Miniature Endplate Currents Research Articles

Prion protein potentiates acetylcholine release at the neuromuscular junction

Cellular prion protein (PrP c), the normal isoform of the pathogenic peptide (PrP sc) responsible of the transmissible spongiform encephalopaties (TSEs), is present in many neural tissues, including neuromuscular junctions (NMJ). To analyze if this protein could influence the synaptic transmission, we performed an electrophysiological approach to study the effect of cellular prion protein on a mammalian neuromuscular junction. The loose patch clamp (LPC) technique enables the study of the whole preparation including the pre- and the post-synaptic domains. In a mouse phrenic–diaphragm preparation, nanomolar concentrations of cellular prion protein were able to induce a very striking potentiation of the acetylcholine (ACh) release. The effect was mainly pre-synaptic with an increase of the amplitude of the miniature end-plate currents, probably calcium dependent. Moreover, an apparent facilitation of the synaptic transmission was noted. The results clearly indicate that cellular prion protein may play a key role in the function of the neuromuscular junction.

Activation of ionotropic glutamate receptors on peripheral axons of primary motoneurons mediates transmitter release at the zebrafish NMJ.

The development and function of the vertebrate neuromuscular junction (NMJ) is continually being redefined. Previous studies have indicated that glutamate may play a role in the development or function of the NMJ by associating with presynaptic receptors. We have used larval zebrafish (Danio rerio) to investigate the presence of presynaptic ionotropic glutamate receptors (iGluRs) at the NMJ in vivo. In whole-mount zebrafish larvae, antibody staining directed to NR2A subunits colocalized with specific staining of motoneuron axon tracts. Whole cell voltage-clamp recordings of miniature endplate currents (mEPCs) from axial white muscle were performed during application of iGluR agonists and antagonists. Local perfusion of the NMJ with iGluR agonists resulted in significant increases in the frequency of spontaneous acetylcholine (ACh) release. These increases were blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist d-(-)-2-amino-5-phosphonopentanoic acid (50 microM) and by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (50 microM). Further pharmacological investigation revealed no effect of the kainate receptor-specific antagonist (2S,4R)-4-methylglutamate (10 microM) on kainate-induced rises in the frequency of spontaneous ACh release. However, these were blocked with the AMPA receptor-specific antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (50 microM). Application of glutamate (1 mM) in the presence of the glutamate uptake inhibitor d-threo-beta-benzyloxyaspartate(200 microM) resulted in a significant increase in the frequency of mEPCs. These results suggest the presence of AMPA and NMDA receptors in association with motoneuron axons of larval zebrafish.

Kiss-and-run quantal secretion in frog nerve-muscle synapse.

Electrophysiological and optical methods were used to study exo- and endocytosis of synaptic vesicles underlying secretion of the neurotransmitter from motor nerve terminals in frog sternocutaneous muscle. Increase in extracellular concentration of K+ or sucrose produced similar increase in the frequency of miniature endplate currents recorded by extracellular microelectrode. Fluorescent microscopy revealed bright spots in nerve terminal during stimulation of secretion with high-potassium solutions in the presence of endocytosis marker FM1-43. These spots corresponded to clusters of synaptic vesicles that passed through the cycles of exo- and endocytosis. Subsequent high-potassium stimulation of exocytosis in normal Ringer solution led to disappearance of marker spots, while in hyperosmotic saline the spots were preserved. No spots were seen after stimulation of neurotransmitter secretion with sucrose in the presence of FM1-43. It is concluded that quantal secretion of the neurotransmitter in frog motor nerve endings can be realized via both complete exocytosis of synaptic vesicles with subsequent endocytosis and kiss-and-run mechanism with the formation of a temporary pore.

Dual action of hydrogen peroxide on synaptic transmission at the frog neuromuscular junction.

There is evidence that reactive oxygen species (ROS) are produced and released during neuromuscular activity, but their role in synaptic transmission is not known. Using a two-electrode voltage-clamp technique, at frog neuromuscular junctions, the action H2O2 on end-plate currents (EPC) was studied to determine the targets for this membrane-permeable ROS. In curarized or cut muscles, micromolar concentrations of H2O2 increased the amplitude of EPCs. Higher (> 30 microM) doses inhibited EPCs and prolonged current decay. These effects were presynaptic since H2O2 did not change the amplitude or duration of miniature EPCs (although it reduced the rate of spontaneous release at high concentrations). Quantal analysis and deconvolution methods showed that facilitation of EPCs was due to increased quantal release, while depression was accompanied by temporal dispersion of evoked release. Extracellular recordings revealed prolonged presynaptic Ca2+ entry in the presence of high H2O2. Both low and high H2O2 increased presynaptic potentiation during high-frequency stimulation. Pro-oxidant Fe2+ did not affect facilitation by low doses of H2O2 but augmented the inhibition of EPCs by high H2O2, indicating involvement of hydroxyl radicals. High Mg2+ and the ROS scavenger N-acetylcysteine eliminated both the facilitatory and depressant effects of H2O2. The facilitatory effect of H2O2 was prevented by protein kinase C (PKC) inhibitors and 4beta-phorbol 12-myristate, 13-acetate (PMA), an activator of PKC. PKC inhibitors but not PMA also abolished the depressant effect of H2O2. Our data suggest complex presynaptic actions of H2O2, which could serve as a fast feedback modulator of intense neuromuscular transmission.

Effects of Hypericum extract on the acetylcholine release: a loose patch clamp approach

The St. John’s Wort ( Hypericum perforatum) extract (Hp) represents one of the most useful natural therapeutic agents in the treatment of moderate and mild depression. The antidepressant effects of Hp are different, by a molecular mechanism point of view, when compared to those of other antidepressant drugs and, we think, a further pharmacological characterization is needed. It is suggested that the neurochemical effects of Hp could be bind either to its activity on the uptake of some mediators in the central nervous system or to the inhibition of some enzymatic activity at the receptor level. The present study carried out with the loose patch clamp (LPC) in the mouse neuromuscular junction, indicates a potentiation of the acetylcholine (ACh) action at the mouse neuromuscular junction. The spontaneous release of ACh was unaffected by Hp indicating that neither presynaptic nor postsynaptic function are modified by Hp. Indeed, both the frequency and the amplitude of the miniature end-plate currents (mepcs) were unmodified by Hp. Furthermore, the mepcs decay time ( τ), i.e. the apparent cholinergic channel life time, was significantly increased after Hp treatment. The other parameter affected was the amplitude of the evoked end-plate currents (epcs) which was constantly and in a dose dependent manner increased by Hp. These findings suggest a possible action of Hp on the acetylcholinesterase (AChE) in terms of a reduction of the degradation rate of ACh.

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.

Transmitter secretion in the frog neuromuscular synapse after prolonged exposure to calcium-free solutions.

Experiments on neuromuscular synapses from frog skin/chest muscle preparations in conditions of extracellular recording addressed changes in the spontaneous and evoked transmitter secretion after long-term (1.5-6 h) maintenance of preparations in calcium-free solution containing EGTA. Use of three microelectrodes for recording of single-quantum postsynaptic signals showed that calcium-free solution altered the characteristic topography of transmitter secretion in nerve terminals, with widening and fusion of groups of transmitter release. These changes persisted after preparations were returned to the initial solution. These data suggest that calcium-free solutions lead to disorganization of the active zones of nerve endings, At initially low extracellular Ca ion concentrations (0.15-0.4 mM), disorganization of active zones induced by prolonged maintenance of preparations in calcium-free solutions led to decreases in the mean amplitude of endplate currents (EPC) because of decreases in their quantum composition, increases in the time course of transmitter secretion, and decreases in the frequency of miniature endplate currents. The relationship between quantum composition of EPC and the extracellular Ca ion concentration showed a sharp displacement towards higher concentrations, without significant changes in the slope of the relationship. At high initial Ca concentrations (1.8 mM), long-term exposure to calcium-free solutions led to a less marked decrease in EPC amplitude. It is suggested that the extra- and intracellular Ca ion concentrations support the maintenance of the characteristic morphofunctional organization of the apparatus responsible for transmitter secretion in frog nerve endings. Disorganization of the active zones leads to disruption of elements involved in transmitter secretion and decreases in the efficiency of secretion.

Thyroid hormones regulate the frequency of miniature end-plate currents in pre- and prometamorphic stages of the tadpole tail.

Thyroid hormones (THs), primarily 3,3',5-triiode-(L)-thyronine (T(3)), have been clearly established as natural inducers of apoptosis during metamorphosis of anuran embryos. We decided to use this phenomenon to test the hypothesis that, prior to genomic activation, T(3) has acute actions in the neuromuscular junction (NMJ) of the tail of amphibian embryos. We detected a dramatic increase in the production of miniature end-plate currents (MEPCs) 2-5 min after continuous application of T(3) (250 nM) using focal recordings under voltage clamp. Furthermore, this increase in the spontaneous release of neurotransmitter, evaluated by the MEPC frequency, was maintained for several hours. Reverse-T(3), the "inhibitory" form of THs, prevented this increase in MEPC frequency, suggesting that this is probably a highly specific action of T(3). In addition, the elevation in MEPC frequency induced by T(3) was unchanged in the presence or absence of extracellular calcium. The T(3)-mediated increase in MEPC frequency was blocked by niflumic acid, a nonsteroidal antinflammatory fenamate used to prevent the apoptotic volume decrease observed in many systems. The present study demonstrated that T(3) induces a remarkable nongenomic action in the NMJ of the tadpole tail at pre- and promatamorphic stages.

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.

Simulation analysis of the effects of the simultaneous release of quanta of acetylcholine on the endplate current at the neuromuscular junction

Arrival of an action potential to a nerve terminal at the neuromuscular junction induces the release of a few hundred quanta of acetylcholine (ACh) into the synaptic cleft, resulting in depolarization of the muscle cell which is observed as the endplate current (EPC). The release of each quantum of ACh invokes the miniature endplate current (MEPC), so that an EPC could be generated by summation of the MEPCs both in time during evolution of the EPC and in space for a certain area of the post-synaptic membrane. In this study, a mathematical model for EPC generation is developed as a reaction–diffusion system (RD system) which represents the dynamic behavior of ACh in the chemical transmission process with the simultaneous quantum release of ACh. The RD system for ACh is mathematically expressed by a two-dimensional diffusion equation with nonlinear reaction terms due to the rate processes for acetylcholinesterase (AChE) and ACh receptor (AChR). Numerical solution of the governing equation with the method of lines and the Gear method yields temporal changes in relative concentrations of the open channel form of AChR which is assumed to be equivalent to the EPC. Analysis of the behavior of the RD system with respect to the various distances between the release sites of ACh on the pre-synaptic membrane demonstrates that the amplitude of EPC is quite sensitive to the distances around 0.5 μm, but independent of the values of the diffusion coefficient of ACh in the synaptic cleft.

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.

Inhibition of skeletal muscle nicotinic receptors by the atypical antipsychotic clozapine

We have previously observed that certain atypical antipsychotic drugs reduce the amplitude and duration of miniature end-plate currents (EPCs) at the frog neuromuscular junction (Effects of atypical antipsychotics on vertebrate neuromuscular transmission, Nguyen, Q.-T., Yang, J., Miledi, R. Neuropharmacology 42, 2002, 670–676), therefore suggesting that these drugs act on nicotinic acetylcholine receptors. In this study we examined the effects of the atypical antipsychotic clozapine on nicotinic receptors of frog neuromuscular end-plates or in Xenopus oocytes expressing the α 1β 1γδ mouse skeletal muscle nicotinic receptor. At neuromuscular junctions, postsynaptic currents were reduced by micromolar concentrations of clozapine. This compound also acted presynaptically by increasing the quantal content of EPCs of muscles without noticeably affecting paired-pulse facilitation. In oocytes, clozapine inhibited α 1β 1γδ receptors with an IC 50 of 10 μM and a Hill coefficient of 1. Blockage of α 1β 1γδ receptors by clozapine bears several hallmarks of open-channel blockers, including faster response decays, strong voltage dependence of the block, large rebound currents upon wash, and reduction of peak responses even at saturating concentrations of acetylcholine. However, clozapine increased the EC 50 for acetylcholine and its blocking effect was enhanced by preincubation. These results suggest that clozapine antagonizes muscle nicotinic receptors by blocking open channels, and possibly also by another mechanism which still remains to be investigated.

Effect of High-Frequency Stimulation on the Time Course of End-Plate Currents

We investigated the effects of repetitive high-frequency (10 sec-1) nerve stimulation on the time course of evoked and miniature end-plate currents (EPC and mEPC, respectively) in the frog neuromuscular junction. The data obtained indicate that at a physiological Ca2+ level in the bath medium, 10 sec-1 stimulation results in prolongation of the growth phase of multiquantum EPC without any effect on the mEPC time course. It is concluded that timing of acetylcholine quantum secretion may be affected by high-frequency stimulation.

Differences between Spontaneous and Evoked Monoquantum Signals in the Frog Neuromuscular Junction

In experiments on the frog cutaneous-pectoris muscle, the amplitude-temporal parameters of monoquantum end-plate currents (EPC) and miniature EPC (mEPC) were investigated using extracellular recording. A significant dependence of the risetime of the signals on their amplitude was found after analyzing mEPC; at the same time, such dependence was absent for EPC. Approaches leading to disorganization of the active zones (AZ) of the nerve ending (NE), prolonged action of a Ca-free solution, and denervation resulted in an increased dependence of the risetime of the monoquantum signals on their amplitude; moreover, these dependences were similar for both mEPC and monoquantum EPC. Mathematical simulation showed that the obtained data could be explained by the spatial heterogeneity of the sites of spontaneous and evoked transmitter release within the regions of the AZ. A new hypothesis interpreting spontaneous and evoked transmitter release is proposed.

Pre- and postsynaptic effects of the calcium channel blocker verapamil at neuromuscular junctions.

Experiments on the frog sartorius muscle were used to study the effects of the L-type calcium channel blocker verapamil on endplate currents. Verapamil had no effect on the amplitudes of miniature and multiple-quantum endplate currents, the synchronicity of transmitter secretion, or repeat activity in nerve endings. Verapamil had no effect on the decay of miniature currents, but accelerated that of multiple-quantum currents. This effect was sharply increased after inhibition of cholinesterase activity. In conditions of inhibited cholinesterase activity, verapamil depressed currents during rhythmic stimulation. This depression was more marked in synapses with high quantal compositions and in conditions of membrane depolarization. Thus, the sensitivity of neuromuscular junction calcium channels to verapamil was unrelated to the release of transmitter from the motor nerve ending either at physiological levels of secretion or when secretion was potentiated by potassium channel blockers. At the postsynaptic level, the effect of verapamil was insignificant in relation to cholinoreceptors in the resting and active states, though verapamil could cooperatively enhance the transition of postsynaptic receptors into the desensitized state in conditions of prolonged transmitter action.

The effects of noradrenaline on the amplitude-time characteristics of multiquantum endplate currents and the kinetics of induced secretion of transmitter quanta.

Experiments on frog neuromuscular junctions using a two-electrode membrane potential clamping method were used to study the effects of noradrenaline on the amplitude-time characteristics of multiquantum endplate current (EPC) parameters and the time course of secretion of transmitter quanta during the process of EPC generation. Noradrenaline (10 microM) induced significant increases in EPC amplitude (by 16%), with a decrease in the ratio of the duration of the leading front of the EPC to the duration of the leading front of the miniature endplate current (mEPC). Analysis of the time course of induced secretion, based on sequential subtraction of signals with displacement on the time scale, showed that noradrenaline induced synchronization of the process of secretion of quanta involved in generating multiquantum EPC, resulting in a 25% decrease in parameter P90, which characterizes the extent of synchronization of quantum release. The quantum composition of EPC, measured by dividing the area of induced and spontaneous signals and by analysis of the time course of the secretion of quanta, showed no changes in response to noradrenaline. Thus, in conditions in which responses to single stimuli applied to the motor nerve results in the release of several tens of quanta, noradrenaline can lead to increases in the amplitude of multiquantum EPC by increasing the level of synchronization of secretion of the transmitter quanta forming this signal.

Effects of Noradrenaline on End-Plate Currents and the Kinetics of Transmitter Release under Long-Lasting Repetitive Stimulation

Noradrenaline causes a significant increase in the amplitude of multiquantum end-plate currents (EPC) and also diminishes the EPC rising phase vs the rising phase of the miniature EPC ratio in the frog neuromuscular junction under conditions of low-frequency long-lasting stimulation of the motor nerve. Noradrenaline changes the kinetics of transmitter release due to synchronization of the quantum transmitter secretion. The synchronizing action of noradrenaline can underlie its de-fatiguing effect in the neuromuscular junction.

Limits to the development of fast neuromuscular transmission in zebrafish.

Zebrafish embryos have small and slow miniature end-plate currents (mEPCs), whereas only a few days later larval mEPCs are an order of magnitude larger and faster, being among the fastest of all neuromuscular synapses. To identify the bases for these changes we compared, in embryos and larvae, the properties and distributions of acetylcholine (ACh) receptors (AChRs) and acetylcholinesterase (AChE) as well as the ultrastructure of the developing neuromuscular junctions (NMJs). To mimic synaptic release, patches of muscle membrane were exposed briefly (for 1 ms) to a saturating concentration (10 mM) of ACh. The AChR deactivation kinetics were twice as slow in embryos compared with larvae. In both embryos and larvae, AChRs demonstrated open channel block by millimolar ACh, and this was detected during mEPCs, indicating that a high concentration of ACh is released at immature and mature NMJs. AChR and AChE distributions were compared using the selective fluorescently conjugated labels alpha-bungarotoxin and fasciculin 2, respectively. In larvae, punctate AChR clusters were detected whereas junctional AChE staining was less intense than that found at adult NMJs. Transmission electron microscopy revealed immature nerve endings in embryos that were closely juxtaposed to the surrounding muscle cells, whereas mature larval NMJs had a wider synaptic cleft with a conspicuous basal lamina over a limited region of synaptic contact. Our results indicate that ACh is released at high concentrations at immature NMJs, but its clearance is prolonged and the AChRs are dispersed, resulting in a slow mEPC time course until a mature cleft appears with densely packed faster AChRs and abundant AChE.