Maria Scuka

Effect of repetitive stimulation on the frog neuromuscular transmission

Presynaptic and postsynaptic effects on the neuromuscular transmission were studied during 20 min of indirect stimulation at 10/s. During the ‘facilitation’ period, there was an increase in the quantal content, in the frequency of miniature endplate potentials and in their amplitude. All these parameters were decreased during the ‘depression’ period. Besides, the end-plate current (e.p.c.), recorded during this high rate of stimulation, increasingly lengthened. The falling phase of the e.p.c. was exponential during facilitation, while marked deviations from the exponential time course were observed during depression. The experiments showed that a possible change in the kinetics between the receptors and the mediator was not responsible for the lengthened time course of the e.p.c. Therefore, it is assumed that either the delayed diffusion of the transmitter from the synaptic cleft or an altered mechanism of the release of acetylcholine may be involved. The latter possibility is supported by a progressive prolongation of the synaptic delay, which was observed during a prolonged repetitive stimulation.

Analysis of the effects of histamine on the end-plate potential.

Abstract Histamine (10 −4 M−5 × 10 −4 M) reduces the amplitude and changes the time course of the end-plate potential, recorded intracellularly in the sartorius muscle of the frog. It was observed that the decreased amplitude of the end-plate potential after histamine is a result of a reduced quantal content and of a reduced sensitivity of the end-plate receptors. An increase in the frequency of miniature end-plate potentials was also observed which suggests that histamine decreases the resting potential of the presynaptic membrane. A shortening of the half decline time, but not of the rise time of the end-plate potential, was observed after histamine. The faster time course of the end-plate potential is not due to a decreased time constant of the muscle fibre, but to a faster time course of the underlying end-plate current.

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 microM 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.

The effect of repetitive neuromuscular activity on the sensitivity of acetylcholine receptors

If skeletal frog muscle is indirectly stimulated at 10 Hz first an increase and later a decrease of the amplitude of miniature end-plate currents (m.e.p.cs) is observed (Ruzzier and Scuka 1979). The underlying mechanism can be a presynaptic change of the quantal size or a postsynaptic change. To distinguish between these possibilities, the neurally evoked end-plate current (e.p.c.n), the ionophoretically evoked end-plate current (e.p.c.i) and the extracellularly recorded miniature end-plate potential (m.e.p.p.e) were studied. It was found that the time constant of decay of m.e.p.p.e did not change during the experiment. The amplitude of the e.p.c.i changed in the same way as the amplitude of the m.e.p.c., it first increased and then decreased. Similar changes of the amplitude of e.p.c.i were observed in the experiments with increased frequency of the nerve stimulation and in those with different increases of the quantal content. It is concluded that during prolonged repetitive stimulation the sensitivity of the end-plate receptors to the released transmitter is modified, probably as a consequence of the cooperative binding of acetylcholine to the receptors.

The effects of pH on the conductance change evoked by iontophoresis in the frog neuromuscular junction

SummaryThe amplitude of the electrophoretically evoked end-plate potential increases with changing the pH of the bathing solution from 9.4 to 5.4 at room temperature. This change is not observed at lower temperature. The underlying current (e.p.c.I) is slightly decreasing at room temperature by lowering the pH. The relationship between the amplitude of the e.p.c.I and membrane potential is highly non-linear at pH 9.4, while it is quite linear at pH 5.4. The time course of the e.p.c.I is changed neither by different pH, nor by different membrane potential. The data suggest that during the e.p.c.I, the mediator (ACh), the receptor (R) and the mediator-receptor complex are in equilibrium: the amplitude of the e.p.c.I will thus depend on the affinity constant of the reversible reaction between ACh and R. It is concluded that by decreasing the pH, the affinity constant is decreased.

On the changes of the time course of the end-plate current during repetitive stimulation

SummaryIn a previous study it was observed that prolonged repetitive stimulation (10.s−1) markedly lengthened the time course of the end-plate current. In order to investigate whether this effect could be due to a more dispersed release of the mediator or to a decreased clearance of the mediator from the synaptic cleft, the rise time of the end-plate current and of the miniature end-plate current were analyzed. The results show that presynaptic factors may be involved.

The effect of anticholinesterase drugs on the ionophoretically-evoked end-plate currents

In order to study the sensitivity of acetylcholine (ACh) receptors exposed to anticholinesterase drugs, the ionophoretically evoked end-plate currents (e.p.c.i) in frog sartorius muscle were studied. During treatment with prostigmine (PST, 3 μM) or methanesulfonyl fluoride (MSF, 1–2 mM), the amplitude of the e.p.c.i evoked by ACh was first increased, reaching its maximum (200–400%) after 20–30 min and then decreased. If these experiments were repeated by applying carbachol (CCh) instead of ACh, an increase, although less pronounced (30–100%), was also observed. The results obtained by applying CCh cannot be ascribed to the inhibition of ACh-esterase, as CCh is not hydrolysed by this enzyme. Therefore, a direct action of both drugs on the ACh-receptor channel complex is suggested. From the comparison of the effects of PST and MSF on the responses to CCh and ACh applications, it is calculated that 20–25% of the increased response to ACh during PST treatment can be ascribed to the direct effect on the ACh-receptor channel complex. During MSF treatment, this amount results in 13–15%. The changes of the time course of the e.p.c.i (evoked by CCh or Ach), which is prolonged in the presence of PST and shortened during the maximum effect of MSF, suggest that the direct action of both studied drugs is different.

Effects of irreversible and reversible cholinesterase inhibitors on single acetylcholine-activated channels

SummaryThe use of cholinesterase (CHE) inhibitors provided valuable information about the mechanism(s) of neuromuscular transmission, but questions on side effects at the level of AChactivated channels were raised. Patch-clamp recording was used to study the effects of prostigmine (PST) and methanesulfonyl fluoride (MSF), a reversible and an irreversible cholinesterase inhibitor, respectively, on ACh-activated channels. We found that these drugs diminish the average dwell time of elementary currents from around 5 msec (control) to less than 1 msec in the presence of PST (20 μm) or MSF (5 mm) (at room temperature). With MSF the ACh-activated channel conductance of the most frequently observed amplitude class decreased from 45 pS (control) to 30 pS, but not in the presence of PST. In control conditions there were also amplitude classes of 60 and 24 pS, with probabilities of occurrence <10%. In the presence of 1.5 mm MSF, where current dwell time was not affected, additional subconductance states of 19 and 36 pS were observed and may be due to partial blockade of the open channel. We conclude that the drug of choice to be used in studies on the role of CHE in the neuromuscular transmission is MSF, because at 20 μm PST, where blockade of ACh-activated channels is significant, cholinesterase was reported to be partially inhibited, whereas at 1 mm MSF it is fully inhibited and the dwell time of ACh-activated channels is not affected.

Effects of vinblastine in the frog neuromuscular junction during repetitive stimulation

Vinblastine (10(-5) to 10(-4) M) applied in a Mg-blocked sciatic nerve-sartorius muscle preparation of the frog at the beginning of 20 min of repetitive stimulation of the nerve at 10 Hz, markedly inhibited the facilitation period. There was a smaller increase of end-plate potential amplitude, of quantal content, and of miniature end-plate potential (MEPP) frequency in comparison with that found in experiments without the drug. This depressant effect was more evident when the preparation was preincubated in vinblastine: synaptic responses often disappeared after a few minutes, whereas MEPP frequency attained a maximum at the 5th min. The results suggest a multiple site of action for vinblastine. First, an alteration of both vesicle and plasmalemma membrane, as revealed by the increase of MEPP frequency and by the change of the binomial release features. Second, the drug could damage the intracellular transport system, interfering with the mobilization of the vesicles toward the nerve endings, as shown by the very low or absent facilitation.

The effect of colchicine on neuromuscular transmission in the frog during repetitive stimulation.

When colchicine 10−4 mol·l−1 was applied at the beginning of repetitive stimulation of the nerve at 10·s−1, the facilitation was markedly inhibited. The quantal content showed a very slight increase and its maximum value was reached later. The maximum frequency of spontaneous release was also reached later in the presence of colchicine than in the absence of the drug. In addition, the synaptic delay was much more pronounced in the presence of colchicine than in the control experiment.The results suggest that a partial block by colchicine of the release process in the nerve terminal occurs. This effect may be due to the action of the drug on the nerve terminal membrane. The results cannot exclude the possibility that colchicine interferes with the transport of vesicles towards the sites of release located on the membrane.