Content Of Endplate Potentials Research Articles

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

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

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

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

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

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

Early Changes of Neuromuscular Transmission in the SOD1(G93A) Mice Model of ALS Start Long before Motor Symptoms Onset

Amyotrophic lateral sclerosis is characterized by a progressive degeneration of the corticospinal tract motor neurons. Growing evidence suggests that degeneration may begin at the distal axon proceeding in a dying-back pattern. It seemed therefore of interest to investigate synaptic transmission at the neuromuscular junction (NMJ) in pre- and symptomatic phases of the disease. Endplate potentials (EPPs), miniatures endplate potentials (MEPPs) and giant MEPPs (GMEPPs) were recorded from innervated diaphragm muscle fibers from 4–6 and 12-15 weeks-old SOD1(G93A) mice and non-transgenic aged-matched littermates (WT). In the pre-symptomatic phase, SOD1(G93A) mice exhibited a significant increase in the mean amplitude of EPPs together with an increase in the mean quantal content of EPPs, suggesting that more acetylcholine is being released into the synaptic cleft. SOD1(G93A) mice presented a higher frequency of GMEPPs, suggestive of intracellular Ca2+ deregulation in nerve terminals. The increase in the mean amplitude of MEPPs and the decreased mean rise-time of MEPPs in SOD1(G93A) mice point to post-synaptic related changes. In the symptomatic phase, electrophysiological data showed evidence for two NMJ groups in SOD1(G93A) mice: SOD1a and SOD1b. SOD1a group presented reduced mean amplitude of both EPPs and MEPPs. The mean rise-time of MEPPs was increased, when compared to WT and to SOD1b group, indicating impairments in the neuromuscular transmission. In contrast, the neuromuscular transmission of SOD1b group was not different from age-matched WT nor pre-symptomatic SOD1(G93A) mice, being somehow in between both groups. Altogether these results show that the neuromuscular transmission of SOD1(G93A) mice is enhanced in the pre-symptomatic phase. In the symptomatic phase our results are consistent with the hypothesis that the diaphragm of SOD1(G93A) mice is undergoing cycles of denervation/re-innervation supported by mixed neuromuscular junction populations. These early changes in the neuromuscular transmission of SOD1(G93A) mice suggest that the ALS associated events start long before symptoms onset.

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd(2+) , the removal of extracellular Ca(2+) and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.

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

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

Short term plasticity caused by Ca 2+ induced Ca 2+ release in the frog motor nerve terminals

The actions of ephedrine were studied using intracellular recording techniques in guinea pig intercostal muscle fiber. After application of ephedrine (10−5 −10−4 M), miniature end-plate potential (MEPP) amplitude was decreased in a concentration-dependent manner. A greater depression in amplitude of end-plate potentials (EPP) was observed in preparations immobilized with d-tuboeurarine. In addition, ephedrine depressed the transient depolarization produced by iontophoretically applied acetylcholine (ACh).In normal Krebs solution and high Mg2+ solution, 10−4 M ephedrine increased MEPP frequency but did not alter the quantal content of EPPs in high Mg2+ blocked muscle. In these preparations, EPP amplitude did not change significantly. These results suggested that in the absence of a postsynaptic receptor blocking agent, a slight presynaptic action of ephedrine may offset the decreased responsiveness of the postsynaptic membrane. In addition, no significant alterations of resting membrane potential or the electrical characteristics of the membrane were recorded.Pretreatment of a mouse phrenic nerve-diaphragm preparation with ephedrine did not protect against the irreversible blockade by α-bungarotoxin. These results suggested that ephedrine may depress ACh potentials, MEPPs, and EPPs in dTc-blocked muscle by acting postsynaptically at a site on or associated with the ACh receptor other than the ACh recognition site.

Physiological and regenerative acetylcholine release form motor nerve: Differential inhibitions by vesamicol and ω-agatoxin IVA

Stimulation of mammalian motor neurons can elicit Ca(2+)-dependent regenerative release of acetylcholine and prolonged endplate depolarization when the enzymatic degradation of the neurotransmitter is inhibited. Unlike physiological phasic release of acetylcholine, the regenerative release is sensitive to L-type Ca2+ channel blockers. We studied the effects of vesamicol (an inhibitor of active transport of acetylcholine into synaptic vesicles) and omega-agatoxin IVA (a blocker of the motor nerve P-type Ca2+ channel) on these two types of acetylcholine release to compare the vesicle pools and Ca2+ channels responsible for the release. When coupled with repetitive stimulations, vesamicol decreased mean amplitude of miniature endplate potentials, resulting in a skewed distribution to lower amplitude, reduced quantal content of endplate potentials and decreased immediate available pool of acetylcholine. omega-Agatoxin IVA had no effect on miniature endplate potential but inhibited quantal content of endplate potential. The mean inhibitory concentration was around 5-10 nM. Vesamicol and omega-agatoxin IVA decreased the probability of triggering regenerative release. However, the magnitude and duration of regenerative release, once triggered, were not depressed by either agent. It appears that the majority of Ca2+ necessary for regenerative release is translocated via omega-agatoxin IVA-insensitive Ca2+ channels, which can be activated by prolonged depolarization of nerve terminals induced by accumulated acetylcholine. The results suggest that different Ca2+ channels are activated in the regenerative (L-type) and phasic (P-type) acetylcholine release, which utilize different pools of synaptic vesicles.

Effects of waglerin-I on neuromuscular transmission of mouse nerve-muscle preparations

The effects of waglerin-I, a toxin from Trimeresurus wagleri, on neuromuscular (NM) transmission were studied on the phrenic nervediaphragm preparation and triangularis sterni nerve-muscle preparation of mice. The toxin (1.2–4.0 μM) reversibly inhibited the indirectly elicited twitch tension of the diaphragm and decreased the ACh-elicited muscle contracture of chronically denervated diaphragm, while the directly elicited twitch tension was not affected. The toxin reversibly decreased the amplitude of miniature endplate potential (MEPP) at 0.52 μM and endplate potential (EPP) at 1.2–4 nM. The toxin (120nM–0.4 μM) also decreased the quantal content of EPP. The perineural waveforms were recorded with an extracellular electrode placed into the perineural sheaths of motor nerves of M. triangularis sterni. The toxin (4 μM) did not alter the amplitudes of waveforms related to sodium and potassium currents of the nerve terminal action potential, while the waveform related to calcium current was decreased. It is concluded that the toxin acts on both presynaptic and postsynaptic sites of the mouse motor endplate, and that the presynaptic effect is apparently more potent than the postsynaptic effect.

Effects of ATP-sensitive K +-channel activators on transmitter release parameters at the frog neuromuscular junction

The frog neuromuscular junction was used to study K ATP channel action and its relation to transmitter release. Diazoxide (10 μM) and cromakalim (300 μM) decreased the amplitude of the end-plate potential (EPP) without significantly affecting the miniature end plate potential (MEPP) amplitude. Thus, there was a significant decrease in the quantal content of EPP after administration of the K ATP channel activators. These agents did not alter MEPP frequency or resting membrane potential (RMP). The diazoxide-induced decrease in EPP amplitude was antagonized by the K ATP blocker glibenclamide (10 μM) suggesting K ATP channel involvement. Glibenclamide (10 μM) by itself caused a significant decrease in the RMP without significantly affecting the parameters of transmitter release. The diazoxide-induced decrease in EPP amplitude was not readily reversible with washing, however, when glibenclamide was added after diazoxide, the effect was easier to reverse. The results indicate that the activators exert their effect predominantly at the presynaptic region.

Multiplicative and additive Ca(2+)-dependent components of facilitation at mouse endplates.

1. Facilitation of endplate potentials (EPPs) and frequency of miniature endplate potentials (MEPPs) were studied, in the presence of low Ca2+/raised Mg2+, in isolated mouse hemidiaphragm, using pseudo-random sequences of nerve stimulation and automated (computer) counting of MEPPs and quantal components of EPPs. 2. The facilitation in quantal content of EPPs (m) produced by one or more antecedent stimuli was accompanied by facilitation of MEPP frequency (fm) that was similar in magnitude and substantially less than expected if facilitation reflects persistent (residual) intraterminal Ca2+. The time course of 'phasic' quantal release, associated with the EPP, was little if at all altered with facilitation. 3. The magnitude and time course of facilitation was consistent with two distinct presynaptic processes, each manifest both in m and fm, (i) an effect to multiply transmitter release, and (ii) residual Ca2+ which adds to Ca2+ brought in by nerve impulses. These have distinct time courses. 4. After loading nerve terminals with bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), facilitation of m and fm became very small. 5. At sufficiently low Ca2+/raised Mg2+ facilitation of m and fm became very small although latency histograms showed clear EPPs. However, the multiplicative component of facilitation became maximal at Ca2+/Mg2+ concentrations giving an average m value less than 0.1, corresponding to about 5% of normal Ca2+ entry per pulse. At lower Ca2+, facilitation was restored when EPPs were made larger using 4-aminopyridine. 6. With EPPs elicited by brief 'direct' nerve terminal depolarizations, facilitation was graded with pulse intensity (and m) and could be much less than with EPPs with similar m evoked by nerve stimuli at lower Ca2+ and/or higher Mg2+. 7. It was concluded that fast facilitation is primarily multiplicative and reflects activity within the nerve terminal of a Ca(2+)-sensitive process distinct from that generating Ca(2+)-dependent release.

Actions of lead on transmitter release at mouse motor nerve terminals

The actions of lead (Pb2+) on transmitter release were studied at neuromuscular junctions in mouse diaphragm in vitro. The quantal content of end-plate potentials (EPPs) was reduced by Pb2+ in a dose-related manner consistent with inhibition of Ca2+ entry into nerve terminals, with a half-maximal effect at 1.4 microM (in 0.5 mM Ca2+ and 2 mM Mg2+). Pb2+ also inhibited the increased frequency of MEPPs (fMEPP where MEPPs denotes miniature EPPs) produced by Ba2+ in the presence of raised K+, blocking the calculated Ba2+ entry half-maximally at 170 microM. However, at concentrations of 50-200 nM, Pb2+ often increased fMEPP in 20 mM K+ in the presence of Ca2+ and acted to promote the irreversible effect of lanthanum (La3+) to raise fMEPP. In nominally Ca(2+)-free solution with 20 mM K+, brief (1 min) application of Pb2+ (20-320 microM) caused rapid dose-dependent reversible rises in fMEPP. With prolonged exposure to Pb2+, fMEPP rose and then slowly declined; after removal of Pb2+, once fMEPP had fallen to low levels, fMEPP responded nearly normally to Ca2+ or ethanol, but not to Pb2+ itself. In 5 mM K+, 0 mM Ca2+ and varied [Pb2+] (where [] denotes concentration), nerve stimulation caused no EPPs, but prolonged tetanic stimulation produced increases in fMEPP graded with [Pb2+] that persisted as a "tail"; results were consistent with growth of fMEPP with the 4th power of intracellular Pb2+ and removal of intracellular Pb2+ with a time constant of about 30 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced safety factor for neuromuscular transmission and abnormal quantal secretion precede neuromuscular weakness induced by dithiobiuret

Daily treatment of rats with 2,4-dithiobiuret (DTB. 1 mg/kg/day, ip) causes a flaccid neuromuscular weakness first observable in the hindlimbs after 5–6 days of treatment. With continued exposure, neuromuscular weakness appears to encompass the other muscles of the body; death is presumed to result from paralysis of respiratory muscles. The purpose of the present study was to investigate the early effects on neuromuscular transmission which precede neuromuscular weakness caused by DTB, particularly as they relate to the apparent differential muscle sensitivity, using conventional intracellular microelectrode recording techniques. Experiments were conducted using the hemidiaphragm muscle isolated from male rats treated for 7–8 days, with 1 mg/kg/day DTB ip, a regimen which resulted in hindlimb, but not diaphragmatic paralysis, or with 0,9% NaCl (1 ml/kg/day) as control. Analysis of quantal content of end plate potentials (EPPs) from hemidiaphragms of DTB-treated rats indicated no difference from control. Exposure of hemidiaphragm preparations from DTB-paralyzed rats to solutions containing elevated Mg 2+ and lowered Ca 2+ concentrations (6 and 1 m m, respectively) resulted in a decreased quantal content of the EPP compared to that of similarly treated control preparations. When miniature EPPs (MEPPs) were evoked from nerve terminals by elevating [K −] e, in the presence of 6 m m Mg 2+ and 1 m m Ca 2+, the mean peak frequency evoked by K +-induced depolarization was reduced in the DTB-treated group; however, the time at which peak frequency was attained was the same for the control and DTB-treated group. Mean MEPP amplitude but not resting MEPP frequency was altered in the presence of high [Mg 2+] for diaphragms of the DTB-treated group. Neither MEPP amplitude nor frequency was altered in diaphragms exposed to normal concentrations of Ca and Mg. Prolongation of rise and decay times of MEPPs occurred from end plates of DTB-treated rats irrespective of whether low [Ca 2/high [Mg 2+] solutions were used. However, these effects were more pronounced when low [Ca 2+/high [Mg 2+] solutions were used. Diaphragm-derived end plates of the DTB-treated group were also characterized frequently by the presence of very large amplitude MEPPs with prolonged decay times. The overall percentage of the total population of MEPPs which these abnormal MEPPs made up in the DTB-treated rats was increased dramatically by exposure to low [Ca 2+]/high [Mg 2+] solutions. Thus, differential muscle sensitivity occurs during paralysis induced by chronic DTB treatment, with the diaphragm being somewhat more resistant than hindlimbs to neuromuscular weakness. This effect appears to be associated with the safety factor associated with acetylcholine release and reflects an impairment of presynaptic processes resulting in a diminished number of quanta liberated in response to motor nerve stimulation. Additional neuromuscular symptoms precede the onset of muscle weakness.

Effects of sodium selenite on neuromuscular junction of the mouse phrenic nerve-diaphragm preparation

The effects of sodium selenite on the neuromuscular junction of the phrenic nerve-diaphragm of the mouse were studied. Nerve-evoked twitches of the diaphragm of the mouse, the frequency of miniature endplate potentials, the quantal content of endplate potentials and the compound action potentials of the axon were measured. Sodium selenite induced a slight increase of the amplitude of the twitch, followed by twitch depression. The amplitude of the twitch, increased by selenite, became more prominent after the suppression of the twitch induced by cadmium ions, d-tubocurarine or magnesium ions. It appeared that the increased amplitude of twitch was due to the facilitation of transmitter release, since selenite significantly increased the frequency of miniature endplate potentials, and the amplitude and quantal content of endplate potentials; the amplitude and half decay time of miniature endplate potentials were unaffected. Twitch depression induced by selenite was enhanced by ammonium ions, high potassium and low magnesium and attenuated by high calcium. During the period of gradual depression of the twitch, selenite decreased the amplitude of compound action potentials of the phrenic nerve axon and caused the disappearance of endplate potentials. Ammonium ions enhanced the blockade of axonal conduction induced by selenite. Moreover, the depolarizing agents, ammonium and high potassium also induced an initial increase of twitch amplitude followed by depression of the twitch. These findings indicate that selenite probably alters the release of the transmitter by depolarizing the nerve membrane. The effects of selenite were antagonized by glutathione and cyanide, suggesting that the binding of selenite to sulfhydryl groups of the membrane was essential for inducing its pharmacological actions.

The nature of the presynaptic effects of (+)-tubocurarine at the mouse neuromuscular junction.

1. The effects of (+)-tubocurarine (TC) on tetanic run-down and quantum content of end-plate potentials (EPPs) were investigated in cut-fibre preparations of mouse diaphragm. 2. (+)-Tubocurarine, 0.15 microM, halved the amplitude of spontaneous miniature EPPs (MEPPs) and steepened the tetanic run-down of EPPs evoked at 10 Hz by increasing the quantum content of the first EPP of the train while having no effect on quantum content of plateau EPPs. With stimulation at 1 Hz, there was little run-down and the quantum content of all EPPs was increased by TC. 3. The use of binomial statistics to analyse release indicated that after TC the increase in the quantum content of the first EPP in the train at 10 Hz was due to an increase in n and that during the run-down there was a decrease in p so that plateau EPP quantum content at 10 Hz was not different from control. 4. To elucidate a possible role of cholinoreceptors in the presynaptic effects of TC, studies were made on the effects of pancuronium or of alpha-bungarotoxin (BTX), with concentrations and exposure times where they had postsynaptic effects equal to 0.15 microM-TC. The run-down of EPPs was unaffected by BTX, while pancuronium steepened it to a lesser extent than TC. 5. The anticholinesterase, ecothiopate, decreased the quantum content of plateau EPPs only at high frequencies of stimulation (50 Hz) and did not affect the presynaptic effects of TC at 10 Hz. 6. At concentrations which reduced MEPP amplitude, atropine (10 microM) or hexamethonium (50 microM) had no effect on EPP run-down. 7. These results indicate that TC could have presynaptic effects via a presynaptic acetylcholine receptor, but that such a receptor may not have the same binding specificities as the postsynaptic receptor.

Multiple potassium conductances at the mammalian motor nerve terminal.

The possibility that multiple K+ conductances are present in mammalian motor nerve terminals was investigated by measuring the differential effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on transmitter release and on nerve terminal excitability in mouse phrenic nerve-diaphram preparations. Neither 4-AP nor TEA alter the spontaneous frequency of miniature end-plate potentials (fmepp) and therefore evidently do not affect calcium channels directly. Both 4-AP and TEA increased the quantal content of end-plate potentials (epps) evoked by nerve stimulation but the effects were not mutually exclusive; TEA continued to act in the presence of a maximally effective concentration of 4-AP. The increase in transmitter release evoked by focal polarisation of the terminal was not affected by 4-AP, whereas TEA exerted an effect consistent with a reduction in membrane conductance. Similarly, threshold for nerve terminal action potential generation was not affected by 4-AP, but TEA caused a reduction in threshold and alteration of the 'accommodation curve' indicative of a reduction of membrane conductance. Under conditions where one would predict no contribution of calcium K+ conductance, i.e., when release was evoked by Ba2+ in the absence of Ca2+, the different and non-competing effects of TEA and 4-AP were still apparent. It is concluded, therefore, that the motor nerve terminal possesses at least two K+ conductances, not including calcium activated gK, which may be distinguished pharmacologically.

Further electrophysiological studies of neuromuscular transmission in an animal model of myasthenia gravis

The development of experimental autoimmune myasthenia gravis (EAMG) in rats produces a significant reduction in the amplitude of spontaneously occurring miniature end-plate potentials (MEPPs) and impulse-evoked end-plate potentials (EPPs). This junctional abnormality, however, does not impair the ability of the affected fibers to produce propagated muscle action potentials of normal amplitude. Quantal content analysis indicates that in Mg 2+-blocked EAMG muscle the mean number of acetylcholine (ACh) quanta released per nerve impulse closely approximates the corresponding normal value. At EAMG end-plates, the depolarization produced by either saturating or nonsaturating doses of carbamylcholine was significantly less than that seen at normal end-plates, suggesting a reduced acetylcholine receptor (AChR) content. When the depolarizing response to 250 μ m carbamylcholine was examined at various EAMG end-plates with different sizes of MEPPs, there was a direct correlation between the carbamylcholine-induced depolarization and the MEPP amplitude; this correlation, however, was much less pronouced in normal end-plates, which supports the concept that the MEPP size in the receptor-immunized muscle reflects the content of the functional postsynaptic AChR. In control solution, the indirect twitch tension produced in EAMG muscle was normal. The twitch tension in EAMG muscle however, was almost completely abolished by a dose of d-tubocurarine that reduced the tension in normal muscle by only 50%. When 4-aminopyridine, a drug known to increase the quantum content of EPPs, was applied to curarized normal or EAMG muscles, normal muscle contraction was restored and the decremental response to repetitive nerve stimulation was abolished. We conclude that the major defect of neuromuscular transmission in EAMG results from postsynaptic abnormalities at the end-plates, presumably secondary to reduction of the number of functional AChRs. Chronic EAMG in rats is a reasonable model of human myasthenia gravis in which a similar defect of neuromuscular transmission is present.

Apparent lack of effect of α-bungarotoxin on the spike-induced release of acetylcholine at the mammalian motor end-plate

During intoxication with α-bungarotoxin (α-BGT) the amplitude of end-plate potentials (EPPs) decreased more slowly than the amplitude of potentials evoked by iontophoretically applied acetylcholine. Mean quantal content of EPPs, however, remained constant. The results do not support the notion that α-BGT influences the spike-evoked transmitter release.

The effect of anticholinesterase drugs on postjunctional potentials of skeletal muscle

1. 1. Quantitative detailed investigation of the effects of seven anticholinesterase (anti-ChE) drugs—organophosphorus inhibitors (OPI) (armin, GD-42, GD-7), carbamates (proserine or neostigmine, eserine), quaternary ammonium salts (ambenonium, edrophonium)—on postjunctional potentials in skeletal muscle was carried out with a reduced quantum content of end plate potentials (EPP). 2. 2. It was established by the method of elimination of other possible causes that the amplitude of miniature end plate potentials (MEPP) and the duration of EPP were increased by inhibitors as a result of their anti-ChE action only. 3. 3. Curare-like action of the inhibitors was analysed. It was found to be related neither to their effect on membrane potential or passive electrical properties of the muscle fibres nor to inhibition of cholinesterases (ChE). 4. 4. It was found that inhibitors, excepting OPI, failed to affect the EPP quantum content and MEPP frequency. OPI affected these parameters in experiments on the frog sartorius muscle but not on the rat diaphragm muscle and only at concentrations of 2 × 10 −5 M and higher. Under these conditions armin decreased the EPP quantum content and the MEPP frequency. GD-42 and GD-7 decreased the EPP quantum content as well but increased MEPP frequency. These variations of parameters were not related to inhibition of ChE activity and were reversible by washout.

Ephedrine: A postsynaptic depressant drug at the mammalian neuromuscular junction

The actions of ephedrine were studied using intracellular recording techniques in guinea pig intercostal muscle fiber. After application of ephedrine (10 −5 −10 −4 M), miniature end-plate potential (MEPP) amplitude was decreased in a concentration-dependent manner. A greater depression in amplitude of end-plate potentials (EPP) was observed in preparations immobilized with d-tuboeurarine. In addition, ephedrine depressed the transient depolarization produced by iontophoretically applied acetylcholine (ACh). In normal Krebs solution and high Mg 2+ solution, 10 −4 M ephedrine increased MEPP frequency but did not alter the quantal content of EPPs in high Mg 2+ blocked muscle. In these preparations, EPP amplitude did not change significantly. These results suggested that in the absence of a postsynaptic receptor blocking agent, a slight presynaptic action of ephedrine may offset the decreased responsiveness of the postsynaptic membrane. In addition, no significant alterations of resting membrane potential or the electrical characteristics of the membrane were recorded. Pretreatment of a mouse phrenic nerve-diaphragm preparation with ephedrine did not protect against the irreversible blockade by α-bungarotoxin. These results suggested that ephedrine may depress ACh potentials, MEPPs, and EPPs in dTc-blocked muscle by acting postsynaptically at a site on or associated with the ACh receptor other than the ACh recognition site.