Frequency Of Miniature End-plate Potentials Research Articles

Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins from Bothrops marajoensis (Marajó Lancehead) Snake Venom.

Neuromuscular preparations exposed to B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.

Beauvericin Inhibits Neuromuscular Transmission and Skeletal Muscle Contractility in Mouse Hemidiaphragm Preparation.

The effects of Beauvericin (BEA) produced by the fungusBeauveria bassianaandFusariumsp. on neuromuscular transmission and contractility were determined in an isolated neuromuscular mouse hemidiaphragm preparation. BEA (5 µM) significantly inhibits indirectly elicited twitch amplitude. At higher concentrations (7.5 and 10 µM), BEA produces a significant reduction of directly elicited, or complete block of indirectly evoked, muscle contraction. BEA also appears to be myotoxic, as indicated by a slowly developing muscle contracture. Development of neuromuscular blockade and contracture is concentration dependent. BEA acted by presynaptically depressing spontaneous acetylcholine release as indicated by the reduction in the frequency of spontaneous miniature endplate potentials (MEPPs), while the membrane potential of muscle fibers remained unchanged. At higher concentrations (7.5 and 10 µM), BEA progressively reduces or completely blocks MEPPs and EPPs amplitudes. Changes in MEPPs and EPPs are associated with substantial depolarization of muscle fibers when exposed to 7.5 and 10 µM of BEA. These results indicate that BEA has neurotoxic and myotoxic effects, which overlap in a narrow range of concentrations.

Age-dependent action of reactive oxygen species on transmitter release in mammalian neuromuscular junctions

Reactive oxygen species (ROS) are implicated in aging, but the neurobiological mechanisms of ROS action are not fully understood. Using electrophysiological techniques and biochemical assays, we studied the age-dependent effect of hydrogen peroxide (H2O2) on acetylcholine release in rat diaphragm neuromuscular junctions. H2O2 significantly inhibited both spontaneous (measured as frequency of miniature end-plate potentials) and evoked (amplitude of end-plate potentials) transmitter release in adult rats. The inhibitory effect of H2O2 was much stronger in old rats, whereas in newborns tested during the first postnatal week, H2O2 did not affect spontaneous release from nerve endings and potentiated end-plate potentials. Proteinkinase C activation or intracellular Ca2+ elevation restored redox sensitivity of miniature end-plate potentials in newborns. The resistance of neonates to H2O2 inhibition was associated with higher catalase and glutathione peroxidase activities in skeletal muscle. In contrast, the activities of these enzymes were downregulated in old rats. Our data indicate that the vulnerability of transmitter release to oxidative damage strongly correlates with aging and might be used as an early indicator of senescence.

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABAB R1 and GABAB R2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. We investigated the effect of gamma-aminobutyric acid (GABA) on neuromuscular transmission. GABA reduced the non-quantal and evoked quantal release of acetylcholine. These effects are mediated by GABAB receptors and are implemented via phospholipase C (PLC) activation. Our findings suggest that in the mammalian neuromuscular synapse, metabotropic GABAB receptors are expressed and their activation results in a reduction in the intensity of acetylcholine release.

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

Homocysteine (HCY) is a pro-inflammatory sulphur-containing redox active endogenous amino acid, which concentration increases in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). A widely held view suggests that HCY could contribute to neurodegeneration via promotion of oxidative stress. However, the action of HCY on motor nerve terminals has not been investigated so far. We previously reported that oxidative stress inhibited synaptic transmission at the neuromuscular junction, targeting primarily the motor nerve terminals. In the current study, we investigated the effect of HCY on oxidative stress-induced impairment of transmitter release at the mouse diaphragm muscle. The mild oxidant H2O2 decreased the intensity of spontaneous quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl D-aspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS.

Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission.

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A(1) receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4-6 weeks old) and symptomatic (12-14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg(2+) paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N (6)-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.

Mechanisms of hydrogen sulfide (H2S) action on synaptic transmission at the mouse neuromuscular junction.

Hydrogen sulfide (H2S) is a widespread gasotransmitter also known as a powerful neuroprotective agent in the central nervous system. However, the action of H2S in peripheral synapses is much less studied. In the current project we studied the modulatory effects of the H2S donor sodium hydrosulfide (NaHS) on synaptic transmission in the mouse neuromuscular junction using microelectrode technique. Using focal recordings of presynaptic response and evoked transmitter release we have shown that NaHS (300 μM) increased evoked end-plate currents (EPCs) without changes of presynaptic waveforms which indicated the absence of NaHS effects on sodium and potassium currents of motor nerve endings. Using intracellular recordings it was shown that NaHS increased the frequency of miniature end-plate potentials (MEPPs) without changing their amplitudes indicating a pure presynaptic effect. Furthermore, NaHS increased the amplitude of end-plate potentials (EPPs) without influencing the resting membrane potential of muscle fibers. L-cysteine, a substrate of H2S synthesis induced, similar to NaHS, an increase of EPC amplitudes whereas inhibitors of H2S synthesis (β-cyano-L-alanine and aminooxyacetic acid) had the opposite effect. Inhibition of adenylate cyclase using MDL 12,330A hydrochloride (MDL 12,330A) or elevation of cAMP level with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (pCPT-cAMP) completely prevented the facilitatory action of NaHS indicating involvement of the cAMP signaling cascade. The facilitatory effect of NaHS was significantly diminished when intracellular calcium (Ca(2+)) was buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM). Activation of ryanodine receptors by caffeine or ryanodine increased acetylcholine release and prevented further action of NaHS on transmitter release, likely due to an occlusion effect. Inhibition of ryanodine receptors by ryanodine or dantrolene also reduced the action of NaHS on EPC amplitudes. Our results indicate that in mammalian neuromuscular synapses endogenously produced H2S increases spontaneously and evoked quantal transmitter release from motor nerve endings without changing the response of nerve endings. The presynaptic effect of H2S appears mediated by intracellular Ca(2+) and cAMP signaling and involves presynaptic ryanodine receptors.

Differential Effects of Mefloquine Enantiomers on a Cholinergic Synapse

Racemic mefloquine activates spontaneous vesicle release (SVR) from motor nerve endings and inhibits endplate acetylcholinesterase (AChE). Such cholinergic effects may reduce mefloquine's safety as an antimalarial drug. We tested the hypothesis that (+) and (‐) mefloquine differentially affect the neuromuscular junction (NMJ). Intracellular recordings at NMJs of mouse Triangularis sterni muscles suggested that 30 min exposure to 10 μM (+) or (‐) enantiomer alters the frequency of miniature endplate potentials (mEPPs). Quantitative comparisons were not possible since (+) reduced mEPP amplitude. Therefore, we recorded miniature endplate currents (mEPCs). (+) and (‐) significantly (p<0.01) increased mEPC frequency to 59.7 ± 20.05 Sec‐1 (mean ± SEM, N=21 NMJs) and 17.2 ± 1.98 Sec‐1 (N=14), respectively, from the control value of 5.6 ± 0.76 Sec‐1 (N=25). (+) also reduced amplitude (p<0.01) to 2.9 ± 0.18 nAmps (N=21) from the control value of 3.4 ± 0.18 nAmps (N=25); (‐) did not affect mEPC amplitude. (+), but not (‐), significantly (p<0.01) prolonged the endplate current decay time constant to 2.5 ± 0.16 mSec (N=12) from the control value of 1.1 ± 0.05 mSec (N=16). Thus, (+) may reduce conductance and prolong open time of the endplate acetylcholine receptor (AChR) as well as activate SVR. Neither (+) nor (‐) altered stimulus‐evoked quantal release. Assay of whole muscle homogenates revealed that while both enantiomers (1 to 100 μM) inhibit AChE, (‐) is more potent. The data suggest that (‐) is a less potent activator of SVR but a more potent inhibitor of AChE than (+) mefloquine. Supported by the Kirby Foundation.

Mechanism of neuromuscular dysfunction in Krabbe disease.

The atrophy of skeletal muscles in patients with Krabbe disease is a major debilitating manifestation that worsens their quality of life and limits the clinical efficacy of current therapies. The pathogenic mechanism triggering muscle wasting is unknown. This study examined structural, functional, and metabolic changes conducive to muscle degeneration in Krabbe disease using the murine (twitcher mouse) and canine [globoid cell leukodystrophy (GLD) dog] models. Muscle degeneration, denervation, neuromuscular [neuromuscular junction (NMJ)] abnormalities, and axonal death were investigated using the reporter transgenic twitcher-Thy1.1-yellow fluorescent protein mouse. We found that mutant muscles had significant numbers of smaller-sized muscle fibers, without signs of regeneration. Muscle growth was slow and weak in twitcher mice, with decreased maximum force. The NMJ had significant levels of activated caspase-3 but limited denervation. Mutant NMJ showed reduced surface areas and lower volumes of presynaptic terminals, with depressed nerve control, increased miniature endplate potential (MEPP) amplitude, decreased MEPP frequency, and increased rise and decay rate constants. Twitcher and GLD dog muscles had significant capacity to store psychosine, the neurotoxin that accumulates in Krabbe disease. Mechanistically, muscle defects involved the inactivation of the Akt pathway and activation of the proteasome pathway. Our work indicates that muscular dysfunction in Krabbe disease is compounded by a pathogenic mechanism involving at least the failure of NMJ function, activation of proteosome degradation, and a reduction of the Akt pathway. Akt, which is key for muscle function, may constitute a novel target to complement in therapies for Krabbe disease.

Effect of 50% enantiomeric excess bupivacaine mixture combined with pancuronium on neuromuscular transmission in rat phrenic nerve-diaphragm preparation; a pilot study.

Background and Aims:Local anaesthetics are drugs that are widely used in clinical practice. However, the effects of these drugs on the neuromuscular junction and their influence on the blockade produced by non-depolarising neuromuscular blocking drugs are still under investigation. The aim of this study was to evaluate, in vitro, the influence of a 50% enantiomeric excess bupivacaine mixture on neuromuscular transmission and neuromuscular block produced by pancuronium.Methods:Rats were distributed into three groups (n = 5) according to the drug studied namely, 50% enantiomeric excess bupivacaine mixture (5 μg/mL); pancuronium (2 μg/mL); 50% enantiomeric excess bupivacaine mixture + pancuronium. The following parameters were evaluated: (1) Effects of a 50% enantiomeric excess bupivacaine mixture on membrane potential (MP) and miniature endplate potentials (MEPPs); (2) amplitude of diaphragmatic response before and 60 min after the addition of a 50% enantiomeric excess bupivacaine mixture; the degree of neuromuscular block with pancuronium and pancuronium combined with a 50% enantiomeric excess bupivacaine mixture.Results:A 50% enantiomeric excess bupivacaine mixture did not alter the amplitude of muscle response (MP) but decreased the frequency and amplitude of MEPP. The block produced by pancuronium was potentiated by a 50% enantiomeric excess bupivacaine mixture.Conclusion:A 50% enantiomeric excess bupivacaine mixture used alone did not affect neuromuscular transmission, but potentiated the neuromuscular block produced by pancuronium. No action was shown on the muscle fibre, and alterations on MEPPs demonstrated a presynaptic action.

The giant miniature endplate potentials frequency is increased in aged rats

At the neuromuscular junction, spontaneous giant events (GMEPPs) are enhanced in different conditions when degenerative and/or remodeling processes take place, but no one investigated their incidence upon aging. In the present work, we evaluated evoked and spontaneous neuromuscular transmission events recorded from single muscle fibers. Phrenic-diaphragm preparations of 3–4, 12–16, 36–40 and 70–80 weeks old rat males were used. We found that the occurrence of GMEPPs significantly increases in aged rats. Moreover, in old rats the neuromuscular transmission was significantly impaired due to a significant decrease in the amplitude and quantal content of evoked endplate potentials. Interestingly, the number of observed EPPs failures were ∼3 times lower than the predicted value based on the quantal content. This discrepancy was not observed in infant or adult rats. The coincidence of a high GMEPPs frequency with a lower than expected EPPs failure rate suggests that GMEPPs events are needed to preserve effective neuromuscular transmission in aged animals.

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.

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.

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.

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.