Amplitude Of Miniature Endplate Currents Research Articles

Regulation of acetylcholinesterase activity by nitric oxide in rat neuromuscular junction via N‐methyl‐d‐aspartate receptor activation

Acetylcholinesterase (AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine, thereby limiting spillover and duration of action. This study demonstrates the existence of an endogenous mechanism for the regulation of synaptic AChE activity. At the rat extensor digitorum longus neuromuscular junction, activation of N-methyl-d-aspartate (NMDA) receptors by combined application of glutamate and glycine led to enhancement of nitric oxide (NO) production, resulting in partial AChE inhibition. Partial AChE inhibition was measured using increases in miniature endplate current amplitude. AChE inhibition by paraoxon, inactivation of NO synthase by N(x)-nitro-L-arginine methyl ester, and NMDA receptor blockade by DL-2-amino-5-phosphopentanoic acid prevented the increase in miniature endplate current amplitude caused by amino acids. High-frequency (10 Hz) motor nerve stimulation in a glycine-containing bathing solution also resulted in an increase in the amplitude of miniature endplate currents recorded during the interstimulus intervals. Pretreatment with an NO synthase inhibitor and NMDA receptor blockade fully eliminated this effect. This suggests that endogenous glutamate, released into the synaptic cleft as a co-mediator of acetylcholine, is capable of triggering the NMDA receptor/NO synthase-mediated pathway that modulates synaptic AChE activity. Therefore, in addition to well-established modes of synaptic plasticity (e.g. changes in the effectiveness of neurotransmitter release and/or the sensitivity of the postsynaptic membrane), another mechanism exists based on the prompt regulation of AChE activity.

Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome.

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with 'limb-girdle myasthenia'

The properties of neuromuscular junctions (NMJs) were studied in motor-point biopsy samples from eight patients with congenital myasthenic syndromes affecting primarily proximal limb muscles ['limb-girdle myasthenia' (LGM)]. All had moderate to severe weakness of the proximal muscles, without short-term clinical fatigability but with marked variation in strength over periods of weeks or months, with little or no facial weakness or ptosis and no ophthalmoplegia. Most had a characteristic gait and stance. All patients showed decrement of the compound muscle action potential (CMAP) on repetitive stimulation at 3 Hz, and increased jitter and blocking was detected by SFEMG, confirming the presence of impaired neuromuscular transmission. None of the patients had serum antibodies against acetylcholine receptors (AChRs). Two of the patients had similarly affected siblings. Intracellular recording from isolated nerve-muscle preparations revealed that the quantal content (the number of ACh quanta released per nerve impulse) was only approximately 50% of that in controls. However, the quantal size (amplitude of miniature end-plate currents) and the kinetic properties of synaptic potentials and currents were similar to control values. The area of synaptic contact and extent of post-synaptic folding were approximately 50% of control values. Thus, the quantal content per unit area of synaptic contact was normal. The number of AChRs per NMJ was also reduced to approximately 50% of normal, so the local AChR density was normal. Immunolabelling studies revealed qualitatively normal distributions and abundance of each of 14 proteins normally concentrated at the NMJ, including components of the basal lamina, post-synaptic membrane and post-synaptic cytoskeleton. DNA analysis failed to detect mutations in the genes encoding any of the following proteins: AChR subunits, rapsyn, ColQ, ChAT or muscle-specific kinase. Response of these patients to treatment was varied: few showed long-term improvement with pyridostigmine and some even deteriorated with treatments, while others had intolerable side-effects. Several patients showed improvement with 3,4-diaminopyridine, but this was generally only transient. Ephedrine was helpful in half of the patients. We conclude that impaired neuromuscular transmission in these LGM patients results from structural abnormalities of the NMJ, including reduced size and post-synaptic folding, rather from any abnormality in the immediate events of neuromuscular transmission.

Dependence of miniature endplate current on kinetic parameters of acetylcholine receptors activation: a model study.

Mathematical modeling was applied to study the dependence of miniature endplate current (MEPC) amplitude and temporal parameters on the values of the rate constants of acetylcholine binding to receptors (k+) when cholinesterase was either active or inactive. The simulation was performed under two different sets of parameters describing acetylcholine receptor activation--one with high and another with low probability (Pohigh and Polow) of receptor transition into the open state after double ligand binding. The dependence of model MEPC amplitudes, rise times, and decay times on k+ differs for set Polow and set Pohigh. The main outcome is that for set Pohigh, the rise time is significantly longer at low values of k+ because of the prolongation of ACh diffusion time to the receptor. For the set Polow, the rise time is shorter at low values of k+, which can be explained by the small probability of AChR forward isomerization after ACh binding and faster MEPC's peak formation.

Reduced neuromuscular quantal content with normal synaptic release time course and depression in canine motor neuron disease.

Hereditary canine spinal muscular atrophy is an autosomal dominant version of motor neuron disease in which motor units exhibit extensive dysfunction before motor terminal or axonal degeneration appear. We showed in a previous paper that motor endplate currents (EPCs) are reduced and that failures of nerve-evoked EPCs appear in the homozygote medial gastrocnemius (MG) muscle in which failing motor units are also found, suggesting a presynaptic deficit of ACh release. To examine this further, we performed a detailed analysis of synaptic release properties in the MG muscle of homozygotes and compared the results with data from genetically normal control animals. We found that the amplitude of miniature EPCs (mEPC) did not differ between homozygote and normal synapses, indicating that quantal content is reduced at homozygote motor terminals. Consistent with this, deconvolution analysis showed that the maximum release rates at homozygote motor terminals were significantly reduced relative to normal. This analysis also demonstrated that the time course of quantal release at homozygote synapses did not differ from normal. The extent of quantal release depression during high-frequency activation in homozygotes did not differ from normal despite the significant reduction of quantal content and maximum release rate. Surprisingly, the absolute amount of posttetanic potentiation was not decreased at homozygotes motor terminals despite the differences in quantal content. We conclude that failure of homozygote motor unit force during repetitive activity is due to a unique combination of low quantal content and normal release depression and suggest that the primary deficit in homozygote motor terminals is a reduced supply of readily releasable quanta.

Active Calcium Accumulation Underlies Severe Weakness in a Panel of Mice with Slow-Channel Syndrome

Mutations affecting the gating and channel properties of ionotropic neurotransmitter receptors in some hereditary epilepsies, in familial hyperekplexia, and the slow-channel congenital myasthenic syndrome (SCCMS) may perturb the kinetics of synaptic currents, leading to significant clinical consequences. Although at least 12 acetylcholine receptor (AChR) mutations have been identified in the SCCMS, the altered channel properties critical for disease pathogenesis in the SCCMS have not been identified. To approach this question, we investigated the effect of different AChR subunit mutations on muscle weakness and the function and viability of neuromuscular synapses in transgenic mice. Targeted expression of distinct mutant AChR subunits in skeletal muscle prolonged the decay phases of the miniature endplate currents (MEPCs) over a broad range. In addition, both muscle strength and the amplitude of MEPCs were lower in transgenic lines with greater MEPC duration. SCCMS is associated with calcium overload of the neuromuscular junctional sarcoplasm. We found that the extent of calcium overload of motor endplates in the panel of transgenic mice was influenced by the relative permeability of the mutant AChRs to calcium, on the duration of MEPCs, and on neuromuscular activity. Finally, severe degenerative changes at the motor endplate (endplate myopathy) were apparent by electron microscopy in transgenic lines that displayed the greatest activity-dependent calcium overload. These studies demonstrate the importance of control of the kinetics of AChR channel gating for the function and viability of the neuromuscular junction.

Active calcium accumulation underlies severe weakness in a panel of mice with slow-channel syndrome.

Mutations affecting the gating and channel properties of ionotropic neurotransmitter receptors in some hereditary epilepsies, in familial hyperekplexia, and the slow-channel congenital myasthenic syndrome (SCCMS) may perturb the kinetics of synaptic currents, leading to significant clinical consequences. Although at least 12 acetylcholine receptor (AChR) mutations have been identified in the SCCMS, the altered channel properties critical for disease pathogenesis in the SCCMS have not been identified. To approach this question, we investigated the effect of different AChR subunit mutations on muscle weakness and the function and viability of neuromuscular synapses in transgenic mice. Targeted expression of distinct mutant AChR subunits in skeletal muscle prolonged the decay phases of the miniature endplate currents (MEPCs) over a broad range. In addition, both muscle strength and the amplitude of MEPCs were lower in transgenic lines with greater MEPC duration. SCCMS is associated with calcium overload of the neuromuscular junctional sarcoplasm. We found that the extent of calcium overload of motor endplates in the panel of transgenic mice was influenced by the relative permeability of the mutant AChRs to calcium, on the duration of MEPCs, and on neuromuscular activity. Finally, severe degenerative changes at the motor endplate (endplate myopathy) were apparent by electron microscopy in transgenic lines that displayed the greatest activity-dependent calcium overload. These studies demonstrate the importance of control of the kinetics of AChR channel gating for the function and viability of the neuromuscular junction.

Effects of atypical antipsychotics on vertebrate neuromuscular transmission

A study was made of the effects of the atypical antipsychotics clozapine, olanzapine, sulpiride and risperidone on nicotinic synaptic transmission at the frog neuromuscular junction. At concentrations higher than 10 μM, these atypical antipsychotics partially reduced the amplitude of miniature end-plate currents (mEPCs) in a dose-dependent and reversible manner. Atypical antipsychotics were, however, less effective than typical neuroleptics of the phenothiazine family at inhibiting mEPCs. In addition to decreasing mEPC amplitude, the atypical antipsychotics reduced the half-decay time of mEPCs. In the case of clozapine, the reduction in mEPC amplitude and duration was not markedly voltage-dependent. Beside their post-synaptic effects, all atypical neuroleptics, except sulpiride, increased the frequency of mEPCs in a concentration-dependent manner, with the strongest effect seen with clozapine. Altogether, these results raise the possibility that atypical neuroleptics could derive some of their therapeutic effects not only from their well-known inhibitory action on dopaminergic receptors, but also from their pre- and post-synaptic modulation of nicotinic neurotransmission.

Desensitization of mutant acetylcholine receptors in transgenic mice reduces the amplitude of neuromuscular synaptic currents.

While the slow onset of desensitization of nicotinic acetylcholine receptors (AChRs), relative to the rate of acetylcholine removal, excludes this kinetic state from shaping synaptic responses in normal neuromuscular transmission, its role in neuromuscular disorders has not been examined. The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder caused by point mutations in the AChR subunit-encoding genes leading to kinetically abnormal (slow) channels, reduced miniature endplate current amplitudes (MEPCs), and degeneration of the postsynaptic membrane. Because of this complicated picture of kinetic and structural change in the neuromuscular junction, it is difficult to assess the importance of the multiple factors that may be responsible for the reduced endplate current amplitudes, and ultimately the clinical syndrome. In order to address this we have used a transgenic mouse model for the SCCMS that has slow AChR ion channels and reduced endplate responsiveness in the absence of any of the degenerative changes. We found that the reduction in MEPC amplitudes in these mice could not be explained by either reduced AChR number or by reduced AChR channel conductance. Rather, we found that the mutant AChRs in situ manifested an activity-dependent reduction in sensitivity that caused diminished MEPC and endplate current amplitude with nerve stimulation. This observation demonstrates that the basis for the reduction in MEPC amplitudes in the SCCMS may be multifactorial. Moreover, these findings demonstrate that, under conditions that alter their rate of desensitization, the kinetic properties of nicotinic AChRs can control the strength of synaptic responses.

Transmitter release differs at snake twitch and tonic endplates during potassium-induced nerve terminal depolarization.

Twitch and tonic muscle fibers of snake skeletal muscle differ in their synpatic as well as mechanical properties. These experiments were aimed at detemining the basis of the difference in vesicular release properties of nerve terminals at twitch and tonic endplates. Miniature endplate currents (MEPCs) were recorded from voltage-clamped garter snake muscle fibers depolarized by high K+ in either a control Ca2+ or high-Ca2+ solution. MEPC frequency increased at twitch and tonic endplates and remained elevated for 8 h during depolarization in control Ca2+. At twitch endplates depolarized in the presence of high Ca2+, an increase in MEPC frequency was followed by a progressive decline. In contrast, MEPC frequency remained elevated in high Ca2+ at tonic endplates. The observed decrease in MEPC frequency at depolarized twitch endplates in high Ca2+ was not a function of the level of depolarization or initial MEPC frequency, nor was it due to a reduction in MEPC amplitude and loss of MEPCs in baseline noise. An optical assay of presynaptic function in which the activity-dependent dye FM1-43 was used confirmed that quantal releases differs at twitch and tonic endplates. Most twitch nerve terminals were labeled by FM1-43 during prolonged depolarization with control Ca2+ or after brief depolarization with high Ca2+. In contrast, the number of twitch nerve terminals and the degree to which they were stained was greatly reduced after prolonged exposure to high K+ and high Ca2+, whereas depolarized tonic endplates were well stained by FM1-43 during brief and prolonged exposure to high Ca2+. FM1-43 staining also revealed variable levels of quantal release between individual boutons at twitch endplates after prolonged depolarization in high-Ca2+ solution. The observed reduction in presynaptic function at twitch nerve terminals after prolonged depolarization in high-Ca2+ solution was reversible and therefore not due to irreversible damage to terminal boutons. MEPC frequency increased at both twitch and tonic endplates when either Sr2+ or Ba2+ was substituted for high Ca2+ during K(+)-induced depolarization. Over time, in Sr2+ or Ba2+ solutions, MEPC frequency remained elevated at tonic endplates but declined at twitch endplates with a time course similar to that observed in high Ca2+. MEPC amplitudes at both endplates remained constant. We conclude that the regulation of quantal release differs in nerve terminals innervating twitch and tonic endplates and postulate that differential intraterminal accumulation of Ca2+ may underlie the observed difference in presynaptic function.

Activation of the protein phosphatase calcineurin during carbachol exposure decreases the extent of recovery from end-plate desensitization.

1. Our previous studies demonstrated that protein kinase C (PKC) activity is required for acetylcholine (ACh) sensitivity to recover fully at snake twitch fiber end plates after prolonged exposure to carbachol. In the present studies, we have investigated whether protein phosphatase(s), activated during carbachol exposure, dephosphorylated critical membrane proteins, which required rephosphorylation by PKC to maintain end-plate sensitivity. End-plate sensitivity was assessed from measurements of miniature end-plate currents (MEPCs) and carbachol-activated currents (EPCCARBS). Conductance of ACh-activated channels was determined from patch-clamp recordings of single-channel currents. 2. Pretreatment of snake muscle preparations with the protein kinase inhibitor staurosporine (0.5 microM), followed by a 10-min exposure to 540 microM carbachol, reduced mean MEPC amplitudes to values 30-40% less than those recorded before carbachol exposure. Conversely, at control end plates exposed to carbachol, the mean MEPC amplitude was reduced by only approximately 5% compared with precarbachol values. This staurosporine-induced decrease in ACh sensitivity could be prevented by pretreatment with the protein phosphatase 2B (calcineurin) inhibitor deltamethrin (0.5 microM), whereas okadaic acid (5 microM) and calyculin A (0.5 microM), inhibitors of protein phosphatases 1 and 2A, had no effect. 3. After a 10-min exposure to 540 microM carbachol, EPCCARB amplitudes (produced by local superfusion with 20 microM carbachol) were significantly smaller at staurosporine-treated end plates than at control end plates. In contrast, the EPCCARB amplitude recorded from end plates pretreated with both deltamethrin and staurosporine was not significantly different from that recorded at control end plates. 4. Substitution of 10 mM Mn2+ for external Ca2+ during the exposure to 540 microM carbachol prevented the decrease in MEPC amplitude recovery at staurosporine-treated end plates. These results suggested that the alteration in sensitivity at staurosporine-treated end plates was calcium dependent. 5. At control end plates, a single population of ACh-activated channels (45-50 pS) is observed both before and after a 10-min exposure to 540 microM carbachol. Conversely, at staurosporine-treated end plates, after exposure to carbachol, a second population of small-conductance (25-30 pS) ACh-activated channels is present in addition to the predominant 45- to 50-pS ACh-activated channels. In preparations pretreated with both deltamethrin and staurosporine, after carbachol exposure, there was a significant decrease in the frequency of small-conductance ACh-activated channels. Deltamethrin treatment alone produced no small-conductance channels before or after a 10-min exposure to 540 microM carbachol. Also, no small-conductance ACh-gated channels were recorded at PKC-inhibited end plates after carbachol exposure either with pretreatment with 10 microM cyclosporin A (another inhibitor of calcineurin) or with the substitution of 10 mM Mn2+ for Ca2+ during the 10-min agonist exposure. 6. We propose that during prolonged exposure to the nicotinic agonist carbachol, calcium influx through ACh-gated channels elevates the level of ionized calcium at the inner surface of the post-junctional membrane and that this local rise in intracellular calcium activates the calcium-dependent phosphatase calcineurin. Dephosphorylation of some key membrane protein by calcineurin leads to a decrease in the extent of recovery from desensitization. Under normal conditions, this process is effectively reversed by PKC activity and end-plate sensitivity recovers fully. However, when PKC is inhibited, the extent of recovery of end-plate sensitivity is decreased, and associated with this decrease is the presence of small-conductance ACh-activated channels not normally recorded at snake twitch fiber end plates.

A transgenic mouse model of the slow-channel syndrome

To investigate the effect of acetylcholine receptor (AChR) mutations on neuromuscular transmission and to develop a model for the human neuromuscular disease, the slow-channel syndrome, we generated transgenic mice with abnormal AChRs using a delta subunit with a mutation in the ion channel domain. In three transgenic lines, nerve-evoked end-plate currents and spontaneous miniature end-plate currents (MEPCs) had prolonged decay phases and MEPC amplitudes were reduced by 33%. Single nerve stimuli elicited repetitive compound muscle action potentials in vivo. Transgenic mice were abnormally sensitive to the neuromuscular blocker, curare. These observations demonstrate that we can predictably alter AChR function, synaptic responses, and muscle fiber excitation in vivo by overexpressing subunits containing well-defined mutations. Furthermore these data support the hypothesis that the electrophysiological findings in the neuromuscular disorder, the slow-channel syndrome, are due to mutant AChRs.

A transgenic mouse model of the slow‐channel syndrome

To investigate the effect of acetylcholine receptor (AChR) mutations on neuromuscular transmission and to develop a model for the human neuromuscular disease, the slow-channel syndrome, we generated transgenic mice with abnormal AChRs using a δ subunit with a mutation in the ion channel domain. In three transgenic lines, nerve-evoked end-plate currents and spontaneous miniature end-plate currents (MEPCs) had prolonged decay phases and MEPC amplitudes were reduced by 33%. Single nerve stimuli elicited repetitive compound muscle action potentials in vivo. Transgenic mice were abnormally sensitive to the neuromuscular blocker, curare. These observations demonstrate that we can predictably alter AChR function, synaptic responses, and muscle fiber excitation in vivo by overexpressing subunits containing well-defined mutations. Furthermore, these data support the hypothesis that the electrophysiological findings in the neuromuscular disorder, the slow-channel syndrome, are due to mutant AChRs. © 1996 John Wiley & Sons, Inc.

Some Effects of d-Tubocurarine Alone and Combined with Halothane or Isoflurane on Neuromuscular Transmission

The mechanisms contributing to the neuromuscular block produced by nondepolarizing muscle relaxants and inhaled anesthetics include:1) receptor blockade, 2) open or closed ion channel block, 3) decreased transmitter release, and 4) receptor desensitization. In this study we investigated the contributions of receptor and ion channel block. We used the two microelectrode voltage clamp to evaluate miniature end-plate currents (MEPCs) for amplitude and time constant of decay (tau) before and after the application of 1) d-Tubocurarine (DTC) (10-7-10-6 M) alone, and then 2) the same concentration of DTC plus 0.5% or 1.0% halothane or isoflurane delivered by passing compressed air through a flow and temperature compensated vaporizer. The electrodes were maintained in the same cell for the entire experiment. DTC alone decreased MEPC amplitude to 91.3% +/- 4.5% and 65.1% +/- 5.6% of control at 10-7 and 10-6 M, respectively. MEPC amplitude with 10-6 M DTC decreased further to 52.4% +/- 7.6% and 37.4% +/- 7.0% of control after the addition of 0.5% and 1.0% halothane, respectively. After the application of DTC 10-7 M tau was 94.7% +/- 3.1% of control and decreased to 73.7% +/- 7.1% of control with 10-6 M DTC. After the application of DTC 10 (-6) M the addition of 0.5% and 1% halothane decreased tau to 52.4% +/- 7.6% and 30.0% +/- 5.9% of control. Isoflurane produced similar changes. This study provides evidence that at least some of the augmentation of nondepolarizing relaxants by inhaled anesthetics can be explained by the additive effect of nondepolarizing muscle relaxants and inhaled anesthetics on MEPC amplitude and tau. (Anesth Analg 1995;81:763-7)

Some effects of d-tubocurarine alone and combined with halothane or isoflurane on neuromuscular transmission.

The mechanisms contributing to the neuromuscular block produced by nondepolarizing muscle relaxants and inhaled anesthetics include: 1) receptor blockade, 2) open or closed ion channel block, 3) decreased transmitter release, and 4) receptor desensitization. In this study we investigated the contributions of receptor and ion channel block. We used the two microelectrode voltage clamp to evaluate miniature end-plate currents (MEPCs) for amplitude and time constant of decay (tau) before and after the application of 1) d-Tubocurarine (DTC) (10(-7)-10(-6) M) alone, and then 2) the same concentration of DTC plus 0.5% or 1.0% halothane or isoflurane delivered by passing compressed air through a flow and temperature compensated vaporizer. The electrodes were maintained in the same cell for the entire experiment. DTC alone decreased MEPC amplitude to 91.3% +/- 4.5% and 65.1% +/- 5.6% of control at 10(-7) and 10(-6) M, respectively. MEPC amplitude with 10(-6) M DTC decreased further to 52.4% +/- 7.6% and 37.4% +/- 7.0% of control after the addition of 0.5% and 1.0% halothane, respectively. After the application of DTC 10(-7) M tau was 94.7% +/- 3.1% of control and decreased to 73.7% +/- 7.1% of control with 10(-6) M DTC. After the application of DTC 10(-6) M the addition of 0.5% and 1% halothane decreased tau to 52.4% +/- 7.6% and 30.0% +/- 5.9% of control. Isoflurane produced similar changes. This study provides evidence that at least some of the augmentation of nondepolarizing relaxants by inhaled anesthetics can be explained by the additive effect of nondepolarizing muscle relaxants and inhaled anesthetics on MEPC amplitude and tau.

Effect of veratridine on miniature endplate current amplitudes at the rat neuromuscular junction and acetylcholine uptake by Torpedo synaptic vesicles

Veratridine produces a marked elevation in spontaneous quantal release from nerve endings through its ability to enhance sodium-channel activity, leading to sustained membrane depolarization. In the course of an electrophysiological investigation into the effects of vesamicol, an inhibitor of the synaptic vesicle acetylcholine transporter, on veratridine-induced acetylcholine release from rat motor nerve terminals we observed that veratridine itself has an effect on miniature endplate current amplitude distributions suggestive of an effect of the compound on the filling of cholinergic synaptic vesicles with acetylcholine. This effect of veratridine is release-dependent, being inhibited by either removal of extracellular calcium ions or by the addition of the sodium channel blocking toxin, tetrodotoxin. Biochemical studies using synaptic vesicles isolated from Torpedo electroplaque confirmed the ability of veratridine to directly inhibit the vesicular transport of acetylcholine. This appears to be a consequence of its ability to dissipate the trans-vesicular membrane proton gradient, which normally drives the active transport of acetylcholine into synaptic vesicles. We discuss how such an action of veratridine could lead to the observed release-dependent effects of the compound on electrophysiologically monitored spontaneous quantal acetylcholine release. The action of veratridine on cholinergic synaptic vesicles could be of considerable import when using this agent to elicit neurotransmitter release from either peripheral or central nerve endings.

Quantal transmitter release at snake twitch and tonic muscle fibres during prolonged nerve terminal depolarization.

1. Miniature endplate currents (MEPCs) were recorded in vitro from voltage-clamped twitch and tonic muscle fibres in costocutaneous muscles of the garter snake, Thamnophis. Recordings were made from fibres in a control sodium-containing solution and then during exposure to an isotonic potassium solution containing either 1.0 mM calcium and 4.2 mM magnesium or 3.6 mM calcium. The experiments were done at two levels of external calcium in order to demonstrate that the change in MEPC frequency was calcium dependent. During the initial exposure to the isotonic potassium solutions, the MEPC frequency was increased manyfold at both twitch and tonic fibres, but it declined progressively with continued exposure. MEPCs were recorded from both fibre types throughout a 20 h exposure to the isotonic potassium solution with 1 mM calcium, but no MEPCs were recorded at most twitch endplates after approximately 6 h in the isotonic potassium solution containing 3.6 mM calcium. In contrast, MEPCs were still present at tonic fibre endplates after 20 h in the isotonic potassium solution containing 3.6 mM calcium. 2. After 30 min in the isotonic potassium solution with 1 mM calcium, the MEPC amplitude recorded from both fibre types was approximately twice that in the control sodium-containing solution. At tonic endplates, the MEPC amplitude was also twofold greater in the isotonic potassium solution with 3.6 mM calcium than in sodium-containing solution. In contrast, after 30 min in the isotonic potassium solution containing 3.6 mM calcium, the MEPC amplitude at twitch endplates was similar to that in control solution. 3. In both fibre types, MEPC amplitude decreased progressively with continued exposure to the isotonic potassium solutions. The progressive decrease in MEPC amplitude was not due to a gradual decrease in postsynaptic sensitivity to acetylcholine. 4. The effects of high potassium were reversible as MEPCs were recorded at twitch fibre endplates in preparations which were returned to the control sodium-containing solution after a 20 h exposure to the isotonic potassium solution containing 3.6 mM calcium. 5. Ultrastructural examination showed that after a 6 h exposure to the isotonic potassium solutions most nerve terminals innervating twitch fibre endplates were devoid of synaptic vesicles whereas at the same time many synaptic vesicles were present in nerve terminals innervating tonic fibre endplates. Surprisingly, numerous synaptic vesicles were present in nerve terminals innervating either fibre type in muscle preparations exposed to the isotonic potassium solutions for 20 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Further evidence for the dynamic formation of transmitter quanta at the neuromuscular junction.

Fatt and Katz (Nature 166:597-598, 1950; J Physiol 117:109-128, 1952) attributed miniature endplate potentials (MEPPs) to the action of a standard quantity of transmitter, the quantum (Del Castillo and Katz, J Physiol 124:560-573, 1954). Quantal packets of transmitter were proposed to be preformed (Del Castillo and Katz, In CNRS Paris (Ed): "Microphysiologie comparée des éléments excitables" 67:245-258, 1957) and stored in large numbers in the motor nerve terminal. Statistical analyses of intervals between MEPPs and numbers of quanta composing small endplate potentials indicated that quantal release was a random process and that release sites functioned independently of each other. With the discovery of synaptic vesicles it was proposed that each contained one quantum of transmitter. The quantal-vesicular hypothesis (Del Castillo and Katz, as cited above) fails, however, to explain amplitude distributions of MEPPs that are skewed and/or that show multiple peaks (Kriebel et al., Brain Res Review 15:167-178, 1990). The drop formation process (Shaw, "The Dripping Faucet as a Model Chaotic System," Santa Cruz, CA: Aerial Press, Inc., 1984) was shown to generate amplitude classes of drops that were similar to classes of MEPPs which suggested that rapid changes in quantal size and ratios of skew- to bell-MEPPs could be explained with a simple dynamic process which determines quantal size at the moment of release (Kriebel et al., as cited above, 1990). Further similarities between miniature endplate currents (MEPCs) and the formation of drops are reported here. We found that rapid changes in MEPC amplitudes and time courses, which accompany an increase in frequency, mimic changes in drop sizes that accompany increases in flow rate. MEPC intervals have a minimum and their distributions are comparable to those of drop intervals. During an increased rate of transmitter release, MEPP amplitudes and intervals were positively correlated. The results suggest that spontaneously released transmitter "packets" are formed at the moment of release and that transmitter supply to the process that forms packets is continuous.

Effects of "non-quantal" acetylcholine on miniature endplate currents in mice

The possibility of postsynaptic potentiation (PSP) and desensitization developing due to nonquantal acetylcholine (ACh) secretion was investigated in mouse diaphragm with reference to time-amplitude relationships of miniature endplate currents (MEPC). The H effect (which characterizes nonquantal secretion (NS) of ACh) fell to zero over 3 h under the action of armine-induced inhibition of acetylcholinesterase (AChE) at a temperature of 20°C. A decline in the decay time constant (τ) of MEPC unaccompanied by observable alteration in MEPC amplitude occurred at the same time. This accelerated decay of MEPC was not observed in the absence of NS (the early stages of denervation). Start of NS did not show any effect on maximum retardation of MEPC decay due to AChE inhibition, indicating that no PSP sets in under the effects of non-quantal secretion. The effect of decline in τ accelerated with a rise in temperature; it could be reproduced with neostigmine replacing armine, while τ remained unchanged in the time spells investigated with AChE in its active state. Non-quantal ACh is not thought to produce substantial retardation of MEPC decay, although it does bring about desensitization, signs of which may be partially masked owing to concurrent onset of PSP.

Time course of neostigmine action on the endplate response

Miniature endplate currents (MEPCs) were focally recorded in frog sartorius muscle. In the presence of 3 μM neostigmine, the amplitude of MEPCs increased, but later, after 45–60 min of treatment, a decrease of the amplitude towards the initial levels appeared. The pronounced lengthening of the falling phase of MEPCs, which accompanied the increased amplitude, suggests that at the beginning the effect is mainly ascribed to cholinesterase inhibition. The final decreased amplitude of MEPC showed a shortening of the time course instead. This suggests that also a direct effect on endplate receptors is involved.