Frequency Of Miniature End-plate Potentials Research Articles

Increased Frequency of Giant Miniature End-Plate Potentials at the Neuromuscular Junction in Diabetic Rats.

There is a need for research addressing the functional characteristics of the motor end-plate in diabetes to identify mechanisms contributing to neuromuscular dysfunction. Here, we investigated the effect of diabetes on spontaneous acetylcholine release in the rat neuromuscular junction. We studied two randomized groups of male Wistar rats (n = 7 per group, 350 ± 50 g, 12-16 weeks of age): one with streptozotocin-induced experimental diabetes, and a healthy control group without diabetes. After 8 weeks of monitoring after diabetes induction, rats in both groups were anesthetized with pentobarbital. Then, the diaphragm muscle was dissected for electrophysiological recordings of miniature end-plate potentials (MEPPs) using a single electrode located at the region of the muscle end-plate. All experiments were conducted at environmental temperature (20-22 °C) in rat Ringer solution with constant bubbling carbogen (95% O2, 5% CO2). Compared to healthy controls, in the diaphragm neuromuscular end-plate derived from diabetic rats, the MEPPs were higher in amplitude and frequency, and the proportion of giant MEPPs was elevated (7.09% vs. 1.4% in controls). Our results showed that diabetes affected the acetylcholine MEPP pattern and increased the number of giant potentials compared to healthy controls.

Morphological and functional alterations of neuromuscular synapses in a mouse model of ACTA1 congenital myopathy.

Mutations in skeletal muscle α-actin (Acta1) cause myopathies. In a mouse model of congenital myopathy, heterozygous Acta1 (H40Y) knock-in (Acta1+/Ki) mice exhibit features of human nemaline myopathy, including premature lethality, severe muscle weakness, reduced mobility, and the presence of nemaline rods in muscle fibers. In this study, we investigated the impact of Acta1 (H40Y) mutation on the neuromuscular junction (NMJ). We found that the NMJs were markedly fragmented in Acta1+/Ki mice. Electrophysiological analysis revealed a decrease in amplitude but increase in frequency of miniature end-plate potential (mEPP) at the NMJs in Acta1+/Ki mice, compared with those in wild type (Acta1+/+) mice. Evoked end-plate potential (EPP) remained similar at the NMJs in Acta1+/Ki and Acta1+/+ mice, but quantal content was increased at the NMJs in Acta1+/Ki, compared with Acta1+/+ mice, suggesting a homeostatic compensation at the NMJs in Acta1+/Ki mice to maintain normal levels of neurotransmitter release. Furthermore, short-term synaptic plasticity of the NMJs was compromised in Acta1+/Ki mice. Together, these results demonstrate that skeletal Acta1 H40Y mutation, albeit muscle-origin, leads to both morphological and functional defects at the NMJ.

Shock waves as treatment of mouse myofascial trigger points.

An abnormal increase in spontaneous neurotransmission can induce subsynaptic knots in the myocyte called myofascial trigger points. The treatment of choice is to destroy these trigger points by inserting needles. However, 10% of the population has a phobia of needles, blood, or injuries. Therefore, the objective of this study is to verify the usefulness of shock waves in the treatment of myofascial trigger points. Two groups of mice have been developed for this: healthy muscles treated with shock waves; trigger points affected muscles artificially generated with neostigmine and subsequently treated with shock waves. Muscles were stained with methylene blue, PAS-Alcian Blue, and labeling the axons with fluorescein and the acetylcholine receptors with rhodamine. Using intracellular recording the frequency of miniature endplate potentials (mEPPs) was recorded and endplate noise was recorded with electromyography. No healthy muscles treated with shock waves showed injury. Twitch knots in mice previously treated with neostigmine disappeared after shock wave treatment. Several motor axonal branches were retracted. On the other hand, shock wave treatment reduces the frequency of mEPPs and the number of areas with endplate noise. Shock waves seem to be a suitable treatment for myofascial trigger points. In the present study, with a single session of shock waves, very relevant results have been obtained, both functional (normalization of spontaneous neurotransmission) and morphological (disappearance of myofascial trigger points). Patients with a phobia of needles, blood, or injuries who cannot benefit from dry needling may turn to noninvasive radial shock wave treatment.

ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses.

The effects of brain-derived neurotrophic factor (BDNF) processing by-products (proBDNF and BDNF prodomain) on the activity of mouse neuromuscular junctions (NMJs) were studied in synapses formed during the reinnervation of extensor digitorum longus muscle (m. EDL) and mature synapses of the diaphragm. The parameters of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were analyzed in presence of each of the BDNF maturation products (both – 1 nM). In newly formed NMJs, proBDNF caused an increase in the resting membrane potential of muscle fibers and a decrease in the frequency of MEPPs, which was prevented by tertiapin-Q, a G-protein-coupled inwardly rectifying potassium channels (GIRK) blocker but not by p75 receptor signaling inhibitor TAT-Pep5. proBDNF had no effect on the parameters of EPPs. BDNF prodomain in newly formed synapses had effects different from those of proBDNF: it increased the amplitude of MEPPs, which was prevented by vesamicol, an inhibitor of vesicular acetylcholine (ACh) transporter; and reduced the quantal content of EPPs. In mature NMJs, proBDNF did not influence MEPPs parameters, but BDNF prodomain suppressed both spontaneous and evoked ACh release: decreased the frequency and amplitude of MEPPs, and the amplitude and quantal content of EPPs. This effect of the BDNF prodomain was prevented by blocking GIRK channels, by TAT-Pep5 or by Rho-associated protein kinase (ROCK) inhibitor Y-27632. At the same time, the BDNF prodomain did not show any inhibitory effects in diaphragm motor synapses of pannexin 1 knockout mice, which have impaired purinergic regulation of neuromuscular transmission. The data obtained suggest that there is a previously unknown mechanism for the acute suppression of spontaneous and evoked ACh release in mature motor synapses, which involves the activation of p75 receptors, ROCK and GIRK channels by BDNF prodomain and requires interaction with metabotropic purinoreceptors. In general, our results show that both the precursor of BDNF and the product of its maturation have predominantly inhibitory effects on spontaneous and evoked ACh release in newly formed or functionally mature neuromuscular junctions, which are mainly opposite to the effects of BDNF. The inhibitory influences of both proteins related to brain neurotrophin are mediated via GIRK channels of mouse NMJs.

Взаимодействие механизмов угнетения квантового выделения ацетилхолина при активации ваниллоидных (TRPV1) и пуриновых рецепторов в нервно-мышечном синапсе мыши

The main goal of the study was to analyze the relationship between the signaling pathways of the regulation of acetylcholine (ACh) quantal release in the peripheral synapse triggered by the activation of vanilloid (TRPV1) and purine receptors. In electrophysiological experiments carried out at the neuromuscular synapses of the m. Levator Auris Longus, it was found that the frequency of miniature endplate potentials (mEPPs) and the quantal content of endplate potentials (EPPs) decrease in the presence of TRPV1 agonist capsaicin. This effect was completely reversed by SB366791, a specific competitive antagonist of TRPV1 receptors. ATP, like capsaicin, decreased the frequency of mEPPs and the EPP quantal content. Against the background of the TRPV1 antagonist, the inhibitory effect of ATP on ACh secretion was realized in full. At the same time, against the background of TRPV1 channels activation by capsaicin, the effect of ATP on both spontaneous and evoked ACh release was absent. It was suggested that the mechanisms of action of ATP and capsaicin may be associated with a change in Ca2+ entry into the nerve ending. To test this hypothesis, experiments were carried out to assess the changes in the presynaptic calcium level (Ca2+ transient) using a fluorescent calcium dye upon nerve stimulation. The amplitude of the calcium transient did not change either with the application of ATP or with the addition of capsaicin. Thus, in the neuromuscular synapse of mammals, along with purinergic pathway of ACh secretion regulation, there is also a mechanism of neurosecretion modulation mediated by the activation of TRPV1 channels. The triggering of these mechanisms leads to the suppression of the processes of both spontaneous and evoked release of ACh quanta from the motor nerve endings. It was demonstrated that both pathways of regulation are not accompanied by a change in the Ca2+ transient, but have a common link of regulation of the quantal neurosecretion.

Effects of a Fat-Rich Diet on the Spontaneous Release of Acetylcholine in the Neuromuscular Junction of Mice.

Western societies are facing a clear increase in the rate of obesity and overweight which are responsible for musculoskeletal pain. Some of the substances described in the environment of myofascial trigger points (MTrPs) are the same as those found in the skeletal muscle of obese people, such as cytokines. Furthermore, elevated neuromuscular neurotransmission has been associated with MTrPs. The main objective of this study is to assess whether obesity or overweight may be a facilitator of myofascial pain. The experiments were performed on male Swiss mice. One experimental group was given a typical “cafeteria” diet and another group a commercial high-fat diet for six weeks. Intramuscular adipocytes were assessed with Sudan III. The functional study was performed with electromyographic recording to determine the plaque noise and intracellular recording of miniature endplate potentials (MEPPs). The intake of a cafeteria diet showed the presence of more adipocytes in muscle tissue, but not with the fat-supplemented diet. Both experimental groups showed an increase in the plaque noise and an increase in the frequency of MEPPs that lasted several weeks after interrupting diets. In summary, the supply of a hypercaloric diet for six weeks in mice increases spontaneous neurotransmission, thus facilitating the development of MTrPs.

Percutaneous Application of Galvanic Current in Rodents Reverses Signs of Myofascial Trigger Points.

An increase in the spontaneous release of acetylcholine (ACh) at the motor endplate is directly related to the generation of myofascial trigger points (MTrPs). In this study, percutaneous electric fields were applied to an animal model of MTrPs with high levels of spontaneous ACh release. All experiments were performed on Swiss mice and Sprague Dawley rats. For evaluating the spontaneous neurotransmission, intracellular recordings were performed, and the frequency of miniature endplate potentials was evaluated. Electromyographic recordings were also conducted to evaluate the endplate noise. Finally, the number and strength of local twitch responses (LTR) were evaluated using ultrasound recordings. The protocols used for the electric currents were 0.4 mA for five seconds and four repetitions (protocol 1), 1.5 mA for five seconds and three repetitions (protocol 2), and 3 mA for three seconds and three repetitions (protocol 3). After a subcutaneous injection of neostigmine (NTG), a great increase was observed in the frequency of mEPPs, together with an elevated endplate noise. Protocols 2 and 3 were the most effective. Protocol 3 could completely reverse the action of NTG at both three hours and 24 hours, respectively. The application of percutaneous currents produced both an increase in the number (144%) and in the speed (230% faster) of LTR compared with dry needling. In conclusion, higher doses of electrical current are more effective for decreasing MTrPs findings in an animal model.

Influência de diferentes anestésicos locais no bloqueio neuromuscular produzido pelo atracúrio em ratos

IntroductionThe association between Local Anesthetics (LAs) and Neuromuscular Blocking (NMB) drugs in clinical practice, and the possibility of interaction between these drugs has been investigated. LAs act on neuromuscular transmission in a dose‐dependent manner and may potentiate the effects of NMB drugs. ObjectiveThe aim of this study was to evaluate, in an experimental model, the effect of lidocaine and racemic bupivacaine on neuromuscular transmission and the influence on neuromuscular blockade produced by atracurium. MethodsMale Wistar rats, weighing from 250 g to 300g were used. The preparation was set up based on a technique proposed by Bülbring. Groups were formed (n = 5) according to the drug studied: lidocaine 20 μg.mL‐1 (Group I); racemic bupivacaine 5 μg.mL‐1 (Group II); atracurium 20 μg.mL‐1 (Group III); atracurium 20 μg.mL‐1 in a preparation previously exposed to lidocaine 20 μg.mL‐1 and racemic bupivacaine 5 μg.mL‐1, Groups IV and V, respectively. The following parameters were assessed: 1) Amplitude of hemi diaphragmatic response to indirect stimulation before and 60 minutes after addition of the drugs; 2) Membrane Potentials (MP) and Miniature Endplate Potentials (MEPPs). ResultsLidocaine and racemic bupivacaine alone did not alter the amplitude of muscle response. With previous use of lidocaine and racemic bupivacaine, the neuromuscular blockade (%) induced by atracurium was 86.66 ± 12.48 and 100, respectively, with a significant difference (p = 0.003), in comparison to the blockade produced by atracurium alone (55.7 ± 11.22). These drugs did not alter membrane potential. Lidocaine initially increased the frequency of MEPPs, followed by blockade. With the use of bupivacaine, the blockade was progressive. ConclusionsLidocaine and racemic bupivacaine had a presynaptic effect expressed by alterations in MEPPs, which may explain the interaction and potentiation of NMB produced by atracurium.

Influence of different local anesthetics on atracurium neuromuscular blockade on rats☆

IntroductionThe association between local anesthetics (LA) and neuromuscular blocking (NMB) drugs in clinical practice, and the possibility of interaction between these drugs has been investigated. LAs act on neuromuscular transmission in a dose-dependent manner and may potentiate the effects of NMB drugs. ObjectiveThe aim of this study was to evaluate, in an experimental model, the effect of lidocaine and racemic bupivacaine on neuromuscular transmission and the influence on neuromuscular blockade produced by atracurium. MethodsMale Wistar rats, weighing from 250 to 300g were used. The preparation was set up based on a technique proposed by Bülbring. Groups were formed (n=5) according to the drug studied: lidocaine 20μg.mL−1 (Group I); racemic bupivacaine 5μg.mL−1 (Group II); atracurium 20μg.mL−1 (Group III); atracurium 20μg.mL−1 in a preparation previously exposed to lidocaine 20μg.mL−1 and racemic bupivacaine 5μg.mL−1, Groups IV and V, respectively. The following parameters were assessed: 1) Amplitude of hemi diaphragmatic response to indirect stimulation before and 60minutes after addition of the drugs; 2) Membrane potentials (MP) and miniature endplate potentials (MEPPs). ResultsLidocaine and racemic bupivacaine alone did not alter the amplitude of muscle response. With previous use of lidocaine and racemic bupivacaine, the neuromuscular blockade (%) induced by atracurium was 86.66±12.48 and 100, respectively, with a significant difference (p=0.003), in comparison to the blockade produced by atracurium alone (55.7±11.22). These drugs did not alter membrane potential. Lidocaine initially increased the frequency of MEPPs, followed by blockade. With the use of bupivacaine, the blockade was progressive. ConclusionsLidocaine and racemic bupivacaine had a presynaptic effect expressed by alterations in MEPPs, which may explain the interaction and potentiation of NMB produced by atracurium.

The Participation of Endocannabinoid Receptors in the Regulation of Spontaneous Synaptic Activity at Neuromuscular Junctions of Mice

The mechanisms underlying changes in spontaneous release of acetylcholine (ACh) induced by exogenous agonists and antagonists of endocannabinoid receptors were investigated by recording miniature endplate potentials (MEPPs) in motor synapses of mouse diaphragm preparations with intracellular microelectrodes. There have not been observed any statistically significant changes either in the membrane potential of muscle fibers or in the frequency, amplitude, and time parameters of the MEPPs under the action of AM-251 (1 μM), antagonist/inverse agonist of CB1 receptors, for 60 min. Activation of CB receptors by their exogenous agonist WIN 55,212-2 (WIN) (20 μM) for 1 h did not cause statistically significant changes in the amplitude of the MEPPs. However, MEPP frequency increased by more than 50% as compared to the control under the action of WIN for 30–60 min. Blocking of CB1 receptors by AM-251 (1 μM) prevented the WIN-induced increase in MEPP frequency. A protein kinase A inhibitor H-89 (1 μM) did not prevent the enhancement of the ACh secretion upon activation of CB receptors by WIN. WIN lost the ability to increase MEPP frequency in the presence of a phospholipase C inhibitor U73122 (5 μM), which did not affect by itself the amplitude and temporal characteristics or MEPP frequency. A protein kinase C inhibitor, chelerythrin (4 μM) or ryanodine, which blocks the ryanodine receptors (RyRs) at a concentration of 5 μM, also prevented the WIN-induced increase in MEPP frequency. Thus, a signaling cascade triggered by stimulation of presynaptic CB1 receptors with subsequent activation of phospholipase C, protein kinase C, and the release of stored calcium, leading to an increase in the frequency of spontaneous quantal release of ACh, has been detected for the first time in mouse motor synapses. This suggests the presence of CB1 receptors on the presynaptic membrane in mouse motor nerve terminals and the possibility of activation of these receptors by endocannabinoids for potentiating the spontaneous secretion of ACh.

Diverse Effects of Noradrenaline and Adrenaline on the Quantal Secretion of Acetylcholine at the Mouse Neuromuscular Junction

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.

Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Regulation of Acetylcholine Quantal Release by Coupled Thrombin/BDNF Signaling in Mouse Motor Synapses.

The aim of this study was to compare the acute effects of thrombin and brain-derived neurotrophic factor (BDNF) on spontaneous miniature endplate potentials (MEPPs) and multiquantal evoked endplate potentials (EPPs) in mouse neuromuscular junctions (NMJs) of m. diaphragma and m. EDL. Intracellular microelectrode recordings of MEPPs and EPPs were used to evaluate the changes in acetylcholine (ACh) release in mature and newly-formed mouse NMJs. Thrombin (1 nM) increased the amplitude of MEPPs and EPPs by 25–30% in mature and newly-formed NMJs. This effect was due to an enhanced loading of synaptic vesicles with ACh and increase of ACh quantal size, since it was fully prevented by blocking of vesicular ACh transporter. It was also prevented by tropomyosin-related kinase B (TrkB) receptors inhibitor ANA12. Exogenous BDNF (1 nM) mimicked thrombin effect and increased the amplitude of MEPPs and EPPs by 25–30%. It required involvement of protein kinase A (PKA) and mitogen-activated protein kinase (MEK1/2)-mediated pathway, but not phospholipase C (PLC). Blocking A2A adenosine receptors by ZM241385 abolished the effect of BDNF, whereas additional stimulation of A2A receptors by CGS21680 increased MEPP amplitudes, which was prevented by MEK1/2 inhibitor U0126. At mature NMJs, BDNF enhanced MEPPs frequency by 30–40%. This effect was selectively prevented by inhibition of PLC, but not PKA or MEK1/2. It is suggested that interrelated effects of thrombin/BDNF in mature and newly-formed NMJs are realized via enhancement of vesicular ACh transport and quantal size increase. BDNF-induced potentiation of synaptic transmission involves the functional coupling between A2A receptor-dependent active PKA and neurotrophin-triggered MAPK pathway, as well as PLC-dependent increase in frequency of MEPPs.

Neurotoxicity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom in vertebrate neuromuscular preparations in vitro and neutralization by antivenom.

We investigated the effect of South American coralsnake (Micrurus lemniscatus lemniscatus) venom on neurotransmission in vertebrate nerve-muscle preparations in vitro. The venom (0.1-30µg/ml) showed calcium-dependent PLA2 activity and caused irreversible neuromuscular blockade in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, contractures to exogenous acetylcholine and carbachol (CCh), but not KCl, were abolished by venom concentrations ≥ 0.3µg/ml; in PND preparations, the amplitude of the tetanic response was progressively attenuated, but with little tetanic fade. In low Ca2+ physiological solution, venom (10µg/ml) caused neuromuscular blockade in PND preparations within ~ 10min that was reversible by washing; the addition of Ca2+ immediately after the blockade temporarily restored the twitch responses, but did not prevent the progression to irreversible blockade. Venom (10µg/ml) did not depolarize diaphragm muscle, prevent depolarization by CCh, or cause muscle contracture or histological damage. Venom (3µg/ml) had a biphasic effect on the frequency of miniature end-plate potentials, but did not affect their amplitude; there was a progressive decrease in the amplitude of evoked end-plate potentials. The amplitude of compound action potentials in mouse sciatic nerve was unaffected by venom (10µg/ml). Pre-incubation of venom with coralsnake antivenom (Instituto Butantan) at the recommended antivenom:venom ratio did not neutralize the neuromuscular blockade in PND preparations, but total neutralization was achieved with a tenfold greater volume of antivenom. The addition of antivenom after 50% and 80% blockade restored the twitch responses. These results show that M. lemniscatus lemniscatus venom causes potent, irreversible neuromuscular blockade, without myonecrosis. This blockade is apparently mediated by pre- and postsynaptic neurotoxins and can be reversed by coralsnake antivenom.

Endogenous purines modulate K+ -evoked ACh secretion at the mouse neuromuscular junction.

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y13 and A1 , A2A , and A3 receptors, respectively. To elucidate the action of endogenous purines on K+ -dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end-plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K+ , the P2Y13 antagonist N-[2-(methylthio)ethyl]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR-C69931MX) increased asynchronous ACh secretion while the A1 , A3 , and A2A antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(±)-dihydropyridine-3,5-, dicarboxylate (MRS-1191), and 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S-(p-nitrobenzyl)-6-thioinosine provoked a reduction of 10 mM K+ -evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K+ , endogenous ATP/ADP and adenosine bind to inhibitory P2Y13 and A1 and A3 receptors since AR-C69931MX, DPCPX, and MRS-1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto-5'-nucleotidase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K+ , suggesting that more adenosine is needed to activate excitatory A2A receptors. At high K+ concentration, the equilibrative transporters appear to be saturated allowing the accumulation of adenosine in the synaptic cleft. In conclusion, when motor nerve terminals are depolarized by increasing K+ concentrations, the ATP/ADP and adenosine endogenously generated are able to modulate ACh secretion by sequential activation of different purinergic receptors.

Protonophoric action of triclosan causes calcium efflux from mitochondria, plasma membrane depolarization and bursts of miniature end-plate potentials

The formerly widely used broad-spectrum biocide triclosan (TCS) has now become a subject of special concern due to its accumulation in the environment and emerging diverse toxicity. Despite the common opinion that TCS is an uncoupler of oxidative phosphorylation in mitochondria, there have been so far no studies of protonophoric activity of this biocide on artificial bilayer lipid membranes (BLM). Yet only few works have indicated the relationship between TCS impacts on mitochondria and nerve cell functioning. Here, we for the first time report data on a high protonophoric activity of TCS on planar BLM. TCS proved to be a more effective protonophore on planar BLM, than classical uncouplers. Correlation between a strong depolarizing effect of TCS on bacterial membranes and its bactericidal action on Bacillus subtilis might imply substantial contribution of TCS protonophoric activity to its antimicrobial efficacy. Protonophoric activity of TCS, monitored by proton-dependent mitochondrial swelling, resulted in Ca2+ efflux from mitochondria. A comparison of TCS effects on molluscan neurons with those of conventional mitochondrial uncouplers allowed us to ascribe the TCS-induced neuronal depolarization and suppression of excitability to the consequences of mitochondrial deenergization. Also similar to the action of common uncouplers, TCS caused a pronounced increase in frequency of miniature end-plate potentials at neuromuscular junctions. Thus, the TCS-induced mitochondrial uncoupling could alter neuronal function through distortion of Ca2+ homeostasis.

Multiple Actions of Rotenone, an Inhibitor of Mitochondrial Respiratory Chain, on Ionic Currents and Miniature End-Plate Potential in Mouse Hippocampal (mHippoE-14) Neurons

Background/Aims: Rotenone (Rot) is known to suppress the activity of complex I in the mitochondrial chain reaction; however, whether this compound has effects on ion currents in neurons remains largely unexplored. Methods: With the aid of patch-clamp technology and simulation modeling, the effects of Rot on membrane ion currents present in mHippoE-14 cells were investigated. Results: Addition of Rot produced an inhibitory action on the peak amplitude of I_Na with an IC₅₀ value of 39.3 µM; however, neither activation nor inactivation kinetics of I_Na was changed during cell exposure to this compound. Addition of Rot produced little or no modifications in the steady-state inactivation curve of I_Na. Rot increased the amplitude of Ca²⁺-activated Cl^- current in response to membrane depolarization with an EC₅₀ value of 35.4 µM; further addition of niflumic acid reversed Rot-mediated stimulation of this current. Moreover, when these cells were exposed to 10 µM Rot, a specific population of ATP-sensitive K⁺ channels with a single-channel conductance of 18.1 pS was measured, despite its inability to alter single-channel conductance. Under current clamp condition, the frequency of miniature end-plate potentials in mHippoE-14 cells was significantly raised in the presence of Rot (10 µM) with no changes in their amplitude and time course of rise and decay. In simulated model of hippocampal neurons incorporated with chemical autaptic connection, increased autaptic strength to mimic the action of Rot was noted to change the bursting pattern with emergence of subthreshold potentials. Conclusions: The Rot effects presented herein might exert a significant action on functional activities of hippocampal neurons occurring in vivo.

Muscle Yap Is a Regulator of Neuromuscular Junction Formation and Regeneration.

Yes-associated protein (Yap) is a major effector of the Hippo pathway that regulates cell proliferation and differentiation during development and restricts tissue growth in adult animals. However, its role in synapse formation remains poorly understood. In this study, we characterized Yap's role in the formation of the neuromuscular junction (NMJ). In HSA-Yap-/- mice where Yap was mutated specifically in muscle cells, AChR clusters were smaller and were distributed in a broader region in the middle of muscle fibers, suggesting that muscle Yap is necessary for the size and location of AChR clusters. In addition, HSA-Yap-/- mice also exhibited remarkable presynaptic deficits. Many AChR clusters were not or less covered by nerve terminals; miniature endplate potential frequency was reduced, which was associated with an increase in paired-pulse facilitation, indicating structural and functional defects. In addition, muscle Yap mutation prevented reinnervation of denervated muscle fibers. Together, these observations indicate a role of muscle Yap in NMJ formation and regeneration. We found that β-catenin was reduced in the cytoplasm and nucleus of mutant muscles, suggesting compromised β-catenin signaling. Both NMJ formation and regeneration deficits of HSA-Yap-/- mice were ameliorated by inhibiting β-catenin degradation, further corroborating a role of β-catenin or Wnt-dependent signaling downstream of Yap to regulate NMJ formation and regeneration.SIGNIFICANCE STATEMENT This paper explored the role of Yes-associated protein (Yap) in neuromuscular junction (NMJ) formation and regeneration. Yap is a major effector of the Hippo pathway that regulates cell proliferation and differentiation during development and restricts tissue growth in adult animals. However, its role in synapse formation remains poorly understood. We provide evidence that muscle Yap mutation impairs both postsynaptic and presynaptic differentiation and function and inhibits NMJ regeneration after nerve injury, indicating a role of muscle Yap in these events. Further studies suggest compromised β-catenin signaling as a potential mechanism. Both NMJ formation and regeneration deficits of HSA-Yap-/- mice were ameliorated by inhibiting β-catenin degradation, corroborating a role of β-catenin or Wnt-dependent signaling downstream of Yap to regulate NMJ formation and regeneration.

Loss of laminin‐a4 results in pre‐ and postsynaptic modifications at the neuromuscular junction

Synaptic basal lamina such as laminin-421 (α4β2γ1) mediate differentiation of the neuromuscular junction (NMJ). Laminins interact with their pre- or postsynaptic receptors to provide stability and alignment of the pre- to postsynaptic specializations. Knockout of the laminin-α4 gene (lama4) does not alter gross NMJ morphogenesis. However, mice deficient in laminin-α4 (lama4-/- mice) display disruptions in the alignment of the active zones and postsynaptic folds at the NMJ, although the physiological consequences of this loss have not been examined. The present study investigated the differences in neurotransmission during the early development and maturation of the NMJ in lama4-/- and wild-type mice. Lama4-/- NMJs demonstrated a decrease in miniature end-plate potential (EPP) frequency and increased amplitude of miniature EPPs and evoked EPPs. Binomial parameters analysis of neurotransmitter release revealed a decrease in quantal release, the result of a decrease in the number of active release sites, but not in the probability of transmitter release. Lama4-/- NMJs displayed higher levels of synaptic depression under high-frequency stimulation and altered facilitation, suggesting compromised delivery of synaptic vesicles. This idea is supported by our molecular investigations of lama4-/- NMJs, where we see altered distribution of Bassoon, a molecular component of active zones, presumably resulting from perturbed neurotransmission.-Chand, K. K., Lee, K. M., Lavidis, N. A., Noakes, P. G. Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Ultrastructural aspects of mouse nerve-muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX-I and Bbil-TX: Unknown myotoxic effects.

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve-muscle preparations exposed to Bothrops venoms and their phospholipase A2 toxins (PLA2 ) can exhibit a neurotoxic pattern as increase in frequency of miniature end-plate potentials (MEPPs) as well as in amplitude of end-plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA2 toxins (BthTX-I and Bbil-TX) in mouse isolated nerve-phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX-I and Bbil-TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic-like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.