Quantal Content Of Endplate Potentials Research Articles

Homeostatic plasticity induced by increased acetylcholine release at the mouse neuromuscular junction

At the neuromuscular junction (NMJ), changes to the size of the postsynaptic potential induce homeostatic compensation. At the Drosophila NMJ, increased glutamate release causes a compensatory decrease in quantal content, but it is unknown if this mechanism operates at the cholinergic mammalian NMJ. We addressed this question by recording endplate potentials (EPP) and muscle contraction in 3-month and 24-month ChAT-ChR2-EYFP mice that overexpress vesicular acetylcholine transporter and release more acetylcholine per vesicle. At 3 months, the quantal content of EPPs from ChAT-ChR2-EYFP mice were not different from WT controls, however tetanic depression was greater, and quantal size during high-frequency stimulation and the size of the readily releasable pool (RRP) were decreased. At 24 months of age, quantal content was reduced in ChAT-ChR2-EYFP mice, which normalized synaptic depression despite smaller RRP. The effect of pancuronium on indirect evoked muscle twitch was not different between groups. These results indicate that an increase in the amount of acetylcholine per vesicle induces two distinct age-dependent homeostatic mechanisms compensating excessive acetylcholine release.

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

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

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

Despite the long history of investigations of adrenergic compounds and their biological effects, specific mechanisms of their action in distinct compartments of the motor unit remain obscure. Recent results have suggested that not only skeletal muscles but also the neuromuscular junctions represent important targets for the action of catecholamines. In this paper, we describe the effects of adrenaline and noradrenaline on the frequency of miniature endplate potentials, the quantal content of the evoked endplate potentials and the kinetics of acetylcholine quantal release in the motor nerve endings of the mouse diaphragm. Noradrenaline and adrenaline decreased the frequency of the spontaneous release of acetylcholine quanta. The effect of noradrenaline was prevented by the β adrenoreceptor blocker propranolol, whereas the action of adrenaline was abolished by the α adrenoreceptor antagonist phentolamine.Noradrenaline did not alter the quantal content of endplate potentials, while adrenaline suppressed the evoked release of acetylcholine. Blocking the α adrenoreceptors prevented the decrease in quantal secretion caused by adrenaline. Quantal release became more asynchronous under noradrenaline, as evidenced by a greater dispersion of real synaptic delays; in contrast, adrenaline synchronized the release process.Our data suggest an involvement of α and β adrenoreceptors in the diverse modulation of the frequency of miniature endplate potentials, the quantal content of the evoked endplate potentials and the kinetics of acetylcholine quantal secretion in the mouse neuromuscular junction. Moreover, the adrenoblockers affected both the evoked and spontaneous quantal release of acetylcholine, suggesting the presence of endogenous catecholamines in the vicinity of cholinergic synapses.

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

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

Dysfunction of Neuromuscular Synapses in the Genetic Model of Alzheimer's Disease.

The function of synaptic transmission and presynaptic vesicular cycle in the neuromuscular synapses of the diaphragm was studied in transgenic APP/PS1 mice (Alzheimer's disease model). The decrease in the quantal content of end-plate potential, intense depression of the amplitude of terminal plate potentials under conditions of lasting high frequency stimulation (50 Hz), a drastic prolongation of the synaptic vesicle recycling time in APP/PS1 mice in comparison with wild type mice were detected. Manifest dysfunction of the neuromuscular synapses, caused by disordered neurosecretion and recycling of the synaptic vesicles in the presynaptic nerve endings, was detected in the Alzheimer's disease model on transgenic APP/PS1 mice. The study supplemented the notions on the pathogenesis of Alzheimer's disease as a systemic disease, while the detected phenomena could just partially explain the development of motor disorders in this disease.

P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction

It is known that adenosine 5′-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12–13 receptors, 2-methylthioadenosine 5′-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12–13 antagonists AR-C69931MX or 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5′-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.

Methods of activation and the role of calcium/calmodulin-dependent protein kinase II in the regulation of acetylcholine secretion in the motor synapses of mice

In electrophysiological studies of neuromuscular junctions of mice, it was demonstrated that evoked release of mediator triggered by calcium influx via P/Q calcium channels in the terminals remains unchanged in the presence of KN-62, a blocker of calcium/calmodulin-dependent protein kinase II (CaMKII). Disinhibition of L-type calcium channels by blockage of phosphatase calcineurin with cyclosporine A (CsA) causes an increase in the quantal content of endplate potentials (EPP’s QC), which can be prevented by the L-type calcium channel blocker nitrendipine, as well as by preliminary inhibition of CaMKII with KN-62. Thus, the acetylcholine (ACh) release is enhanced with involvement of activated CaMKII only after disinhibition of L-type calcium channels. When disinhibition of L-type calcium channels was induced by suppression of calcium-activated BK-type potassium channels with paxilline, an increase in EPP’s QC was also observed. The conclusion was drawn that in motor synapses of mice, calcium influx via P/Q-type calcium channels cannot provide selective activation of CaMKII to facilitate transmission. However, disinhibition of L-type calcium channels results in the activation of CaMKII, which is accompanied by a persistent increase in evoked ACh release.

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.

Involvement of Basal and Calcium-Activated Protein Kinase C in Neurotransmitter Secretion in Mouse Motor Synapses

Blocker of presynaptic protein kinase C isoforms, GF109203X, reduced quantal content of single and rhythmic evoked end-plate potentials. The increase in quantal content of single potentials under the effect of 4- aminopyridine was neutralized by 75% under the effect of L-type Ca(2+)-channel blocker nitrendipine and completely returned to the control level after protein kinase C inhibition with chelerythrine. Neither nitrendipine, nor GF109203X affected the potentiating effect of tetraethylammonium on quantal content of end-plate potentials. Thus, we discovered basal activity of presynaptic protein kinase C under normal conditions that is aimed at the maintenance of quantal content of evoked release. It has been concluded that 4-aminipyridine, but not tetraethylammonium, triggers Ca(2+) entry into the terminal, which activates protein kinase C and enhanced the evoked acetylcholine release.

Mechanism of Inhibition of Acetylcholine Secretion in Newly Formed Mouse Synapses Involving Ca2+-Dependent Kinases and Voltage-Gated K+-Channels

Nifedipine, a blocker of L-type Ca(2+)-channels, increased quantal content of endplate potentials in newly formed nerve-muscle synapses, while R 24571 (calmodulin inhibitor) and KN 62 (inhibitor of calmodulin-dependent kinase II) did not change it. KN 62 suppressed the increase in quantal content of endplate potentials evoked by nifedipine. Similar to nifedipine, chelerythrine and bisindolylmaleimide I (blockers of protein kinase C) increased quantal content of endplate potentials. In the presence of chelerythrine, nifedipine lost its ability to facilitate secretion of neurotransmitter. 4-Aminopyridine, a blocker of voltage-gated potassium channels, increased quantal content of endplate potentials. In the presence of chelerythrine, 4-aminopyridine induced no additional increase in the quantal content of endplate potentials. In its turn, chelerythrine induced no extra facilitation of secretion in the presence of 4-aminopyridine. It is hypothesized that Ca(2+)-dependent inhibition of secretion results from suppression of calmodulin-dependent kinase II and activation of protein kinase C, which potentiates the work of voltage-gated K(+)-channels.

Mutations in MUSK causing congenital myasthenic syndrome impair MuSK–Dok-7 interaction

We describe a severe congenital myasthenic syndrome (CMS) caused by two missense mutations in the gene encoding the muscle specific receptor tyrosine kinase (MUSK). The identified MUSK mutations M605I and A727V are both located in the kinase domain of MuSK. Intracellular microelectrode recordings and microscopy studies of the neuromuscular junction conducted in an anconeus muscle biopsy revealed decreased miniature endplate potential amplitudes, reduced endplate size and simplification of secondary synaptic folds, which were consistent with postsynaptic deficit. The study also showed a striking reduction of the endplate potential quantal content, consistent with additional presynaptic failure. Expression studies in MuSK deficient myotubes revealed that A727V, which is located within the catalytic loop of the enzyme, caused severe impairment of agrin-dependent MuSK phosphorylation, aggregation of acetylcholine receptors (AChRs) and interaction of MuSK with Dok-7, an essential intracellular binding protein of MuSK. In contrast, M605I, resulted in only moderate impairment of agrin-dependent MuSK phosphorylation, aggregation of AChRs and interaction of MuSK with Dok-7. There was no impairment of interaction of mutants with either the low-density lipoprotein receptor-related protein, Lrp4 (a co-receptor of agrin) or with the mammalian homolog of the Drosophila tumorous imaginal discs (Tid1). Our findings demonstrate that missense mutations in MUSK can result in a severe form of CMS and indicate that the inability of MuSK mutants to interact with Dok-7, but not with Lrp4 or Tid1, is a major determinant of the pathogenesis of the CMS caused by MUSK mutations.

Resistance to d-Tubocurarine of the Rat Diaphragm as Compared to a Limb Muscle

The diaphragm is resistant to competitive neuromuscular blocking agents, as compared to peripheral muscles. The basis of this difference may be a higher concentration of acetylcholine released or higher number of postsynaptic nicotinic acetylcholine receptors in diaphragmatic neuromuscular junctions. Nerve-evoked twitch-tension was measured in rat hemidiaphragm as was Extensor digitorum longus (EDL) nerve-muscle preparation to determine the effective D-tubocurarine concentration that decreased twitch responses by 50%. The mean quantal content of endplate potentials was determined in single junctions in a low-Ca(2+), high-Mg(2+) Krebs-Ringer medium. Strips of hemidiaphragm and EDL muscle, containing the endplate regions, were used to determine the number of nAChR nicotinic acetylcholine receptor binding sites with the aid of radiolabeled [(125)I]alpha-bungarotoxin. The effective D-tubocurarine concentration that decreased twitch responses by 50% (median [interquartile range]) was seven-fold higher in the hemidiaphragm than in the EDL (1.82 microm [1.43-2.20] vs. 0.26 microm [0.23-0.29], P < 0.01). The median of the mean quantal content was higher in the hemidiaphragm than in the EDL (0.57 [0.44-0.84] vs. (0.14 [0.11-0.19], P < 0.01). The number of specific [(125)I]alpha-bungarotoxin binding sites to junctional nicotinic acetylcholine receptors was higher in the diaphragm than in the EDL (1.15 fmol/mg [0.48-1.70] vs. 0.55 fmol/mg [0.23-0.70 ], P < 0.05). The current study indicates that the resistance of the diaphragm to neuromuscular blocking agents can be explained by both a higher mean quantal content of endplate potentials and a higher number of nicotinic acetylcholine receptor binding sites than in the peripheral EDL muscle.

Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome

Background:We describe a severe form of congenital myasthenic syndrome (CMS) associated with congenital nephrosis and ocular malformations caused by two truncating mutations in the gene encoding the laminin β2 subunit...

Acetylcholine Release at Neuromuscular Junctions of Adult Tottering Mice Is Controlled by N-(Cav2.2) and R-Type (Cav2.3) but Not L-Type (Cav1.2) Ca2+Channels

The mutation in the alpha(1A) subunit gene of the P/Q-type (Ca(v)2.1) Ca(2+) channel present in tottering (tg) mice causes ataxia and motor seizures that resemble absence epilepsy in humans. P/Q-type Ca(2+)channels are primarily involved in acetylcholine (ACh) release at mammalian neuromuscular junctions. Unmasking of L-type (Ca(v)1.1-1.2) Ca(2+) channels occurs in cerebellar Purkinje cells of tg mice. However, whether L-type Ca(2+) channels are also up-regulated at neuromuscular junctions of tg mice is unknown. We characterized thoroughly the pharmacological sensitivity of the Ca(2+) channels, which control ACh release at adult tg neuromuscular junctions. Block of N- and R-type (Ca(v)2.2-2.3), but not L-type Ca(2+) channels, significantly reduced quantal content of end-plate potentials in tg preparations. Neither resting nor KCl-evoked miniature end-plate potential frequency differed significantly between tg and wild type (WT). Immunolabeling of Ca(2+) channel subunits alpha(1A), alpha(1B), alpha(1C), and alpha(1E) revealed an apparent increase of alpha(1B), and alpha(1E) staining, at tg but not WT neuromuscular junctions. This presumably compensates for the deficit of P/Q-type Ca(2+)channels, which localized presynaptically at WT neuromuscular junctions. No alpha(1C) subunits juxtaposed with pre- or postsynaptic markers at either WT or tg neuromuscular junctions. Thus, in adult tg mice, immunocytochemical and electrophysiological data indicate that N- and R-type channels both assume control of ACh release at motor nerve terminals. Recruitment of alternate subtypes of Ca(2+) channels to control transmitter release seems to represent a commonly occurring method of neuronal plasticity. However, it is unclear which conditions underlie recruitment of Ca(v)2 as opposed to Ca(v)1-type Ca(2+) channels.

Presynaptic failure of neuromuscular transmission and synaptic remodeling in EA2.

To further investigate the basis of abnormal neuromuscular transmission in two patients with congenital myasthenic syndrome associated with episodic ataxia type 2 (EA2) using stimulated single fiber EMG (SFEMG) and in vitro microelectrode studies. Two patients with genetically characterized EA2 previously shown to have abnormal neuromuscular transmission by voluntary SFEMG were studied with stimulated SFEMG and anconeus muscle biopsy with microelectrode studies and electron microscopy of the neuromuscular junction. In vivo stimulated SFEMG showed signs of presynaptic failure, with jitter and blocking that improved with increased stimulation frequency. Additional evidence of presynaptic failure was provided by the in vitro microelectrode studies, which showed marked reduction of the end plate potential quantal content in both patients. Of note, the end plate potentials showed high sensitivity to N-type blockade with omega-conotoxin not seen in controls. The ultrastructural studies revealed some evidence of small nerve terminals apposed to normal or mildly overdeveloped postsynaptic membranes, suggesting an ongoing degenerative process. The authors demonstrated presynaptic failure of neurotransmission in patients with heterozygous nonsense mutations in CACNA1A. The contribution of non-P-type calcium channels to the process of neurotransmitter release in these patients likely represents a compensatory mechanism, which is insufficient to restore normal neuromuscular transmission.

Mitochondrial Ca2+ uptake prevents desynchronization of quantal release and minimizes depletion during repetitive stimulation of mouse motor nerve terminals.

We investigated how inhibition of mitochondrial Ca2+ uptake affects transmitter release from mouse motor terminals during brief trains of action potentials (500 at 50 Hz) in physiological bath [Ca2+]. When mitochondrial Ca2+ uptake was inhibited by depolarizing mitochondria with antimycin A1 or carbonyl cyanide m-chlorophenyl-hydrazone, the stimulation-induced increase in cytosolic [Ca2+] was greater (> 10 microM, compared to < or = 1 microM in control solution), the quantal content of the endplate potential (EPP) depressed more rapidly (approximately 84 % depression compared to approximately 8 % in controls), and asynchronous release during the stimulus train reached higher frequencies (peak rates of approximately 6000 s-1 compared to approximately 75 s-1 in controls). These effects of mitochondrial depolarization were not accompanied by a significant change in EPP quantal content or the rate of asynchronous release during 1 Hz stimulation, and were not seen in oligomycin, which blocks mitochondrial ATP synthesis without depolarizing mitochondria. Inhibition of endoplasmic reticular Ca2+ uptake with cyclopiazonic acid also had little effect on stimulation-induced changes in cytosolic [Ca2+] or EPP amplitude. We hypothesize that the high rate of asynchronous release evoked by stimulation during mitochondrial depolarization was produced by the elevation of cytosolic [Ca2+], and contributed to the accelerated depression of phasic release by reducing the availability of releasable vesicles. During mitochondrial depolarization, the post-tetanic potentiation of the EPP observed under control conditions was replaced by a post-tetanic depression with a slow time course of recovery. Thus, mitochondrial Ca2+ uptake is essential for sustaining phasic release, and thus neuromuscular transmission, during and following tetanic stimulation.

Iberiotoxin-induced block of Ca2+-activated K+ channels induces dihydropyridine sensitivity of ACh release from mammalian motor nerve terminals.

The role which Ca(2+)-activated K(+) (K(Ca)) channels play in regulating acetylcholine (ACh) release was examined at mouse motor nerve terminals. In particular, the ability of the antagonist iberiotoxin to recruit normally silent L-type Ca(2+) channels to participate in nerve-evoked release was examined using conventional intracellular electrophysiological techniques. Incubation of cut hemidiaphragm preparations with 10 microM nimodipine, a dihydropyridine L-type Ca(2+) channel antagonist, had no significant effect on quantal content of end-plate potentials. Nevertheless, 1 microM S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K 8644) enhanced quantal content to 134.7 +/- 3.5% of control. Iberiotoxin (150 nM) increased quantal content to 177.5 +/- 9.9% of control, whereas iberiotoxin plus nimodipine increased quantal content to only 145.7 +/- 10.4% of control. Coapplication of 1 microM Bay K 8644 with iberiotoxin did not significantly increase quantal content further than did treatment with iberiotoxin alone. The effects of iberiotoxin and nimodipine alone or in combination on the miniature end-plate potential (MEPP) frequency following KCl-induced depolarization were examined using uncut hemidiaphragm preparations. Nimodipine alone had no effect on MEPP frequency from preparations incubated in physiological saline containing 5 to 20 mM KCl. Moreover, iberiotoxin alone or combined with nimodipine also had no effect on MEPP frequency in physiological salines containing 5 to 15 mM KCl. At 20 mM KCl, however, iberiotoxin significantly increased MEPP frequency to 125.6% of iberiotoxin-free values; combined treatment with nimodipine and iberiotoxin prevented this increase in MEPP frequency. Thus, loss of functional K(Ca) channels unmasks normally silent L-type Ca(2+) channels to participate in ACh release from motor nerve terminals, particularly under conditions of intense nerve terminal depolarization.

Adenosine depresses a Ca(2+)-independent step in transmitter exocytosis at frog motor nerve terminals.

The depressant action of adenosine on acetylcholine release at frog motor nerve terminals was studied by intracellular recording and Ca(2+)-imaging techniques. Adenosine (200 microm) quickly and reversibly decreased the amplitude and quantal content of end-plate potentials (EPPs) with no change in quantal size in a low-Ca(2+), high-Mg(2+) solution, and EPP amplitude in normal Ringer containing d-tubocurarine. Likewise, adenosine (200 microm) reduced miniature EPP (MEPP) frequency, but not amplitude, in a high-K(+) (6 mm) solution. Adenosine (40-200 microm), however, did not affect single or repetitive impulse(s)-induced rises in Ca(2+) in the nerve terminals or its basal level. Adenosine (100-200 microm) reduced the Ca(2+)-independent enhancement of MEPP frequency caused by hypertonicity. EPPs induced by tetanic stimulation (33 Hz) in Ringer with d-tubocurarine initially increased in amplitude within 10 stimuli and then declined to the minimum. Adenosine (200 microm) decreased EPP amplitude in the initial phase of the tetanus, but enhanced it in the middle phase, thus prolonging the decay of EPP amplitude. The total sum of these EPPs, reflecting the readily releasable pool of vesicles and its refilling, however, was not changed. The results suggest that adenosine inhibits a Ca(2+)-independent step of transmitter exocytosis at frog motor nerve terminals.

Regulation of acetylcholine release by muscarinic receptors at the mouse neuromuscular junction depends on the activity of acetylcholinesterase.

Muscarinic acetylcholine receptors (mAChRs) play an important role in regulating the release of acetylcholine (ACh) in various tissues. We used subtype-specific antibodies and a fluorescent-labelled muscarinic toxin to demonstrate that mammalian neuromuscular junction expresses mAChR subtypes M1 to M4, and that localization of all subtypes is highly restricted to the innervated part of the muscle. To elucidate the roles of the mAChR subtypes regulating ACh release, we measured the mean quantal content of endplate potentials in isolated mouse phrenic--hemidiaphragm preparations in which release was reduced by a low Ca2+/high Mg2+ medium. Muscarine decreased evoked ACh release in normal junctions but, depending on the concentration, reduced or increased transmitter release in collagen Q-deficient junctions completely lacking acetylcholinesterase (AChE). Both effects were also seen in normal junctions when AChE was inhibited by various doses of fasciculin-2. Block of mAChRs by atropine had no effect on evoked release at normal junctions, but decreased release at junctions lacking AChE. The muscarine-elicited depression of ACh release in normal junctions was completely abolished by pertussis toxin or methoctramine pretreatment, but was not affected by muscarinic toxin MT-3, thus indicating the involvement of the M2 mAChR. The muscarine-induced increase of ACh release in AChE-deficient junctions was not affected by pertussis toxin, but was completely blocked by MT-7, a specific M1 mAChR antagonist. Our results show that the M1 and M2 mAChRs have opposite presynaptic functions in modulating quantal ACh release, and that regulation of release by the two receptor subtypes depends on the functional state of AChE at the neuromuscular junction.

An electrophysiological study on the effects of Pa-1G (a phospholipase A 2) from the venom of king brown snake, Pseudechis australis, on neuromuscular function

The effects of Pa-1G, a phospholipase A 2 (PLA 2) from the venom of the Australian king brown snake ( Pseudechis australis) were determined on the release of acetylcholine, muscle resting membrane potential and motor nerve terminal action potential at mouse neuromuscular junction. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations revealed that Pa-1G (800 nM) significantly reduced the amplitude of endplate potentials within 10 min exposure. The quantal content of endplate potentials was decreased to 58±6% of control after 30 min exposure to 800 nM Pa-1G. The toxin also caused a partial depolarisation of mouse muscle fibres within 60 min exposure. Extracellular recording of action potentials at motor nerve terminals showed that Pa-1G reduced the waveforms associated with both sodium and potassium conductances. To investigate whether this was a direct or indirect effect of the toxin on these ionic currents, whole cell patch clamp experiments were performed using human neuroblastoma (SK-N-SH) cells and B82 mouse fibroblasts stably transfected with rKv1.2. Patch clamp recording experiments confirmed that potassium currents sensitive to α-dendrotoxin recorded from B82 cells and sodium currents in SK-N-SH cells were not affected by the toxin. Since neither facilitation of acetylcholine release at mouse neuromuscular junction nor depression of potassium currents in B82 cells has been observed, the apparent blockade of potassium currents at mouse motor nerve endings induced by the toxin is unlikely to be due to a selective block of potassium channels.