Miniature End-plate Potentials Research Articles

LRP4 is critical for neuromuscular junction maintenance.

The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.

Quantal Basis of Secretory Granule Biogenesis and Inventory Maintenance: the Surreptitious Nano-machine Behind It.

Proteins are molecular machines with the capacity to perform diverse physical work as response to signals from the environment. Proteins may be found as monomers or polymers, two states that represent an important subset of protein interactions and generate considerable functional diversity, leading to regulatory mechanisms closely akin to decision-making in service systems. Polymerization is not unique to proteins. Other cell compartments (e.g. secretory granules) or tissue states (e.g. miniature end plate potential) are associated with polymerization of some sort, leading to information transport. This data-processing mechanism has similarities with (and led us to the investigation of) granule homotypic polymerization kinetics. Using information theory, we demonstrate the role played by the heterogeneity induced by polymerization: granule size distribution and the stealthy machine behind granule life cycle increase system entropy, which modulates the source/receiver potential that affects communication between the cell and its environment. The granule inventory management by the same nano-machine is discussed.

Adenosine A2A receptors activation facilitates neuromuscular transmission in the pre-symptomatic phase of the SOD1(G93A) ALS mice, but not in the symptomatic phase.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to motor neuron dysfunction resulting in impairment of neuromuscular transmission. A2A adenosine receptors have already been considered as a potential therapeutical target for ALS but their neuromodulatory role at the neuromuscular junction in ALS remains to be clarified. In the present work, we evaluated the effects of A2A receptors on neuromuscular transmission of an animal model of ALS: SOD1(G93A) mice either in the pre-symptomatic (4–6 weeks old) or in the symptomatic (12–14 weeks old) stage. Electrophysiological experiments were performed obtaining intracellular recordings in Mg2+ paralyzed phrenic nerve-hemidiaphragm preparations. Endplate potentials (EPPs), quantal content (q. c.) of EPPs, miniature endplate potentials (MEPPs) and giant miniature endplate potential (GMEPPs) were recorded. In the pre-symptomatic phase of the disease (4–6 weeks old mice), the selective A2A receptor agonist, CGS 21680, significantly enhanced (p<0.05 Unpaired t-test) the mean amplitude and q.c. of EPPs, and the frequency of MEPPs and GMEPPs at SOD1(G93A) neuromuscular junctions, the effect being of higher magnitude (p<0.05, Unpaired t-test) than age-matched control littermates. On the contrary, in symptomatic mice (12–14 weeks old), CGS 21680 was devoid of effect on both the amplitude and q.c. of EPPs and the frequency of MEPPs and GMEPPs (p<0.05 Paired t-test). The results herein reported clearly document that at the neuromuscular junction of SOD1(G93A) mice there is an exacerbation of A2A receptor-mediated excitatory effects at the pre-symptomatic phase, whereas in the symptomatic phase A2A receptor activation is absent. The results thus suggest that A2A receptors function changes with ALS progression.

Effects of the ß2-Adrenoceptor Agonist, Albuterol, in a Mouse Model of Anti-MuSK Myasthenia Gravis

The β2-adrenergic receptor agonist, albuterol, has been reported beneficial in treating several forms of congenital myasthenia. Here, for the first time, we examined the potential benefit of albuterol in a mouse model of anti-Muscle Specific Kinase (MuSK) myasthenia gravis. Mice received 15 daily injections of IgG from anti-MuSK positive patients, which resulted in whole-body weakness. At neuromuscular junctions in the tibialis anterior and diaphragm muscles the autoantibodies caused loss of postsynaptic acetylcholine receptors, and reduced the amplitudes of the endplate potential and spontaneous miniature endplate potential in the diaphragm muscle. Treatment with albuterol (8 mg/kg/day) during the two-week anti-MuSK injection series reduced the degree of weakness and weight loss, compared to vehicle-treated mice. However, the compound muscle action potential recorded from the gastrocnemius muscle displayed a decremental response in anti-MuSK-injected mice whether treated with albuterol or vehicle. Ongoing albuterol treatment did not increase endplate potential amplitudes compared to vehicle-treated mice nor did it prevent the loss of acetylcholine receptors from motor endplates. On the other hand, albuterol treatment significantly reduced the degree of fragmentation of endplate acetylcholine receptor clusters and increased the extent to which the remaining receptor clusters were covered by synaptophysin-stained nerve terminals. The results provide the first evidence that short-term albuterol treatment can ameliorate weakness in a robust mouse model of anti-MuSK myasthenia gravis. The results also demonstrate that it is possible for albuterol treatment to reduce whole-body weakness without necessarily reversing myasthenic impairment to the structure and function of the neuromuscular junction.

Neuromuscular Activity of Micrurus laticollaris (Squamata: Elapidae) Venom in Vitro

In this work, we have examined the neuromuscular activity of Micrurus laticollaris (Mexican coral snake) venom (MLV) in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations, the MLV induced an irreversible concentration- and time-dependent (1–30 µg/mL) neuromuscular blockade, with 50% blockade occurring between 8 and 30 min. Muscle contractures evoked by exogenous acetylcholine were completely abolished by MLV, whereas those of KCl were also significantly altered (86% ± 11%, 53% ± 11%, 89% ± 5% and 89% ± 7% for one, three, 10 and 30 µg of venom/mL, respectively; n = 4; p < 0.05). In mouse phrenic nerve-diaphragm preparations, MLV (1–10 µg/mL) promoted a slight increase in the amplitude of twitch-tension (3 µg/mL), followed by neuromuscular blockade (n = 4); the highest concentration caused complete inhibition of the twitches (time for 50% blockade = 26 ± 3 min), without exhibiting a previous neuromuscular facilitation. The venom (3 µg/mL) induced a biphasic modulation in the frequency of miniature end-plate potentials (MEPPs)/min, causing a significant increase after 15 min, followed by a decrease after 60 min (from 17 ± 1.4 (basal) to 28 ± 2.5 (t15) and 12 ± 2 (t60)). The membrane resting potential of mouse diaphragm preparations pre-exposed or not to d-tubocurarine (5 µg/mL) was also significantly less negative with MLV (10 µg/mL). Together, these results indicate that M. laticollaris venom induces neuromuscular blockade by a combination of pre- and post-synaptic activities.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

BackgroundRhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity.MethodsThe neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.ResultsVenom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively).ConclusionThese results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.

Reduced Expression of the Vesicular Acetylcholine Transporter and Neurotransmitter Content Affects Synaptic Vesicle Distribution and Shape in Mouse Neuromuscular Junction

In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KDHOM) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KDHOM mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1–43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KDHOM neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KDHOM exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape.

Evidence of presynaptic and postsynaptic action of local anesthetics in rats

To assess the probable actions of ropivacaine, 50% enantiomeric excess bupivacaine mixture (S75-R25) and levobupivacaine on neuromuscular transmission in vitro. Thirty rats were distributed into groups (n=5) according to the drug used: ropivacaine, bupivacaine (S75-R25) and levobupivacaine. The concentration used for the three local anesthetics (LA) was 5 µg.mL. (-1)The following parameters were evaluated: 1) LA effects on membrane potential (MP) and miniature end plate potential (MEPP). A chick biventer cervicis preparation was also used to evaluate LA effects on the contracture response to acetylcholine. LA did not alter MP values and decreased the frequency and amplitude of MEPP. In a chick biventer cervicis preparation, bupivacaine (S75-R25) and levobupivacaine decreased the contracture response to acetylcholine with statistical significance, in comparison to ropivacaine. In the concentrations used, levobupivacaine and bupivacaine (S75-R25) exhibited presynaptic and postsynaptic actions evidenced by alterations in miniature end plate potentials and contracture response to acetylcholine. Ropivacaine only had a presynaptic action.

Neuromuscular blocking activity of pinnatoxins E, F and G

Pinnatoxins are produced by dinoflagellates and belong to the cyclic imine family of toxins. They are fast-acting and highly toxic when administered in vivo in rodent bioassays, causing death by respiratory depression within minutes. Studies have revealed that some cyclic imine toxins cause their toxicity by antagonizing both muscle type and heteromeric and homomeric neuronal nicotinic acetylcholine receptors (nAChRs). Pinnatoxins E, F and G all display potent toxicity in in vivo bioassays, with symptoms of toxicity similar to other cyclic imine toxins. However, very little work has been done on the mechanism of action of these pinnatoxin isomers. Thus the aim of the current study was to investigate the rank order of potency and mechanism of action of pinnatoxins E, F and G. The effects of pinnatoxin E, F and G on in vitro rat hemidiaphragm preparations were investigated using twitch tension and electrophysiological techniques to determine the effects of these toxins on cholinergic transmission at the neuromuscular junction. Pinnatoxins E, F and G all produced concentration-dependent reductions in the nerve evoked twitch response of the rat hemidiaphragm, with IC50 values ranging from 11 to 53 nM and a rank order of potency of F > G > E. Only complete washout of pinnatoxin E was evident, with pinnatoxins F and G displaying slow and incomplete washout profiles. Pinnatoxins F and G also reduced the amplitudes of spontaneous miniature endplate potentials and evoked endplate potentials at the neuromuscular junction, without affecting miniature endplate potential frequency or the resting membrane potential of the muscle fibres. These results show that pinnatoxins E, F and G are all potent neuromuscular blocking agents and cause toxicity by acting as antagonists at muscle type nicotinic acetylcholine receptors.

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na+ channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca2+ because its effect was abolished in Ca2+-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca2+ channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca2+ channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.

Cyclooxygenase‐2, prostaglandin E2 glycerol ester and nitric oxide are involved in muscarine‐induced presynaptic enhancement at the vertebrate neuromuscular junction

Previous work has demonstrated that activation of muscarinic acetylcholine receptors at the lizard neuromuscular junction (NMJ) induces a biphasic modulation of evoked neurotransmitter release: an initial depression followed by a delayed enhancement. The depression is mediated by the release of the endocannabinoid 2-arachidonylglycerol (2-AG) from the muscle and its binding to cannabinoid type 1 receptors on the motor nerve terminal. The work presented here suggests that the delayed enhancement of neurotransmitter release is mediated by cyclooxygenase-2 (COX-2) as it converts 2-AG to the glycerol ester of prostaglandin E2 (PGE2-G). Using immunofluorescence, COX-2 was detected in the perisynaptic Schwann cells (PSCs) surrounding the NMJ. Pretreatment with either of the selective COX-2 inhibitors, nimesulide or DuP 697, prevents the delayed increase in endplate potential (EPP) amplitude normally produced by muscarine. In keeping with its putative role as a mediator of the delayed muscarinic effect, PGE2-G enhances evoked neurotransmitter release. Specifically, PGE2-G increases the amplitude of EPPs without altering that of spontaneous miniature EPPs. As shown previously for the muscarinic effect, the enhancement of evoked neurotransmitter release by PGE2-G depends on nitric oxide (NO) as the response is abolished by application of either N(G)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthesis, or carboxy-PTIO, a chelator of NO. Intriguingly, the enhancement is not prevented by AH6809, a prostaglandin receptor antagonist, but is blocked by capsazepine, a TRPV1 and TRPM8 receptor antagonist. Taken together, these results suggest that the conversion of 2-AG to PGE2-G by COX-2 underlies the muscarine-induced enhancement of neurotransmitter release at the vertebrate NMJ.

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd(2+) , the removal of extracellular Ca(2+) and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.

Membrane cholesterol regulates different modes of synaptic vesicle release and retrieval at the frog neuromuscular junction

We investigated the effects of cholesterol removal on spontaneous and KCl-evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl-β-cyclodextrin (MβCD) induced an increase in the frequency of miniature end-plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1-43. Treatment with MβCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MβCD were larger than those from control. In addition, treatment with MβCD reduced the fusion of synaptic vesicles that are mobilized during KCl-evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MβCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl-evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.

A mouse model of the slow channel myasthenic syndrome: Neuromuscular physiology and effects of ephedrine treatment

In the slow channel congenital myasthenic syndrome mutations in genes encoding the muscle acetylcholine receptor give rise to prolonged ion channel activations. The resulting cation overload in the postsynaptic region leads to damage of synaptic structures, impaired neuromuscular transmission and fatigable muscle weakness. Previously we identified and characterised in detail the properties of the slow channel syndrome mutation εL221F. Here, using this mutation, we generate a transgenic mouse model for the slow channel syndrome that expresses mutant human ε-subunits harbouring an EGFP tag within the M3–M4 cytoplasmic region, driven by a ~1500bp region of the CHRNB promoter. Fluorescent mutant acetylcholine receptors are assembled, cluster at the motor endplates and give rise to a disease model that mirrors the human condition. Mice demonstrate mild fatigable muscle weakness, prolonged endplate and miniature endplate potentials, and variable degeneration of the postsynaptic membrane. We use our model to investigate ephedrine as a potential treatment. Mice were assessed before and after six weeks on oral ephedrine (serum ephedrine concentration 89±3ng/ml) using an inverted screen test and in vivo electromyography. Treated mice demonstrated modest benefit for screen hang time, and in measures of compound muscle action potentials and mean jitter that did not reach statistical significance. Ephedrine and salbutamol show clear benefit when used in the treatment of DOK7 or COLQ congenital myasthenic syndromes. Our results highlight only a modest potential benefit of these β2-adrenergic receptor agonists for the treatment of the slow channel syndrome.

Presynaptic pH and vesicle fusion in Drosophila larvae neurones

Both intracellular pH (pHi) and synaptic cleft pH change during neuronal activity yet little is known about how these pH shifts might affect synaptic transmission by influencing vesicle fusion. To address this we imaged pH- and Ca2+-sensitive fluorescent indicators (HPTS, Oregon green) in boutons at neuromuscular junctions. Electrical stimulation of motor nerves evoked presynaptic Ca2+i rises and pHi falls (∼0.1 pH units) followed by recovery of both Ca2+i and pHi. The plasma-membrane calcium ATPase (PMCA) inhibitor, 5(6)-carboxyeosin diacetate, slowed both the calcium recovery and the acidification. To investigate a possible calcium-independent role for the pHi shifts in modulating vesicle fusion we recorded post-synaptic miniature end-plate potential (mEPP) and current (mEPC) frequency in Ca2+-free solution. Acidification by propionate superfusion, NH4+ withdrawal, or the inhibition of acid extrusion on the Na+/H+ exchanger (NHE) induced a rise in miniature frequency. Furthermore, the inhibition of acid extrusion enhanced the rise induced by propionate addition and NH4+ removal. In the presence of NH4+, 10 out of 23 cells showed, after a delay, one or more rises in miniature frequency. These findings suggest that Ca2+-dependent pHi shifts, caused by the PMCA and regulated by NHE, may stimulate vesicle release. Furthermore, in the presence of membrane permeant buffers, exocytosed acid or its equivalents may enhance release through positive feedback. This hitherto neglected pH signalling, and the potential feedback role of vesicular acid, could explain some important neuronal excitability changes associated with altered pH and its buffering. Synapse 67:729–740, 2013.

Why does acetylcholine exacerbate myasthenia caused by anti‐MuSK antibodies?

According to current knowledge, MuSK is a transmembrane protein with four immunoglobulin-like domains and a cysteine-rich domain extracellularly and an intracellular kinase domain. It is expressed on the surface of non-innervated myotubes and on the postsynaptic membrane of the neuromuscular junction (NMJ). Activation of MuSK is a prerequisite for local concentration of acetylcholine receptor (AChR) molecules via rapsyn. In non-innervated myotubes, MuSK is activated by cytosolic Dok-7 to form primitive AChR clusters. At the innervated NMJ, MuSK is activated by neural agrin through interaction with its co-receptor, the transmembrane lipoprotein receptor-related protein LRP4. At the NMJ, MuSK also participates in anchoring the collagenic tail of AChE, collagen Q (ColQ), in the synaptic space. By interacting with additional postsynaptic proteins that include Dok-7, MuSK emerges as a master organizer of postsynaptic development of the NMJ (Wu et al. 2010). Most patients suffering from autoimmune myasthenia gravis (MG) harbour anti-AChR antibodies (AChR-MG) but 5–15% do not. Clinical interest in MuSK arose when enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation experiments revealed that a high proportion of AChR-seronegative myasthenic sera immunoreacted with MuSK (Hoch et al. 2001). Subsequent studies of MuSK antibody-positive myasthenia (MuSK-MG) showed it differs from AChR-MG in several respects. First, the acetylcholinesterase (AChE) inhibitor pyridostigmine, which is effective in AChR-MG, is ineffective in, or worsens, MuSK-MG. Second, MuSK-MG selectively affects bulbar, cervical and respiratory muscles whereas generalized AChR-MG also affects limb muscles. Third, intercostal muscle NMJs in AChR-MG are depleted of AChR, have low-amplitude miniature endplate potentials (MEPPs) and show signs of complement-mediated lysis of the junctional folds whereas in MuSK-MG, NMJs of intercostal or biceps brachii muscles have normal AChR content, generate normal-amplitude MEPPs and have well-preserved junctional folds that bind little or no complement. The reasons for the differences between AChR-MG and MuSK-MG are now reasonably well understood. First, the pathogenic MuSK immunoglobulin is predominantly of class 4 that does not fix complement. Second, combined results of a number of studies show that passive transfer to mice of immunoglobulin G (IgG) isolated from sera of MuSK-MG patients as well as active immunization of mice with rat MuSK causes muscle weakness, reduces MuSK expression by dispersal of synaptic AChR and decreases the synaptic response to spontaneous or evoked release of ACh. Importantly, the deleterious effects on the NMJ are greatest in cranial, paraspinal and masseter muscles that express lower levels of MuSK and MuSK RNA than the less affected muscles (Punga et al. 2011). Finally, it was proposed that pyridostigmine is generally ineffective or harmful in MuSK-MG because MuSK autoantibodies interfere with binding of ColQ to MuSK. This would reduce AChE in the synaptic space and result in cholinergic overactivity at the NMJ. Examination of intercostal muscle NMJs in human MuSK-MG did not show reduced AChE expression, but NMJs of more severely affected muscles were not examined (Kawakami et al. 2011). In this issue of the Journal of Physiology, Morsch and co-workers (2013) report that in the antibody-induced mouse model of MuSK-MG, relevant doses of pyridostigmine worsen the postsynaptic depletion of AChR and reduce the synaptic response to ACh. In contrast, 3,4-diaminopyridine (3,4-DAP), which augments quantal release by nerve impulse, improves neuromuscular transmission without worsening the loss of AChR from the NMJ. What are the possible reasons for this paradox? A partial explanation would be that pyridostigmine increases the lifetime of ACh in the synaptic space by a few milliseconds, during which ACh continues to bind to multiple AChR molecules as it exits the synaptic space by diffusion. In contrast, 3,4-DAP increases quantal release, but the lifetime of ACh in the synaptic space is less than 0.1 ms, during which ACh binds to AChR; and when ACh is released from the receptor over an exponentially distributed period, it is instantly destroyed by AChE. It is also well recognized that the agrin–LRP4–MuSK–Dok-7 signalling pathway promotes AChR clustering, whereas ACh activates an opposite pathway involving subsynaptic AChRs, inositol trisphosphate (IP3) receptors, calcium and cyclin-dependent kinase-5 to disassemble AChR clusters (Misgeld et al. 2005; Zhu et al. 2011). But is the increased exposure to ACh in the model animals sufficient to enhance the synaptic injury? In the experiments of Morsch and co-workers, treatment with conventional doses of pyridostigmine lasted only 7–9 days whereas in other studies chronic treatment of normal animals with high doses of neostigmine was required to cause disintegration of the synaptic folds with loss of AChR. This strongly implies that the enhanced toxicity of ACh in the model animals as well as in human MuSK-MG is caused by impaired cluster stabilization by MuSK. This, in turn, could be due to antibody-dependent reduced expression of MuSK, or is caused by direct interference of anti-MuSK antibodies with MuSK signalling, or both.

Adenosine A1 and A2A receptor‐mediated modulation of acetylcholine release in the mice neuromuscular junction

Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1 R and A2 A R, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 μm adenosine reduces (50%) acetylcholine release in high Mg(2+) or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1 R or A2 A R subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1 Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1 R and A2 A R modulators, which suggests that both receptors need to collaborate. Thus, A1 R and A2 A R might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity.

The effects of hydrogen sulfide on the processes of exo- and endocytosis of synaptic vesicles in the mouse motor nerve endings

The effects of sodium hydrosulfide (NaHS), the donor of hydrogen sulfide (H2S), on the exo/endocytosis cycle of synaptic vesicles in the motor nerve ending of the mouse diaphragm were studied using intracellular microelectrode technique and fluorescent microscopy. NaHS increased the frequency of miniature end-plate potentials (MEPPs), without changing their amplitude-time parameters. NaHS also increased the amplitude of the evoked postsynaptic responses during single stimulation (0.3 Hz), which was the evidence of the enhanced synaptic vesicle exocytosis. During high-frequency stimulation (50 Hz), NaHS induced more significant decline of neurotransmitter release, probably due to the lower rate of synaptic vesicle mobilization from recycling pool to exocytic sites. NaHS also decreased the uptake of the fluorescent endocytic dye FM 1–43, which indicated the reduced endocytosis of synaptic vesicles. Thus, the H2S donor increases exocytosis and decreases the processes of synaptic vesicle endocytosis and mobilization in the mouse motor nerve ending.

Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems

Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.

Exome sequencing detection of two untranslated GFPT1 mutations in a family with limb‐girdle myasthenia

The term 'limb-girdle myasthenia' (LGM) was first used to describe three siblings with proximal limb weakness without oculobulbar involvement, but with EMG decrement and responsiveness to anticholinesterase medication. We report here that exome sequencing in the proband of this family revealed several sequence variations in genes linked to proximal limb weakness. However, the only mutations that cosegregated with disease were an intronic IVS7-8A>G mutation and the previously reported 3'-UTR c.*22C>A mutation in GFPT1, a gene linked to LGM. A minigene assay showed that IVS7-8A>G activates an alternative splice acceptor that results in retention of the last seven nucleotides of intron 7 and a frameshift leading to a termination codon 13 nucleotides downstream from the new splice site. An anconeus muscle biopsy revealed mild reduction of the axon terminal size and postsynaptic fold simplification. The amplitudes of miniature endplate potentials and quantal release were also diminished. The DNA of the mildly affected father of the proband showed only the intronic mutation along with sequence variations in other genes potentially relevant to LGM. Thus, this study performed in the family originally described with LGM showed two GFPT1 untranslated mutations, which may cause disease by reducing GFPT1 expression and ultimately impairing protein glycosylation.