Miniature End-plate Potentials Research Articles

Effects and Mechanisms of Action of Nitric Oxide on Transmitter Release in Mouse Motor Nerve Terminals

We examined the mechanisms of NO action on transmitter release in neuromuscular preparations of the mouse diaphragm muscle using a standard microelectrode technique. A donor of NO (SNAP, 100 μM) and a substrate for NO synthesis (L-arginine, 100 μM) reduced the evoked transmitter release from motor nerve endings. At the same time, SNAP did not change the frequency and amplitude of miniature end-plate potentials, while an inhibitor of NO synthase, L-NAME, exerted no effect on evoked and spontaneous transmitter release. Inhibition of soluble guanylate cyclase by ODQ (2.5 μM) abolished the effect of NO on the evoked transmitter release, while the elevation of cGMP-level by its membranepenetrating analog 8BrcGMP did not prevent such effect. The elevation of intracellular concentration of cAMP by 100 μM of its analog 8(4CPT) cAMP or inhibition of phosphodiesterase (PDE) by the action of 100 μM IBMX eliminated NO effects on transmitter release. It is concluded that NO activates soluble guanylate cyclase and intensifies the cGMP synthesis. Activation of the PDE II via an increase in the cGMP level with consequent reduction of the level of intracellular cAMP and decrease in the activity of PKA reduced transmitter release from mouse motor nerve ending.

Toxicity of the synthetic polymeric 3-alkylpyridinium salt (APS3) is due to specific block of nicotinic acetylcholine receptors

The in vivo and in vitro toxic effects of the synthetic polymeric 3-alkylpyridinium salt (APS3), from the Mediterranean marine sponge Reniera sarai, were evaluated on mammals, with emphasis to determine its mode of action. The median lethal doses of APS3 were 7.25 and higher that 20mg/kg in mouse and rat, respectively. Intravenous administration of 7.25 and 20mg/kg APS3 to rat caused a significant fall followed by an increase in mean arterial blood pressure accompanied by tachycardia. In addition, cumulative doses of APS3 (up to 60mg/kg) inhibited rat nerve-evoked skeletal muscle contraction in vivo, with a median inhibitory dose (ID50) of 37.25mg/kg. When administrated locally by intramuscular injection to mouse, APS3 decreased the compound muscle action potential recorded in response to in vivo nerve stimulation, with an ID50 of 0.5mg/kg. In vitro experiments confirmed the inhibitory effect of APS3 on mouse hemidiaphragm nerve-evoked muscle contraction with a median inhibitory concentration (IC50) of 20.3μM, without affecting directly elicited muscle contraction. The compound inhibited also miniature endplate potentials and nerve-evoked endplate potentials with an IC50 of 7.28μM in mouse hemidiaphragm. Finally, APS3 efficiently blocked acetylcholine-activated membrane inward currents flowing through Torpedo nicotinic acetylcholine receptors (nAChRs) incorporated to Xenopus oocytes, with an IC50 of 0.19μM. In conclusion, our results strongly suggest that APS3 blocks muscle-type nAChRs, and show for the first time that in vivo toxicity of APS3 is likely to occur through an antagonist action of the compound on these receptors.

The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC50=0.74μM), without affecting directly-elicited twitch tension up to 2.72μM. The compound (0.007–3.40μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68μM, without affecting their frequency. Full size endplate potentials, recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC50=0.36μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α12β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC50=0.0005μM), indicating a higher affinity of the compound for Torpedo (α12β1γδ) than for the mouse (α12β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction.

Muscle specific kinase autoantibodies cause synaptic failure through progressive wastage of postsynaptic acetylcholine receptors

In myasthenia gravis muscle weakness is caused by autoantibodies against components of the neuromuscular junction. Patient autoantibodies against muscle specific kinase (MuSK) deplete MuSK from the postsynaptic membrane and reproduce signs of myasthenia gravis when injected into mice. Here we have examined the time-course of structural and functional changes that lead up to synaptic failure. C57Bl6J mice received daily injections of anti-MuSK patient IgG for 15days. Mice began to lose weight from day 12 and demonstrated whole-body weakness by day 14. Electromyography indicated synaptic impairment from day 6 in the gastrocnemius muscle and from day 10 in the diaphragm muscle. Confocal microscopy revealed linear declines in the area and density of postsynaptic acetylcholine receptors (3–5% per day) from day 1 through day 15 of the injection series in all five muscles examined. Intracellular recordings from the diaphragm muscle revealed comparable progressive declines in the amplitude of the endplate potential and miniature endplate potential of 3–4% per day. Neither quantal content nor the postsynaptic action potential threshold changed significantly over the injection series. The inverse relationship between the quantal amplitude of a synapse and its quantal content disappeared only late in the injection series (day 10). Our results suggest that the primary myasthenogenic action of anti-MuSK IgG is to cause wastage of postsynaptic acetylcholine receptor density. Consequent reductions in endplate potential amplitudes culminated in failure of neuromuscular transmission.

Neuromuscular transmission modulation by adenosine upon aging

In infant rats adenosine A2A receptor-mediated modulation of neuromuscular transmission predominates over A1 receptor-mediated neuromodulation. We investigated whether aging affects this A2A/A1 receptor balance. Evoked (EPPs) and miniature end plate potentials (MEPPs) were recorded from single fibers of (weeks-old) infant (3–4), young adult (12–16), older (36–38), and aged (80–90) male rat-diaphragm. The non A1/A2A selective agonist, 2-chloroadenosine (CADO; 30 nM) and the adenosine kinase inhibitor, iodotubericidin (ITU; 10 μM) increased mean amplitude and quantal content of EPPs in infant, young adult, and older adult rats, but not in aged rats. The facilitatory effects were prevented by the A2A receptor antagonist, ZM241385 (50 nM) and mimicked by the A2A receptor agonist, CGS21680 (10 nM). The A1 receptor agonist, 6-cyclopentyladenosine (CPA; 100 nM), decreased EPPs amplitude in all age groups. It is concluded that aging differently influences adenosine A1 receptor and A2A receptor-mediated presynaptic modulation of neuromuscular transmission, so that the facilitatory influence decreases upon aging, whereas the inhibitory influence remains unchanged in aged animals. The reduction of adenosine A2A receptors upon aging may contribute to the age-related changes in neuromuscular function.

Passive and active immunization models of MuSK-Ab positive myasthenia: Electrophysiological evidence for pre and postsynaptic defects

Antibodies directed against the post-synaptic neuromuscular junction protein, muscle specific kinase (MuSK) are found in a small proportion of generalized myasthenia gravis (MuSK-MG) patients. MuSK is a receptor tyrosine kinase which is essential for clustering of the acetylcholine receptors (AChRs) at the neuromuscular junction, but the mechanisms by which MuSK antibodies (MuSK-Abs) affect neuromuscular transmission are not clear. Experimental models of MuSK-MG have been described but there have been no detailed electrophysiological studies and no comparisons between the MuSK-MG and the typical form with AChR-Abs (AChR-MG). Here we studied the electrophysiology of neuromuscular transmission after immunization against MuSK compared with immunization against AChR, and also after passive transfer of IgG from MuSK-MG or AChR-MG patients. Overt clinical weakness was observed in 6/10 MuSK-immunized and 3/9 AChR-immunized mice but not in those injected with patients' IgG. Miniature endplate potentials (MEPPS) were reduced in all weak mice consistent with the reduction in postsynaptic AChRs that was found. However, whereas there was an increase in the quantal release of acetylcholine (ACh) in the weak AChR-immunized mice, no such increase was found in the weak MuSK-immunized mice. Similar trends were found after the passive transfer of purified IgG antibodies from MuSK-MG or AChR-MG patients. Preliminary results showed that MuSK expression was considerably higher at the neuromuscular junctions of the masseter (facial) than in the gastrocnemius (leg) with no reduction in MuSK immunostaining at the neuromuscular junctions. Overall, these results suggest that MuSK antibodies act in at least two ways. Firstly by indirectly affecting MuSK's ability to maintain the high density of AChRs and secondly by interfering with a compensatory presynaptic mechanism that regulates quantal release and helps to preserve neuromuscular function. These results raise questions about how MuSK is involved in retrograde signaling, and the combination of post-synaptic defects with lack of presynaptic compensation may begin to explain the more severe disease in MuSK-MG patients.

Role of Stored Calcium in the Regulation of Neurotransmitter Quantum Size

Release of stored calcium ions during activation of ryanodine receptors with ryanodine or caffeine elevates the mean amplitude of spontaneous miniature end-plate potentials. Blockade of these receptors with selective antagonists abolishes this effect. Preliminary loading of the motor nerve terminals with intracellular calcium buffer EGTA-AM, but not with BAPTA-AM, also completely prevented the ryanodine-induced increment of miniature end-plate potential amplitude probably induced by the release of stored calcium. Vesamicol, a selective blocker of acetylcholine transport into vesicles, prevented the ryanodine-induced increment of the mean amplitude of miniature end-plate potentials. This increment was 2-fold more pronounced after preliminary blockade of protein kinase C with chelerythrine and was completely abolished by blockade of protein kinase A with H-89.

Effect of 2-Nitropropane on Chemical Neurotransmission, Spontaneous and Evoked, in the Sartorius Muscle of the Frog

n this project we studied the effect of 2-nitropropane in spontaneous and evoked chemical neurotransmission, using as a model the frog neuromuscular junction, in vitro. The experiments were carried out in the sciatic nerve- sartorius muscle preparation. Our results show that neurotransmission was inhibited, manifesting as a decrease in the amplitude and instantaneous frequency of miniature end-plate potentials and the reduction of end-plate potentials amplitude of the neuromuscular junction, evoked by nerve stimulation. Because 2-nitropropane showed inhibitory effects at peripheral level, we recommend that in work environments where there is exposure to organic solvents, neurological clinical examinations must be performed to detect abnormalities or neurological damage the earlie

Quantal Analysis of Endplate Potentials in Mouse Flexor Digitorum Brevis Muscle.

The isolated flexor digitorum brevis (FDB) muscle from mice is extremely well suited to rapid acquisition of data and analysis of neurotransmitter release and action at neuromuscular junctions, because the muscle and its tibial nerve supply are simple to dissect and its constituent muscle fibers are short (<1 mm) and isopotential along their length. Methods are described here for dissection of FDB, stimulation of the tibial nerve, microelectrode recording from individual muscle fibers, and quantal analysis of endplate potentials (EPPs) and miniature endplate potentials (MEPPs). Curr. Protoc. Mouse Biol. 1:429-444 © 2011 by John Wiley & Sons, Inc.

Lidocaine treatment during synapse reformation periods permanently inhibits NGF-induced excitation in an identified reconstructed synapse of Lymnaea stagnalis

Nerve growth factor (NGF) has been reported to affect synaptic transmission and cause neuropathic pain. In contrast, lidocaine has been used to reduce neuropathic pain; however, the effect of NGF and lidocaine on spontaneous transmitter release and synapse excitation has not been fully defined. Therefore, the effect of NGF and lidocaine on nerve regeneration, synapse reformation, and subsequent spontaneous transmitter release was investigated. We used Lymnaea stagnalis soma-soma-identified synaptic reconstruction to demonstrate that a transient increase in both frequency and amplitude of spontaneous events of miniature endplate potentials (MEPPs) occurs following NGF treatment and a short burst of action potentials in the presynaptic cell; in addition, the effect of lidocaine on NGF-induced synapse reformation was investigated. Using a cell culture and electrophysiological and FM-143 imaging techniques for exocytosis on unequivocally identified presynaptic visceral dorsal 4 (VD4) and postsynaptic somata left pedal (LPeE) neurons from the mollusc Lymnaea stagnalis, the effects of NGF and lidocaine on nerve regeneration, synapse reformation, and its electrophysiological spontaneous synaptic transmission between cultured neurons were described. NGF increased axonal growth, frequency, and amplitudes of MEPPs. Lidocaine exposure during synapse reformation periods was drastically and permanently reduced axonal growth and the incidence of synapse excitation by NGF. NGF increased amplitudes and frequencies of MEPPs and induced synaptic excitation by increasing axonal growth and exocytosis. Lidocaine exposure during synapse reformation periods permanently suppressed NGF-induced excitation by suppressing axonal growth and exocytosis of presynaptic neurons in the identified reconstructed synapse of L. stagnalis.

Nonextensivity and self-affinity in the mammalian neuromuscular junction

We study time series and the spontaneous miniature end-plate potentials (MEPPs) of mammals recorded at neuromuscular junctions using two different approaches: generalized thermostatistics and detrended fluctuation analysis (DFA). Classical concepts establish that the magnitude of these potentials is characterized by Gaussian statistics and that their intervals are randomly displayed. First we show that MEPP distributions adequately satisfy the q-Gaussian distributions that maximize the Tsallis entropy, indicating their nonextensive and nonequilibrium behavior. We then examine the intervals between the miniature potentials via DFA, where the profile of the intervals between events configures a deviation from the expected random behavior. Some possible physiological substrates for these findings are discussed.

Role of acetylcholinesterase on the structure and function of cholinergic synapses: insights gained from studies on knockout mice.

Electrophysiological and ultrastructural studies were performed on phrenic nerve-hemidiaphragm preparations isolated from wild-type and acetylcholinesterase (AChE) knockout (KO) mice to determine the compensatory mechanisms manifested by the neuromuscular junction to excess acetylcholine (ACh). The diaphragm was selected since it is the primary muscle of respiration, and it must adapt to allow for survival of the organism in the absence of AChE. Nerve-elicited muscle contractions, miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were recorded by conventional electrophysiological techniques from phrenic nerve-hemidiaphragm preparations isolated from 1.5- to 2-month-old wild-type (AChE(+/+)) or AChE KO (AChE(-/-)) mice. These recordings were chosen to provide a comprehensive assessment of functional alterations of the diaphragm muscle resulting from the absence of AChE. Tension measurements from AChE(-/-) mice revealed that the amplitude of twitch tensions was potentiated, but tetanic tensions underwent a use-dependent decline at frequencies below 70 Hz and above 100 Hz. MEPPs recorded from hemidiaphragms of AChE(-/-) mice showed a reduction in frequency and a prolongation in decay (37%) but no change in amplitude compared to values observed in age-matched wild-type littermates. In contrast, MEPPs recorded from hemidiaphragms of wild-type mice that were exposed for 30 min to the selective AChE inhibitor 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW284C51) exhibited a pronounced increase in amplitude (42%) and a more marked prolongation in decay (76%). The difference between MEPP amplitudes and decays in AChE(-/-) hemidiaphragms and in wild-type hemidiaphragms treated with BW284C51 represents effective adaptation by the former to a high ACh environment. Electron microscopic examination revealed that diaphragm muscles of AChE(-/-) mice had smaller nerve terminals and diminished pre- and post-synaptic surface contacts relative to neuromuscular junctions of AChE(+/+) mice. The morphological changes are suggested to account, in part, for the ability of muscle from AChE(-/-) mice to function in the complete absence of AChE.

APP and APLP2 are essential at PNS and CNS synapses for transmission, spatial learning and LTP

Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsα knock-in (KI) mice expressing solely the secreted ectodomain APPsα. Here, we generated double mutants (APPsα-DM) by crossing APPsα-KI mice onto an APLP2-deficient background and show that APPsα rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsα-DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsα-DM muscle showed fragmented post-synaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsα-DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABA(A) receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.

Endplate structure and parameters of neuromuscular transmission in sporadic centronuclear myopathy associated with myasthenia

Centronuclear myopathy is a pathologically diagnosed congenital myopathy. The disease genes encode proteins with membrane modulating properties ( MTM1, DNM2, and BIN1) or alter excitation–contraction coupling ( RYR1). Some patients also have myasthenic symptoms but electrodiagnostic and endplate studies in these are limited. A sporadic patient had fatigable weakness and a decremental EMG response. Analysis of centronuclear myopathy disease- and candidate-genes identified no mutations. Quantitative endplate electron microscopy studies revealed simplified postsynaptic regions, endplate remodeling with normal nerve terminal size, normal synaptic vesicle density, and mild acetylcholine receptor deficiency. The amplitude of the miniature endplate potential was decreased to 60% of normal. Quantal release by nerve impulse was reduced to 40% of normal due to a decreased number of releasable quanta. The safety margin of neuromuscular transmission is compromised by decreased quantal release by nerve impulse and by a reduced postsynaptic response to the released quanta.

Protection of human muscle acetylcholinesterase from soman by pyridostigmine bromide

Pretreatment with pyridostigmine bromide (PB) of human intercostal muscle fibers exposed to the irreversible acetylcholinesterase (AChE) inhibitor soman was investigated. Muscles were pretreated with 3 × 10(-6) M PB or saline for 20 minutes, then exposed to 10(-7) M soman for 10 minutes. AChE of muscles treated with soman alone was inhibited >95%. In contrast, PB pretreatment of soman-exposed bundles protected 20% of AChE activity. AChE of bundles exposed to PB alone recovered after 4 hours, but bundles exposed to both PB and soman did not. Soman-induced reduction of resting membrane potentials and increment of amplitudes and decay times of miniature endplate potentials (MEPPs) were partially corrected by PB pretreatment. In vitro pretreatment of human muscles with PB protected up to 20% of muscle AChE and ameliorated some deleterious effects on endplate physiology induced by soman.

Transmitter release in the neuromuscular synapse of the protein kinase C theta‐deficient adult mouse

We studied structural and functional features of the neuromuscular junction in adult mice (P30) genetically deficient in the protein kinase C (PKC) theta isoform. Confocal and electron microscopy shows that there are no differences in the general morphology of the endplates between PKC theta-deficient and wild-type (WT) mice. Specifically, there is no difference in the density of the synaptic vesicles. However, the myelin sheath is not as thick in the intramuscular nerve fibers of the PKC theta-deficient mice. We found a significant reduction in the size of evoked endplate potentials and in the frequency of spontaneous, asynchronous, miniature endplate potentials in the PKC theta-deficient neuromuscular preparations in comparison with the WT, but the mean amplitude of the spontaneous potentials is not different. These changes indicate that PKC theta has a presynaptic role in the function of adult neuromuscular synapses.

Increased Neurotransmitter Release at the Neuromuscular Junction in a Mouse Model of Polyglutamine Disease

In Huntington's disease (HD), the expansion of polyglutamine (polyQ) repeats at the N terminus of the ubiquitous protein huntingtin (htt) leads to neurodegeneration in specific brain areas. Neurons degenerating in HD develop synaptic dysfunctions. However, it is unknown whether mutant htt impacts synaptic function in general. To investigate that, we have focused on the nerve terminals of motor neurons that typically do not degenerate in HD. Here, we have studied synaptic transmission at the neuromuscular junction of transgenic mice expressing a mutant form of htt (R6/1 mice). We have found that the size and frequency of miniature endplate potentials are similar in R6/1 and control mice. In contrast, the amplitude of evoked endplate potentials in R6/1 mice is increased compared to controls. Consistent with a presynaptic increase of release probability, synaptic depression under high-frequency stimulation is higher in R6/1 mice. In addition, no changes were detected in the size and dynamics of the recycling synaptic vesicle pool. Moreover, we have found increased amounts of the synaptic vesicle proteins synaptobrevin 1,2/VAMP 1,2 and cysteine string protein-α, and the SNARE protein SNAP-25, concomitant with normal levels of other synaptic vesicle markers. Our results reveal that the transgenic expression of a mutant form of htt leads to an unexpected gain of synaptic function. That phenotype is likely not secondary to neurodegeneration and might be due to a primary deregulation in synaptic protein levels. Our findings could be relevant to understand synaptic toxic effects of proteins with abnormal polyQ repeats.

Congenital myasthenic syndrome associated with epidermolysis bullosa caused by homozygous mutations in PLEC1 and CHRNE

Mutations in the plectin gene (PLEC1) cause epidermolysis bullosa simplex (EBS), which may associate with muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA). The association of EBS with congenital myasthenic syndrome (CMS) is also suspected to result from PLEC1 mutations. We report here a consanguineous patient with EBS and CMS for whom mutational analysis of PLEC1 revealed a homozygous 36 nucleotide insertion (1506_1507ins36) that results in a reduced expression of PLEC1 mRNA and plectin in the patient muscle. In addition, mutational analysis of CHRNE revealed a homozygous 1293insG, which is a well-known low-expressor receptor mutation. A skin biopsy revealed signs of EBS, and an anconeus muscle biopsy showed signs of a mild myopathy. Endplate studies showed fragmentation of endplates, postsynaptic simplification, and large collections of thread-like mitochondria. Amplitudes of miniature endplate potentials were diminished, but the endplate quantal content was actually increased. The complex phenotype presented here results from mutations in two separate genes. While the skin manifestations are because of the PLEC1 mutation, footprints of mutations in PLEC1 and CHRNE are present at the neuromuscular junction of the patient indicating that abnormalities in both genes contribute to the CMS phenotype.

Excitatory effect of the A 2A adenosine receptor agonist CGS-21680 on spontaneous and K +-evoked acetylcholine release at the mouse neuromuscular junction

The mechanism of action of the A 2A adenosine receptor agonist 2- p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) in the facilitation of spontaneous (isotonic and hypertonic condition) and K +-evoked acetylcholine (ACh) release was investigated in the mouse diaphragm muscles. At isotonic condition, the CGS-21680-induced excitatory effect on miniature end-plate potential (MEPP) frequency was not modified in the presence of CdCl 2 and in a medium free of Ca 2+ (0Ca 2+-EGTA), but it was abolished after buffering the rise of intracellular Ca 2+ with 1,2-bis-(2-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid tetra(acetoxy-methyl) (BAPTA-AM) and when the Ca 2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca 2+ stores. CGS-21680 did not have a direct effect on the Ca 2+-independent neurotransmitter-releasing machinery, since the modulatory effect on the hypertonic response was also occluded by BAPTA-AM and thapsigargin. CGS-21680 facilitation on K +-evoked ACh release was not altered by the P/Q-type voltage-dependent calcium channel (VDCC) blocker ω-Agatoxin IVA, but it was completely prevented by both, the L-type VDCC blocker nitrendipine (which is known to immobilize their gating charges), or thapsigargin, suggesting that the effects of CGS-21680 on L-type VDCC and thapsigargin-sensitive internal stores are associated. We found that the VDCC pore blocker Cd 2+ (2 mM Ca 2+ or 0Ca 2+-EGTA) failed to affect the CGS-21680 effect in high K + whereas nitrendipine in 0Ca 2+-EGTA+Cd 2+ occluded its action. The blockade of Ca 2+ release from endoplasmic reticulum with ryanodine antagonized the facilitating effect of CGS-21680 in control and high K + concentration. It is concluded that, at the mouse neuromuscular junction, activation of A 2A receptors facilitates spontaneous and K +-evoked ACh release by an external Ca 2+-independent mechanism but that involves mobilization of Ca 2+ from internal stores: during spontaneous ACh release stimulating directly the ryanodine-sensitive stores and, at high K +, probably modulating the L-type VDCCs which may cause the opening of the ryanodine receptors that would be directly coupled to the channels. In both cases, Ca 2+ released from the endoplasmic reticulum would be capable of activating the exocytotic machinery, thus producing facilitation of ACh release.

Pre‐ and postsynaptic neuromuscular junction abnormalities in musk myasthenia

Autoantibodies to muscle-specific kinase (MuSK) can cause myasthenia gravis (MG). The pathophysiological mechanism remains unknown. We report in vitro electrophysiological and histological studies of the neuromuscular junction in a MuSK MG patient. Low levels of presynaptic acetylcholine release and small miniature endplate potentials were found. This combination of pre- and postsynaptic abnormalities was supported by histology, revealing partially denervated postsynaptic areas, and some degeneration of postsynaptic folds. Results suggest that anti-MuSK antibodies reduce the stability of muscle-nerve contact.