Miniature currents of the endplates of the muscle fibers of the diaphragm of the rat after inhibition of acetylcholinesterase with galanthamine

Miniature end-plate currents (MEPC) in rat diaphragm were studied with voltage-clamp technique when synaptic acetylcholinesterase (AChE) was inhibited with different concentrations of galanthamine. The MEPC amplitude and time course were increased progressively with galanthamine concentrations in the range of 3.16 X 10(-8) - 10(-6) g/ml. The decay of MEPC was always exponential. The input resistance of muscle fibres increased. Galanthamine (10(-5) g/ml) produced a curare-like action: the amplitude and duration of MEPC were less as compared with those at galanthamine concentration 10(-6) g/ml, the decay of MEPC became biphasic. During washing out of the drug, the duration of MEPC began to increase and then to diminish, returning to the initial value 3 hours later. The decay of MEPC became exponential. A positive correlation was found between half-decay time and amplitude of MEPC both in the presence and in the absence of anticholinesterase. It is supposed that the functional role of synaptic AChE in limiting the postsynaptic effect of acetylcholine is not so significant as it is usually considered, therefore it is possible to use the parameters of MEPC for the estimation of functional AChE activity.     

Effect of magnesium ions on the functional state of postsynaptic membrane by modulation of the level of non-quantal secretion of the mediator]

The role of non-quantal secretion (NS) of acetylcholine (ACh) in shortening of miniature end-plate currents (MEPC) after initial prolongation of MEPC due to acetylcholinesterase (AChE) inhibition was studied in the mouse diaphragm muscle under the voltage clamp conditions. Elevation of NS level by removal of magnesium ions from solution accelerated the shortening effect. After elimination of NS due to an increase of magnesium concentration up to 3 mmol/l the shortening of MEPC was absent. It was suggested that after AChE inhibition the magnesium ions in physiological range of concentrations can modulate the desensitization development on postsynaptic membranes by NS level regulation.     

Postsynaptic potentiation of miniature currents of muscle fiber endplates in the rat diaphragm. The effect of an acetylcholinesterase inhibitor, temperature and curare

Miniature end-plate currents (MEPC) were recorded in voltage-clamped end-plates of the rat diaphragm. A positive correlation between the amplitude and half-decay time of individual MEPC was found in control, after acetylcholinesterase (AChE) inhibition the correlation increased: correlation coefficients--0.29 and 0.49, respectively (28 degrees C). Addition of curare after the AChE inhibition caused a decrease in the amplitude and duration of MEPC, but had no effect on the correlation between these parameters. Cooling down to 18 degrees C led to the essential reduction of this correlation in control as well as after the AChE inhibition. Effect of the prolongation of the half-decay time due to the AChE inhibition was more significant at 28 degrees C than at 18 degrees C. The Q10 value for duration of the rising MEPC phase (about 2) was less than that for the half-decay time (about 3). The role of factors determining the MEPC duration is discussed. The results are explained by the postsynaptic potentiation in terms of the cooperative action of agonists on cholinoreceptors and of peculiarities of the acetylcholine diffusion from the synaptic cleft.     

Features of the postsynaptic potentials and ion currents in synapses of fast and slow rat muscle fibers

The time course of spontaneous miniature end-plate potentials (MEPP) and currents (MEPC) was compared in fast and slow muscle fibres using extracellular focal recording or voltage-clamp technique. The mean values of the rise and decline half-time of MEPP and MEPC in slow fibres was 1.3-1.4 times as much as those in fast ones. A smaller difference was observed after AChE inhibition. Decay of the end-plate currents remained generally exponential and its rate depended on the membrane potential level. Distribution of different muscle fibres according to the time course of MEPP and MEPC in fast and slow muscles correlates with the content of the different fibre types identified on other properties. The possible factors controlling the time course of postsynaptic responses (AChE activity, the length of diffusional pathways, differences in the membrane passive electrical properties) and their significance in synapses of different types are discussed.     

Effect of the quantal composition, membrane potential and cholinoreceptor density on the temporal flow of the end plate current in the rat under conditions of acetylcholinesterase inhibition]

The factors determining the decay of multiquantal end plate currents (EPC) were studied in the diaphragm muscle of rat by the comparison of EPC and miniature EPC (MEPC) amplitude--temporal characteristics. The decay of EPC (quantal content 25-100) was 1.2 times slower than the decay of MEPC when AChE was active. The AChE inhibition by armine or neostigmine made this difference 10-100 times higher. In most synapses the decay of multiquantal EPC can be approximated by a sum of two or three exponents. It depended on the quantal content and 3-exponential EPC could be transformed in 2-exponential and later to monoexponential ones if increasing concentration of magnesium ions. A slow component of EPCs (but not of MEPC) decay was highly sensitive to concentration of magnesium ions and had 3 times higher dependence of the membrane potential value than that one of MEPC. The irreversible blocking of receptors by alpha-bungarotoxin (alpha-BuTX) accelerated the decay of MEPC but the decay of multiquantal EPC changed in two phases: it was prolonged at the beginning of alpha-BuTX action followed by its acceleration, but never the time of the decay of EPC had achieved the apparent open time of ACh-activated ionic channels. It is suggested that during the multiquantal EPC generation not only the synchronization of opening but the kinetic of ACh-activated channels is changed, probably by blocking of this channels by high concentrations of endogenous ACh.     

Effects of "non-quantal" acetylcholine on the sensitivity of the postsynaptic membrane: action of ouabain and imitation of these effects by exogenous acetylcholine]

Development of postsynaptic potentiation (PSP) and desensitization (DS) caused by "non-quantal" acetylcholine after acetylcholinesterase inhibition was studied by means of ouabain, an agent known to modulate (initially increase and then decrease) the level of non-quantal secretion of ACh. Ouabain had no effect on the MEPC parameters when AChE was active. After AChE inhibition ouabain initially increased the decay time constant of MEPC (tau), i.e. caused postsynaptic potentiation (PSP). This effect of ouabain grew with time between inhibition of AChE and application of ouabain. The PSP stage was followed by shortening of MEPCs decay, due to the development of desensitization (DS), and that process was more pronounced than in control. Applied before AChE inhibition, ouabain had no effect on tau. Thus neither PSP nor DS developed under those conditions. Exogenous ACh (20 nmol/l) applied simultaneously with inhibitor of AChE partially prevented the shortening of MEPCs decay, but decreased the amplitude of MEPC. Applied after MEPCs shortening, exogenous ACh (50 nmol/l) tended to return the initial value of tau. It is concluded that nonquantal ACh produces PSP and DS on the postsynaptic membrane after inhibition of ACh and that the DS persists after cessation of nonquantal secretion for a long time.     

Features of ion currents induced by acetylcholine in the muscle fibers of the lamprey

Spontaneous miniature end-plate currents (MEPC) and acetylcholine-induced noise were recorded in muscle fibres of m. longitudinalis linguae of lamprey. The MEPC decay time constant was 5.3 ms and amplitude 2.2 nA. The mean channel life time was 3.6 ms and conductance 13.6 pS. Ionic channels in synaptic and extrasynaptic zones had identical kinetics. Inhibition of acetylcholinesterase induced a 2.2-fold prolongation of MEPC, but did not affect the channel life time.     

The postsynaptic effects of substance P in the frog neuromuscular synapse]

The effect of substance P on the end-plate currents (EPC) and miniature EPC (MEPC) was studied in the "cut" sartorius muscle of the frog using voltage-clamp technique after acetylcholinesterase inhibition. Substance P in the concentration 5.10(-7)-1.10(-6) mol/l had no effect on the amplitude and time course of the single EPC and MEPC, but promoted significant prolongation of EPC decay during repetitive nerve stimulation (10/s), which indicated development of postsynaptic potentiation. Elevation of the substance P concentration to 5.10(-6) mol/l has led to the shortening of single EPS decay and more significant depression of the EPC amplitude in trains. This effect was connected with a decrease of the postsynaptic membrane sensitivity to acetylcholine, i. e. development of desensitization.     

Effects of the quinoline derivatives quinine, quinidine, and chloroquine on neuromuscular transmission

The quinoline derivatives quinine, its stereoisomer quinidine, and chloroquine may worsen or provoke disorders of neuromuscular transmission. In this study, we investigate effects of these drugs on neuromuscular transmission by conventional microelectrode as well as patch-clamp techniques. At 5 X 10⁻⁵ M, quinine, quinidine, and chloroquine reduced the quantal content of the end-plate potential by 37–45%. Between 10⁻⁶ and 10⁻⁴ M, all 3 drugs progressively decreased the amplitude and decay time constant of miniature end-plate potential (MEPP) and miniature end-plate current (MEPC); at 5 X 10⁻³ M, the MEPP became undetectable. The effect on the MEPP was not reversed by 1 μg/ml neostigmine. Single-channel patch-clamp analysis of the effects of quinine showed that this agent causes a long-lived open-channel as well as a closed-channel block of AChR. Tests for competitive inhibition or desensitization of the acetylcholine receptor (AChR) by quinine in concentrations that had a marked effect on the MEPC and on single-channel open and closed intervals were negative. Because quinoline drugs adversely affect both presynaptic and postsynaptic aspects of neuromuscular transmission at concentrations close to those employed in clinical practice, they should not be used, or used with caution, in disorders that compromise the safety margin of neuromuscular transmission.     

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.     

Biochemical and physiological consequences of an age-related increase in acetylcholinesterase activity at the rat neuromuscular junction

Acetylcholinesterase (AChE) specific activity was assayed using diaphragm muscles obtained from mature adult (10 months) and aged (25-27 months) rats. Biochemical assays indicated significant age-related increases in the AChE specific activity of both noninnervated and innervated tissue. The different molecular forms of AChE were separated by velocity sedimentation and were further assayed. The age-related increase was manifest primarily in the 10S (G4) form in both noninnervated and innervated tissue and also the 16S (A12) form of the noninnervated samples. To ascertain more conclusively whether AChE activity in the end-plate junctional region of innervated tissue changed in the older rats, miniature end-plate currents (m.e.p.c.s) were recorded under voltage-clamp conditions before and after AChE inhibition. When AChE activity was inhibited by 10 microM echothiopate or 1 mM methanesulfonyl fluoride, m.e.p.c. amplitudes and decay time constants increased in both age groups. The magnitude of these increases was larger in the older animals. However, calculations of the relative change in m.e.p.c. amplitudes after AChE inhibition indicated that less ACh was hydrolyzed by AChE in the older animals. Inhibition of AChE did not affect mean channel open time, which was estimated from spectral analyses of ACh-induced membrane noise. These data indicate that the prolonged decay times in the older rats following AChE inhibition is quite likely due to an expanded field of postsynaptic ACh receptors and not exclusively to a change in junctional AChE.     

Changes in the properties of synaptic channels opened by acetylcholine in denervated frog muscle

Acetylcholine (ACh)-activated channels in end-plates of frog sartorius muscle were studied at various times after denervation. Mean open times of the synaptic membrane channels were derived from the time constant of decay of miniature end-plate currents (tau MEPC) evoked by ACh quanta released from Schwann cells, which replace the motor nerve terminals after these degenerate. Membrane current noise, elicited by iontophoretic application of ACh to voltage-clamped end-plates, was also used to determine mean open time (tau noise) and conductance of the ion channels. About 1 week after denervation, soon after Schwann cell MEPCs appeared, they had a tau similar to that of the neural MEPC in innervated end-plates. However, 5-6 weeks after denervation tau MEPC was increased by a factor of about 5. Circa 4 weeks after denervation, cholinesterase activity of the denervated muscle decreased to 76% of that in the contralateral, innervated muscle, and even 4 months after the operation it was still 64%. Thus, it is unlikely that a change in acetylcholinesterase activity is the main factor responsible for the increase in tau of Schwann cell MEPC. About 1 week after denervation tau noise was close to that in innervated end-plates (about 2 ms). Twelve to 24 days after denervation the average channel open time was 4.5 +/- 1.0 ms, with some end-plates still showing normal 'fast' channels. However, in muscles denervated for 47-113 days the open time was 12.9 +/- 1.9 ms. In the early and intermediate periods, ACh-induced noise spectra with two components were obtained from many end-plates, indicating the simultaneous activation of two different types of channels. At some end-plates during the early and intermediate periods after denervation, but not after about 5 weeks, neostigmine caused the appearance of a component, which was as fast as that of normal end-plate channels. In other experiments small doses of alpha-bungarotoxin were applied in order to predominantly block extra-junctional receptors. In the early period of denervation, when two components were present in the noise spectra, alpha-bungarotoxin eliminated the slow component leaving channels as fast as in innervated end-plates. After prolonged denervation, a component with tau of about 5.5 ms was occasionally disclosed by application of alpha-bungarotoxin. tau noise and tau MEPC from the same end-plate closely agreed. Our results indicate that at frog end-plates the open time of the majority of the synaptic channels opened by ACh becomes longer with increasing time after denervation.     

Reactivating and cholinolytic action of dipyroxime in the myoneural synapse of warm-blooded animals

Reactivating (RA) and cholinolytic (Chl) effects of dipyroxime in soleus and diaphragm muscles of the rat were estimated by amplitudes and durations of miniature end-plate potentials (MEPP) and currents (MEPC). After armin-induced inhibition of acetylcholinesterase (AchE), the action of dipyroxime in concentrations of 5.10(-6)-5.10(-4) mol/l on the amplitude and duration of MEPPs and MEPCs represents the combination of RA and Chl effects. Separate study of the RA effect (after washing out of the reactivator) has shown that this effect increased within the whole range of concentrations used. Complete reactivation of phosphorylated AchE was achieved at dipyroxime concentrations of 2-5.10(-4) mol/l. Separate analysis of Chl effect (in voltage-clamp experiments with intact AchE) has shown that dipyroxime suppressed acetylcholine-induced responses due to a block of cholinoreceptors in the open conformation. This block was characterized by prolongation of MEPCs decay without affecting its exponential nature. It is concluded that dipyroxime is a "very fast blocker".     

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.     

An attempt to estimate various characteristics of neuro-muscular transmission from the rising phase of miniature end-plate currents

A simple expression describing the relation between the duration of rising phase and amplitude of miniature end-plate currents (MEPC) is suggested. Parameters of MEPC of the rat diaphragm with different degrees of acetylcholinesterase inhibition by galanthamine were used to estimate the lower levels of acetylcholine diffussion coefficient (0.86.10-6 cm2/s) and channel opening rate constant (21000 s-1).     

Analysis of anticholinesterase-induced neuromuscular transmission failure

To define the underlying mechanism of neuromuscular transmission failure induced by anticholinesterases, we simultaneously performed surface recordings of compound muscle action potentials (CMAPs) and intracellular recordings of miniature end-plate potentials (MEPPs), miniature end-plate current (MEPCs), and end-plate potential (EPPs) in rat diaphragms exposed in vitro to 1 × 10^?4 to 2 × 10^?2 mmol/L neostigmine methylsulfate. At low concentrations of neostigmine, repetitive stimulation of the phrenic nerve resulted in decrement followed by complete recuperation of CMAP amplitudes. This bimodal pattern was associated with maximal end-plate depolarization at the beginning of the stimulation period, increased MEPP amplitudes, and prolonged time constants of MEPC decays. Higher concentrations of neostigmine resulted in a unimodal decline of amplitudes of CMAPs and EPPS, reduced MEPP amplitudes, and a double exponential time course of MEPC decays. These results indicate that low concentrations of anticholinesterases impaired neuromuscular transmission by producing transient depolarization of the end-plate region. Higher concentrations induced desensitization and direct blockade of the end-plate receptor channel, probably in its open conformation. © 1993 John Wiley & Soncs, Inc.     

Analysis of a model of the miniature endplate current

The numerical model of the miniature end-plate current MEPC described earlier was systematically analyzed to obtain an optimal set of parameters. This set permits simulating a number of experimental effects: time course of normal MEPC, cholinesterase inhibition, alpha-bungarotoxin action, potential dependence of MEPC, etc. The time course of the simulated "giant" MEPC fitted the experimental data only partially. A good correspondence between the model and experimental data underlies the conclusion that this model reflects well the relative contribution of several processes to MEPC generation.     

Factors determining the duration of a single postsynaptic response in neuromuscular junctions

The miniature end-plate currents (MEPCs) and acetylcholine-induced current fluctuations were compared in experiments on fast and slow muscle fibres of lamprey, frog, chicken and rat. Simultaneous analysis of MEPCs decay time constant and mean life-time of single ionic channels suggests that the former is the main but not the sole factor determining the MEPCs duration. Additional factors, in particular, the relative insufficiency of acetylcholinesterase activity (AChE) are more important for the generation of the usual MEPSs in slow muscle fibres or the "giant" MEPCs in fast ones. Low activity of AChE favours the prominent asynchrony of interaction between acetylcholine molecules and cholinoreceptors causing prolongation of the time course of synaptic response. The mechanisms of MEPCs decay shortening induced by alpha-bungarotoxin or d-tubocurarine and the nonuniformity of different active spots of junctions are discussed.     

Effect of hyaluronidase on the end-plate miniature currents and potentials in the frog

The effect of 0.1% hyaluronidase on miniature end-plate potentials and currents (MEPP and MEPC) was studied in the frog cutaneous-pectoris muscle. The action of hyaluronidase on armin-pretreated muscles caused a decrease in the amplitude, duration of half-decay time and rising phase of MEPPs and MEPCs. The positive correlation between the amplitude and half-decay time of MEPPs and MEPCs was diminished. Hyaluronidase treatment of preparations with active acetylcholinesterase caused the half-life time of MEPCs to increase without any changes in the amplitude and rising phase of MEPCs. It is suggested that enzymatic destruction of a part of the glycocalix of cells forming the neuromuscular junction and of a part of the extracellular matrix results in a weakening of the nonspecific acetylcholine binding, thus facilitating the acetylcholine diffusion into the synaptic cleft.     

The effect of "nonquantal" acetylcholine on miniature end-plate currents in the mouse

The development of postsynaptic potentiation (PSP) and desensitization (DS) under the nonquantal secretion (NS) of acetylcholine (ACh) was studied using characteristics of miniature end-plate currents (MEPCs) in mice diaphragm muscle. The H-effect (the measure of the NS level) fell to zero during 3 hours after acetylcholinesterase (AChE) inhibition by armine at 20 degrees C. The MEPCs decay time constant (tau) decreased in parallel with the H-effect, though there was no reduction of the MEPCs amplitude in this case; tau did not change when NS was absent (early denervation). The maximal prolongation of tau after AChE inhibition was the same both in innervated (NS was normal) and denervated (NS was absent) muscle. The reduction of tau accelerated with the temperature rise and occurred after AChE inhibition by neostigmine. There was no changes in tau when AChE was active. It is suggested that nonquantal ACh decreases tau due to the DS development. NS induces no significant PSP after the AChE inhibition, but can do it later according to DS development, so that signs of DS may partially be masked by PSP.