This activity may lessen the need for primary muscle relaxants. Moreover, its capacity to potentially reduce the total amount of main aesthetic agents needed emphasizes its function in maximizing anesthesia dosage, ensuring sufficient depth while perhaps potentially reducing adverse effects linked with increased dosages. MgSO4's adaptable qualities present a viable path for improving anesthetic outcomes, possibly improving patient safety and improving surgical results.

Dietary NMN supplementation enhances motor and NMJ function in ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that causes the degeneration of motor neurons in the motor cortex and spinal cord. Patients with ALS experience muscle weakness and atrophy in the limbs which eventually leads to paralysis and death. NAD+ is critical for energy metabolism, such as glycolysis and oxidative phosphorylation, but is also involved in non-metabolic cellular reactions. In the current study, we determined whether the supplementation of nicotinamide mononucleotide (NMN), an NAD+ precursor, in the diet had beneficial impacts on disease progression using a SOD1G93A mouse model of ALS. We found that the ALS mice fed with an NMN-supplemented diet (ALS+NMN mice) had modestly extended lifespan and exhibited delayed motor dysfunction. Using electrophysiology, we studied the effect of NMN on synaptic transmission at neuromuscular junctions (NMJs) in symptomatic of ALS mice (18 weeks old). ALS+NMN mice had larger end-plate potential (EPP) amplitudes and maintained better responses than ALS mice, and also had restored EPP facilitation. While quantal content was not affected by NMN, miniature EPP (mEPP) amplitude and frequency were elevated in ALS+NMN mice. NMN supplementation in diet also improved NMJ morphology, innervation, mitochondrial structure, and reduced reactive astrogliosis in the ventral horn of the lumbar spinal cord. Overall, our results indicate that dietary consumption of NMN can slow motor impairment, enhance NMJ function and improve healthspan of ALS mice.

Development of abnormalities at the neuromuscular junction in the SOD1-G93A mouse model of ALS: dysfunction then disruption of postsynaptic structure precede overt motor symptoms.

The ultimate deficit in amyotrophic lateral sclerosis (ALS) is neuromuscular junction (NMJ) loss, producing permanent paralysis, ultimately in respiratory muscles. However, understanding the functional and structural deficits at NMJs prior to this loss is crucial for therapeutic strategy design. Should early interventions focus on reversing denervation, or supporting largely intact NMJs that are functionally impaired? We therefore determined when functional and structural deficits appeared in diaphragmatic NMJs relative to the onset of hindlimb tremor (the first overt motor symptoms) in vivo in the SOD1-G93A mouse model of ALS. We employed electrophysiological recording of NMJ postsynaptic potentials for spontaneous and nerve stimulation-evoked responses. This was correlated with fluorescent imaging microscopy of the postsynaptic acetylcholine receptor (AChR) distribution throughout the postnatal developmental timecourse from 2 weeks to early symptomatic ages. Significant reduction in the amplitudes of spontaneous miniature endplate potentials (mEPPs) and evoked EPPs emerged only at early symptomatic ages (in our colony, 18-22 weeks). Reductions in mEPP frequency, number of vesicles per EPP, and EPP rise time were seen earlier, at 16weeks, but this reversed by early symptomatic ages. However, the earliest and most striking impairment was an inability to maintain EPP amplitude during a 20 Hz stimulus train, which appeared 6 weeks before overt in vivo motor symptoms. Despite this, fluorescent α-bungarotoxin labelling revealed no systematic, progressive changes in 11 comprehensive NMJ morphological parameters (area, shape, compactness, number of acetylcholine receptor, AChR, regions, etc.) with disease progression. Rather, while NMJs were largely normally-shaped, from 16 weeks there was a progressive and substantial disruption in AChR concentration and distribution within the NMJ footprint. Thus, NMJ functional deficits appear at least 6 weeks before motor symptoms in vivo, while structural deficits occur 4 weeks later, and predominantly within NMJs. These data suggest initial therapies focused on rectifying suboptimal NMJ function could produce effective relief of symptoms of weakness.

Novel Organoruthenium(II) Complex C1 Selectively Inhibits Butyrylcholinesterase without Side Effects on Neuromuscular Transmission.

Enzyme butyrylcholinesterase (BChE) shows increased activity in some brain regions after progression of Alzheimer's disease and is therefore one of the therapeutic targets for symptomatic treatment of this neurodegenerative disorder. The organoruthenium(II) complex [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1) was designed based on the results of our previous structure-activity studies. Inhibitory activity toward cholinesterase enzymes shows that this complex selectively, competitively, and reversibly inhibits horse serum BChE (hsBChE) with an IC50 value of 2.88 µM. When tested at supra-pharmacological concentrations (30, 60, 90, and 120 µM), C1 had no significant effect on the maximal amplitude of nerve-evoked and directly elicited single-twitch and tetanic contractions. At the highest tested concentration (120 µM), C1 had no effect on resting membrane potential, but significantly decreased the amplitude of miniature end-plate potentials (MEPP) without reducing their frequency. The same concentration of C1 had no effect on the amplitude of end-plate potentials (EPP), however it shortened the half-decay time of MEPPs and EPPs. The decrease in the amplitude of MEPPs and shortening of the half-decay time of MEPPs and EPPs suggest a possible weak inhibitory effect on muscle-type nicotinic acetylcholine receptors (nAChR). These combined results show that, when applied at supra-pharmacological concentrations up to 120 µM, C1 does not importantly affect the physiology of neuromuscular transmission and skeletal muscle contraction.

Presynaptic Acetylcholine Receptors Modulate the Time Course of Action Potential-Evoked Acetylcholine Quanta Secretion at Neuromuscular Junctions.

For effective transmission of excitation in neuromuscular junctions, the postsynaptic response amplitude must exceed a critical level of depolarization to trigger action potential spreading along the muscle-fiber membrane. At the presynaptic level, the end-plate potential amplitude depends not only on the acetylcholine quanta number released from the nerve terminals in response to the nerve impulse but also on a degree of synchronicity of quanta releases. The time course of stimulus-phasic synchronous quanta secretion is modulated by many extra- and intracellular factors. One of the pathways to regulate the neurosecretion kinetics of acetylcholine quanta is an activation of presynaptic autoreceptors. This review discusses the contribution of acetylcholine presynaptic receptors to the control of the kinetics of evoked acetylcholine release from nerve terminals at the neuromuscular junctions. The timing characteristics of neurotransmitter release is nowadays considered an essential factor determining the plasticity and efficacy of synaptic transmission.

Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function.

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10-25mM) reduced endplate potential (EPP) amplitudes by 10-40% and enhanced depression during repetitive (2-20Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole-cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74mM. Cyclohexanol (10-20mM) also caused prolonged episodes of reduced-current, multi-channel bursting in outside-out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (-6kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α-subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (-3.8kcal/mol) to voltage-sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. KEY POINTS: Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+ -channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function.

The Safety Factor for Neuromuscular Transmission: Effects of Dimethylsulphoxide, Cannabinoids and Synaptic Homeostasis.

BackgroundIn myasthenia gravis, impaired postsynaptic sensitivity to acetylcholine results in failure of neuromuscular transmission and fatiguing muscle weakness.ObjectiveDevelop an ex vivo muscle contraction assay to test cannabinoids and other substances that might act on the myasthenic neuromuscular junction to restore control of the muscle.MethodsTubocurarine was added to an ex vivo, mouse phrenic nerve-hemidiaphragm muscle preparation to reduce acetylcholine sensitivity. This produced a myasthenia-like decrement in twitch force during a train of 10 nerve impulses (3 / sec). Endplate potential (EPP) recordings were used to confirm and extend the findings.ResultsSurprisingly, addition to the bath of dimethylsulphoxide (DMSO), at concentrations as low as 0.1%(v/v), partially reversed the decrement in nerve-evoked force. Intracellular electrophysiology, conducted in the presence of tubocurarine, showed that DMSO increased the amplitudes of both the spontaneous miniature EPP (MEPP) and the (nerve-evoked) EPP. In the absence of tubocurarine (synaptic potentials at physiological levels), an adaptive fall in quantal content negated the DMSO-induced rise in EPP amplitude. The effects of cannabinoid receptor agonists (solubilized with DMSO) in the contraction assay do not support their further exploration as useful therapeutic agents for myasthenia gravis. CP 55,940 (a dual agonist for cannabinoid receptor types 1 and 2) reversed the beneficial effects of DMSO.Conclusions:We demonstrate a powerful effect of DMSO upon quantal amplitude that might mislead pharmacological studies of synaptic function wherever DMSO is used as a drug vehicle. Our results also show that compounds targeting impaired neuromuscular transmission should be tested under myasthenic-like conditions, so as to avoid confounding effects of synaptic homeostasis.

A comparison of acute mouse hindlimb injuries between tourniquet- and femoral artery ligation-induced ischemia-reperfusion

The tourniquet or femoral artery ligation is widely used to stop extremity hemorrhage or create a bloodless operating field in the combat scenario and civilian setting. However, these procedures with subsequent reperfusion also induce ischemia-reperfusion (IR) injuries. To fully evaluate animal models of limb IR injuries, we compared tourniquet- and femoral artery ligation-induced IR injuries in the hindlimb of mice. In C57/BL6 mice, 3 h of unilateral hindlimb ischemia was induced by placement of a rubber band at the hip joint or a surgical ligation of the femoral artery. The tourniquet or femoral artery ligation was then released, allowing for 24 h of reperfusion. Compared to the femoral artery ligation/IR, the tourniquet/IR induced more severe skeletal muscle damage, including muscle necrosis and interruption of muscle fibers. There was no gastrocnemius muscle contraction in tourniquet/IR, while femoral artery ligation/IR markedly weakened gastrocnemius muscle contraction. Motor nerve terminals disappeared, and endplate potentials (EPPs) were undetectable in tourniquet/IR, whereas femoral artery ligation/IR only induced mild impairment of motor nerve terminals and decreased the amplitude of EPPs. Additionally, western blot data showed that proinflammatory cytokine levels (IL-1β and TNF-α) were higher in the tourniquet/IR than that in femoral artery ligation/IR. Moreover, tourniquet/IR caused significant tissue edema and dilation of lymphatic vessels in the hindlimb, compared to femoral artery ligation/IR. The above data demonstrated that tourniquet/IR-induced acute hindlimb injuries are more severe than those induced by femoral artery ligation/IR. This suggests that future investigators should determine which hindlimb IR model (tourniquet/IR or femoral artery ligation/IR) is optimal depending on the purpose of their study.

Metformin Increases Sarcolemma Integrity and Ameliorates Neuromuscular Deficits in a Murine Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease characterized by progressive muscle weakness and wasting. Stimulation of AMP-activated protein kinase (AMPK) has been demonstrated to increase muscle function and protect muscle against damage in dystrophic mice. Metformin is a widely used anti-hyperglycemic drug and has been shown to be an indirect activator of AMPK. Based on these findings, we sought to determine the effects of metformin on neuromuscular deficits in mdx murine model of DMD. In this study, we found metformin treatment increased muscle strength accompanied by elevated twitch and tetanic force of tibialis anterior (TA) muscle in mdx mice. Immunofluorescence and electron microscopy analysis of metformin-treated mdx muscles revealed an improvement in muscle fiber membrane integrity. Electrophysiological studies showed the amplitude of miniature endplate potentials (mEPP) was increased in treated mice, indicating metformin also improved neuromuscular transmission of the mdx mice. Analysis of mRNA and protein levels from muscles of treated mice showed an upregulation of AMPK phosphorylation and dystrophin-glycoprotein complex protein expression. In conclusion, metformin can indeed improve muscle function and diminish neuromuscular deficits in mdx mice, suggesting its potential use as a therapeutic drug in DMD patients.

Dexamethasone ameliorates recovery process of neuromuscular junctions after tourniquet-induced ischemia-reperfusion injuries in mouse hindlimb

As a primary tool in first-line treatment of severe extremity hemorrhage, tourniquet and subsequent reperfusion also induce ischemia-reperfusion (IR) injuries including severe dysfunction of the neuromuscular junction (NMJ). Here, we observed the effect of dexamethasone (Dex) on NMJs suffering from IR-cause damage in mouse hindlimb. Unilateral hindlimb of mice was subjected to 3 h of tourniquet application by placing a rubber band, and then releasing the rubber band for reperfusion during different periods of time (1, 2, 4, and 6 weeks). Dex treatment (1 mg/kg/day, i.p.) began on the day of tourniquet-IR induction and lasted for 7 days. During tourniquet-induced IR, NMJs in gastrocnemius muscles showed significant morphological and functional changes. These changes include that motor nerve terminals gradually regenerated, even reaching that seen in sham mice; nicotinic acetylcholine receptor (nAChR) clusters were gradually fragmented with prolongation of reperfusion; and the amplitude of endplate potentials (EPPs) gradually increased, but it did not restore to the sham level at 6 weeks of tourniquet-induced IR. IL-1β and TNFα were over-produced in gastrocnemius muscles at 1 week, gradually decreased to the sham level at 4 weeks, and returned back to a high level at 6 weeks of tourniquet-induced IR. Treatment with Dex mitigated fragmentation of nAChR clusters, increased the amplitude of EPPs, and decreased levels of TNFα and IL-1β during the first two weeks of tourniquet-induced IR. The present study suggests that anti-inflammation is a potentially important strategy for promoting the morphological and functional recovery processes of NMJs after tourniquet-induced IR injuries.

Muscle BDNF: a potential therapeutic target for Kennedy's disease.

Muscle BDNF: a potential therapeutic target for Kennedy's disease.

Delayed increase of acetylcholine quantal size induced by the activity-dependent release of endogenous CGRP but not ATP in neuromuscular junctions.

In mouse motor synapses tetanic neuromuscular activity (30Hz, 2min) led to a delayed posttetanic potentiation of amplitude and duration of spontaneous miniature endplate potentials (MEPPs). Microelectrode recordings of MEPPs before and after nerve stimulation showed an increase in MEPP amplitude and time course by 30% and 15%, respectively, without changes in their frequency. Peak effect was detected 20min after tetanic activity and progressively faded throughout the next 40min of recording. The revealed potentiation of MEPPs was fully preserved in preparations from pannexin 1 knockout mice. It means, that myogenic ATP released via pannexin 1 channels from contracting muscle fibers is not likely to participate in the described phenomenon. But posttetanic potentiation of MEPPs was fully prevented by competitive antagonist of calcitonin gene-related peptide (CGRP) receptors CGRP8-37 , ryanodine receptors inhibitor ryanodine and by vesicular acetylcholine transporter inhibitor vesamicol. It is suggested that the combination of intensive synaptic and contractile activity in neuromuscular junctions is required to induce Ca2+ -dependent exocytosis of endogenous CGRP. The accumulation of CGRP in the synaptic cleft and its presynaptic activity may induce posttetanic potentiation of MEPP amplitude due to CGRP-stimulated acetylcholine loading into vesicles and subsequent increase of quantal size.

Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug

In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibit several medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact with muscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission and muscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η6-p-cymene)Ru(nitrophen)Cl]Cl; C22H21Cl2N3O2Ru (C1-Cl) was synthesized, structurally characterized and evaluated in vitro for its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinant acetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings. The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrations producing 50 % inhibition (IC50) of enzyme activity ranging from 16 to 26 μM. Moreover, C1-Cl inhibited the nerve-evoked isometric muscle contraction (IC50 = 19.44 μM), without affecting the directly-evoked muscle single twitch up to 40 μM. The blocking effect of C1-Cl was rapid and almost completely reversed by neostigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a concentration-dependent manner (IC50 = 7.6 μM) without any significant change in the resting membrane potential of muscle fibers up to 40 μM. Finally, C1-Cl (5–40 μM) decreased (i) the amplitude of miniature endplate potentials until a complete block by concentrations higher than 25 μM and (ii) their frequency at 10 μM or higher concentrations. The compound C1-Cl reversibly blocked the neuromuscular transmission in vitro by a non-depolarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may be therefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thus myorelaxant, drug.

Dysfunction of Neuromuscular Synaptic Transmission and Synaptic Vesicle Recycling in Motor Nerve Terminals of mSOD1 Transgenic Mice with Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive incurable neurodegenerative disease with selective loss of lower and upper motoneurons. Dysfunction and destruction of neuromuscular synapses leading to skeletal muscle denervation is one of the early and major events in ALS pathogenesis. Despite of the presence of studies devoted to investigation of neuromuscular transmission in ALS mouse models, no detailed information about molecular mechanisms underlying synaptic dysfunction in ALS and their temporal dynamics during ALS progression is provided. The goal of present work was to study the processes of neurotransmitter release and presynaptic vesicle recycling in neuromuscular synapses of mutated SOD1 (mSOD1) transgenic mice at different clinical stages of disease. Utilizing combination of electrophysiological recording and FM 1–43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide) fluorescent imaging, we found that mSOD1 mice at symptomatic and terminal stages of disease showed decreased baseline quantal content of end-plate potentials and prolonged synaptic vesicle recycling time comparing to wild-type mice. Despite the decrease of end-plate potential (EPP) quantal content, studied mSOD1 mice groups showed unchanged dynamics of EPP relative amplitude comparing to WT mice. We also found an increase of miniature end-plate potential amplitude in mSOD1 mice at symptomatic stage, which may reflect compensatory mechanism that alleviates reduction of EPP amplitude. Thus, we provided one of the first detailed characteristics of presynaptic dysfunction at neuromuscular junction in ALS model. Obtained data expand our understanding of the ALS pathogenesis and contribute to stage- and localization-specific description of ALS pathogenetic mechanisms.

Role of different nicotinic acetylcholine receptor subtypes in skeletal muscles in sepsis-related nerve-muscle dysfunction in septic rats: neuroelectrophysiology

Objective To evaluate the role of different nicotinic acetylcholine receptor (nAChR) subtypes in skeletal muscles in sepsis-related nerve-muscle dysfunction in septic rats by neuroelectrophysiology. Methods SPF adult male Sprague-Dawley rats, weighing 200-220 g, aged 2-3 months, were studied.Sepsis was induced by cecal ligation and puncture in chloral hydrate-anesthetized rats.Twelve portions of sciatic nerve-tibialis anterior muscles taken at day 3 after successful establishment of the sepsis model served as group S. Another 12 portions of sciatic nerve-tibialis anterior muscles of normal rats served as group C. The endplate potential (EPP) was monitored by intracellular microelectrode recording.Six portions of nerve-muscle samples were incubated with the adult nAChR (e-nAChR) specific antagonist waglerin-1 at a concentration of 1 μmol/L, the fetal nAChR (γ-nAChR) specific antagonist αA-OIVA at a concentration of 250 nmol/L and neuronal nAChR (α7-nAChR) antagonist methyllycaconitine (MLA) at a final concentration of 6 nmol/L.The amplitude of EPP was recorded at 15 min of incubation, and the difference in the amplitude of EPP (ΔEPP) and changing rate of EPP (ΔEPP%) were calculated.Another 6 portions of samples were incubated for 15 min with the Kreb mixture of waglerin-1 and 250 nmol/L αA-OIVA at a final concentration of 1 μmol/L, the Kreb mixture of waglerin-1 and 6 nmol/L MLA at a final concentration of 1 μmol/L, and the Kreb mixture of αA-OIVA and 6 nmol/L MLA at a final concentration of 250 nmol/L, and then the amplitude of EPP was recorded.Washout was performed with antagonist free Kreb solution for 15 min between treatments with different blockers. Results Compared with group C, ΔEPP and ΔEPP% were significantly decreased after antagonizing e-nAChR, ΔEPP% was decreased after antagonizing γ-nAChR, ΔEPP and ΔEPP% were increased after antagonizing α7-nAChR, the amplitude of EPP was increased after antagonizing e-nAChR+ γ-nAChR and e-nAChR+ α7-nAChR, and the amplitude of EPP was decreased after antagonizing γ-nAChR+ α7-nAChR (P<0.05). ΔEPP and ΔEPP% were significantly higher after antagonizing α7-nAChR than after antagonizing γ-nAChR in group S (P<0.05). The EPP was significantly lower after antagonizing e-nAChR+ α7-nAChR than after antagonizing e-nAChR+ γ-nAChR (P<0.05). Conclusion Weakened function of e-nAChR and enhanced function of α7-nAChR in the membrane of the end-plate in skeletal muscle are the main reasons for sepsis-related nerve-muscle dysfunction in septic rats, and the role is comparable between them. Key words: Sepsis; Neuromuscular diseases; Receptors, cholinergic

Ryanodine- and CaMKII-dependent release of endogenous CGRP induces an increase in acetylcholine quantal size in neuromuscular junctions of mice.

ObjectiveThe aim of this study was to identify the mechanism responsible for an increase in miniature endplate potentials (MEPPs) amplitude, induced by ryanodine as an agonist of ryanodine receptors in mouse motor nerve terminals.MethodsUsing intracellular microelectrode recordings of MEPPs and evoked endplate potentials (EPPs), the changes in spontaneous and evoked acetylcholine release in motor synapses of mouse diaphragm neuromuscular preparations were studied.ResultsRyanodine (0.1 μM) increased both the amplitudes of MEPPs and EPPs to a similar extent (up to 130% compared to control). The ryanodine effect was prevented by blockage of receptors of calcitonin gene‐related peptide (CGRP) by a truncated peptide CGRP 8‐37. Endogenous CGRP is stored in large dense‐core vesicles in motor nerve terminals and may be released as a co‐transmitter. The ryanodine‐induced increase in MEPPs amplitude may be fully prevented by inhibition of vesicular acetylcholine transporter by vesamicol or by blocking the activity of protein kinase A with H‐89, suggesting that endogenous CGRP is released in response to the activation of ryanodine receptors. Activation of CGRP receptors can, in turn, upregulate the loading of acetylcholine into synaptic vesicles, which will increase the quantal size. This new feature of endogenous CGRP activity looks similar to recently described action of exogenous CGRP in motor synapses of mice. The ryanodine effect was prevented by inhibitors of Ca/Calmodulin‐dependent kinase II (CaMKII) KN‐62 or KN‐93. Inhibition of CaMKII did not prevent the increase in MEPPs amplitude, which was caused by exogenous CGRP.ConclusionsWe propose that the activity of presynaptic CaMKII is necessary for the ryanodine‐stimulated release of endogenous CGRP from motor nerve terminals, but CaMKII does not participate in signaling downstream the activation of CGRP‐receptors followed by quantal size increase.

Natural polymeric 3-alkylpyridinium salt affects vertebrate skeletal muscle contractility by preferentially blocking neuromuscular transmission

The effects of natural polymeric alkylpyridinium salt (nPoly-3-APS), a potent acetylcholinesterase inhibitor isolated from the marine sponge Reniera sarai, were studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations using electrophysiological approaches. nPoly-3-APS inhibited nerve-evoked isometric muscle twitch and tetanic contraction in a concentration-dependent manner (IC50=29.4μM and 18.5μM, respectively) and produced a 30–44% decrease of directly muscle-elicited twitch and tetanus amplitudes at 54.4μM. Additionally, nPoly-3-APS (9.1–27.2μM) markedly decreased the amplitude of miniature endplate potentials, while their frequency was only affected at the highest concentration used. Endplate potentials were also inhibited by nPoly-3-APS in a concentration-dependent manner (IC50=20.1μM), without significant change in the resting membrane potential of muscle fibers (up to 54.4μM). In conclusion, our results show, for the first time, that nPoly-3-APS preferentially blocks the neuromuscular transmission, in vitro, by a non-depolarizing mechanism. This strongly suggests that the in vivo toxicity of nPoly-3-APS mainly occurs through an antagonist action of the compound on nicotinic acetylcholine receptors of skeletal muscles.

Morphological Regeneration and Functional Recovery of Neuromuscular Junctions after Tourniquet-Induced Injuries in Mouse Hindlimb

Tourniquet application and its subsequent release cause serious injuries to the skeletal muscle, nerve, and neuromuscular junction (NMJ) due to mechanical compression and ischemia-reperfusion (IR). Monitoring structural and functional repair of the NMJ, nerve, and skeletal muscle after tourniquet-induced injuries is beneficial in exploring potential cellular and molecular mechanisms responsible for tourniquet-induced injuries, and for establishing effective therapeutic interventions. Here, we observed long-term morphological and functional changes of the NMJ in a murine model of tourniquet-induced hindlimb injuries. Unilateral hindlimbs of C57/BL6 mice were subjected to 3 h of tourniquet by placing an orthodontic rubber band, followed by varied periods of tourniquet release (1 day, 3 days, 1 week, 2 weeks, 4 weeks, and 6 weeks). NMJ morphology in the gastrocnemius muscle was imaged, and the endplate potential (EPP) was recorded to evaluate NMJ function. In NMJs, nicotinic acetylcholine receptor (nAChR) clusters normally displayed an intact, pretzel-like shape, and all nAChR clusters were innervated (100%) by motor nerve terminals. During 3 h of tourniquet application and varied periods of tourniquet release, NMJs in the gastrocnemius muscle were characterized by morphological and functional changes. At 1 day and 3 days of tourniquet release, nAChR clusters retained normal, pretzel-like shapes, whereas motor nerve terminals were completely destroyed and no EPPs recorded. From 1 to 6 weeks of tourniquet release, motor nerve terminals gradually regenerated, even reaching that seen in sham mice, whereas nAChR clusters were gradually fragmented with prolongation of tourniquet release. Additionally, the amplitude of EPPs gradually increased with prolongation of tourniquet release. However, even at 6 weeks after tourniquet release, the amplitude of EPPs did not restore to the level seen in sham mice (13.9 ± 1.1 mV, p < 0.05 vs. sham mice, 29.8 ± 1.0 mV). The data suggest that tourniquet application and subsequent release impair the structure and function of NMJs. Morphological change in motor nerve terminals is faster than in nAChR clusters in NMJs. Slow restoration of fragmented nAChR clusters possibly dampens neuromuscular transmission during the long phase following tourniquet release.

Morphological regeneration and functional recovery of neuromuscular junction after ischemic/reperfusion injury induced by tourniquet in mouse hind limb

BackgroundTourniquet‐induced ischemia and subsequent reperfusion cause serious acute ischemia‐reperfusion (IR) injury in the neuromuscular junction (NMJ) and skeletal muscle. These injuries are associated with permanent neurological deficiency, and long‐term skeletal muscle contractile dysfunction, even complete limb paralysis. Monitoring structural and functional repair of the NMJ and skeletal muscle after tourniquet‐induced IR is beneficial to exploring the mechanisms responsible for tourniquet‐induced IR injury and finding effective therapeutic interventions. Here, we observed long‐term morphological and functional alterations of the NMJ in tourniquet‐induced hindlimb IR.MethodUnilateral hindlimb of C57/BL6 mice was subjected to 3 hours of ischemia by placing an orthodontic rubber band and followed different periods of reperfusion (1 day, 3 days, 1 weeks, 2 wekks, 4 weeks, and 6 weeks). At each time point of reperfusion, the gastrocnemius muscle was isolated for measurement of the NMJ morphology and function. Motor nerve terminals and nicotinic acetylcholine receptors (nAChRs) in the NMJ were measured by immunofluorescence staining. The end plate potential (EPP) was recorded by the intracellular recording technique.ResultsIn the NMJ of sham mice, the structure of the nAChR clusters remained intact pretzel‐like shape and motor nerve terminals innervated the nAChR clusters to form synapses for signal transmission. The number of fragments in one nAChR cluster was 1.83 ± 0.07 and all nAChR clusters were innervated by motor nerve terminals (100%). The amplitude of EPPs was 29.84 ± 1.02 mV. At 1 day and 3 days of reperfusion, the motor nerve terminals disappeared, the AChR clusters kept normal shape, and no EPPs were recorded. From 1 week to 6 weeks of reperfusion, the motor nerve terminals gradually reinnervated (46.88%, 88.05%, 99.2%, and 100% for 1, 2, 4, and 6 weeks of reperfusion, respectively). However, the AChR clusters were gradually fragmented and the number of fragments in one nAChR cluster increased with longer period of reperfusion (2.92 ± 0.14, 5.50 ± 0.30, 8.94 ± 0.67, and 6.88 ± 0.50 for for 1, 2, 4, and 6 weeks of reperfusion, respectively; p&lt;0.05 vs. sham). The amplitude of EPPs also increased gradually with longer period of reperfusion, but the amplitude of EPPs at 6 weeks of reperfusion didn't restore towards the level seen in sham mice (13.92 ± 1.11 mV, p&lt;0.05 vs. sham).ConclusionTourniquet‐induced ischemia and subsequent reperfusion impaired the NMJ. The morphological change of motor nerve terminals was faster than that of the nAChR clusters. The slow recovery of fragmented nAChR clusters could attenuate neuromuscular transmission.

24S-Hydroxycholesterol enhances synaptic vesicle cycling in the mouse neuromuscular junction: Implication of glutamate NMDA receptors and nitric oxide

24S-hydroxycholesterol (24S-HC) is a brain-derived product of lipid metabolism present in the systemic circulation, where its level can change significantly in response to physiological and pathophysiological conditions. Here, using electrophysiological and optical approaches, we have found a high sensitivity to 24S-HC of the synaptic vesicle cycle at the mouse neuromuscular junctions. Treatment with 24S-HC increased the end plate potential amplitude (EPP) in response to a single stimulus and attenuated the EPP amplitude rundown during high frequency (HF) activity but had no influence on miniature EPP amplitude or frequency. The effects on evoked responses were associated with enhanced FM1-43 dye loading and unloading by endo- and exocytosis. Comparison of electrophysiological and optical data revealed an increase in the rate of vesicular cycling. The impact of 24S-HC was abolished or potentiated by stimulation or inhibition of NMDA-receptors respectively. Moreover, 24S-HC, acting in the same manner as the endothelial NO synthase (eNOS) inhibitor cavtratin, suppressed an increase in NO-sensitive dye fluorescence during HF stimulation, while l-glutamate had the opposite effect. Inhibitors of NOS (l-NAME and cavtratin, but not the neuronal NOS inhibitor TRIM), a scavenger of extracellular NO and a protein kinase G blocker all had stimulatory effects, similar to those of 24S-HC, on exocytosis induced by HF activity and completely masked the effect of 24S-HC. The data suggest that 24S-HC enhances synaptic vesicle cycling due to an attenuation of retrograde NO signaling that depends on eNOS. In this regard, 24S-HC counteracts the effects of NMDA-receptor stimulation at mouse neuromuscular junctions.