Mepp Amplitude Research Articles

Electrophysiology of the neuromuscular junction of the laminin-2 (merosin) deficient C57 BL/6J dy 2J/dy 2J dystrophic mouse

The C57 BL/6J dy 2J/dy 2J dystrophic mouse expresses an abnormal truncated form of the α2 subunit of the protein laminin-2 (or merosin), which is unable to form a stable link between the extracellular matrix and the dystrophin-associated proteins, resulting in muscular dystrophy. Morphological abnormalities of the peripheral nervous system and neuromuscular junction have also been reported. The electrophysiological properties of the neuromuscular junctions of diaphragm, extensor digitorum longus (EDL), and soleus from C57 BL/6J dy 2J/dy 2J mice and controls are described. No evidence for the presence of denervated fibres were found. Mean MEPP amplitudes were significantly increased in EDL and soleus but reduced in the diaphragm from affected mice. Mean MEPP frequencies were raised in all the dy 2J/dy 2J muscles studied. dy 2J/dy 2J muscles were paralysed by low concentrations of μ-conotoxin suggesting that embryonic (tetrodotoxin and μ-conotoxin resistant) sodium channels are not widespread on dy 2J/dy 2J muscle as has previously been reported. EPP latencies were significantly prolonged in the diaphragm and EDL but not soleus from dy 2J/dy 2J mice. Quantal contents were higher in all dy 2J/dy 2J muscles. In the dy 2J/dy 2J diaphragm failures in neurotransmission occurred and a faster rate of rundown of EPPs was apparent. Some changes appear from a direct effect of dystrophy, whilst increased MEPP frequency and quantal content, and failures in neurotransmission indicate neuronal abnormalities.

Amyotrophic lateral sclerosis: serum factors enhance spontaneous and evoked transmitter release at the neuromuscular junction.

Sera from 30 patients with sporadic amyotrophic lateral sclerosis (ALS) were tested to determine their effects at the neuromuscular junction. Spontaneous transmitter release was significantly increased, as evidenced by a 151% increase in MEPP frequency, by sera from 16 ALS patients. In addition, 16 patients' sera elevated EEP quantal content by an average of 89%. Eleven sera produced both effects. There was no consistent change in MEPP amplitude or time course, indicating the absence of a humoral effect on postjunctional ACh receptors or endplate membrane function. These results suggest that a portion of the sporadic ALS patient population possess serum factors that can alter presynaptic function of the motor nerve terminal. Evidence from the present experiments indicates that alterations at the neuromuscular junction are a result of a combination of increased Ca2+ influx into the cell and an independent increase in intracellular calcium concentration.

Non-quantal acetylcholine release in the mouse diaphragm after phrenic nerve crush and during recovery.

The progressive decline and recovery of spontaneous quantal acetylcholine (ACh) release (miniature endplate potentials, MEPPs) and the H-effect were measured in the mouse diaphragm after nerve crush and during regeneration. The H-effect is the hyperpolarization of the muscle fibre membrane produced by the addition of (+)tubocurarine, which indicates non-quantal ACh release. One hour after nerve crush the H-effect had declined to 50% of control values and 4 h later the H-effect disappeared completely. There were no substantial changes in the MEPP frequency and amplitude during the first 4 h after denervation. MEPP frequency then increased, but after 6 h of denervation it decreased and after 16 h no MEPPs were found in any of the muscle fibres. The times of onset of these denervation changes in the proximal, central and distal parts of diaphragm were similar. During reinnervation, the H-effect was detectable in all muscle parts 3 days before the reappearance of MEPPs. The H-effect developed first on day 8 in the proximal endplates and then, with a delay of 3 and 6 days, in the central and distal areas, respectively. During axonal regrowth the non-quantal release was restored before detectable quantal release. Non-quantal release is the first indication of the ability of the nerve terminal to release ACh in the process of reinnervation.

Differential effects of an L-type Ca2+ channel antagonist on activity- and phosphorylation-enhanced release of acetylcholine at the neuromuscular junction of the frog in vitro.

The effects of the selective L-type Ca(2+)-channel antagonist nimodipine on changes in the electrophysiological correlates of acetylcholine release induced by activity in the motor nerve or by inhibition of protein dephosphorylation were studied in the isolated sartorius muscle of the frog. Nimodipine (1 microM) had no effect on basal miniature endplate potential (mEPP) frequency or on the quantal content of the endplate potential (EPP) evoked at 0.33 Hz. Stimulation of the motor nerve at frequencies of 20, 40 and 50 Hz progressively increased the quantal content and at 50 Hz caused an increase in mEPP frequency measured at the end of the train. Nimodipine (1 microM) had no effect on the change in either parameter caused by activity in the motor nerve. The L-channel agonist BAYK 8644 had inconsistent effects, causing at 0.33 Hz an increase in quantal content at 55% of the neuromuscular junctions examined. Application of the phosphatase inhibitor okadaic acid (1 microM) caused an increase in miniature endplate potential and/or current [mEPP(C)] frequency but not in quantal content. The increase in spontaneous frequency was reduced by the Ca(2+)-channel blockers nimodipine (1 microM) and cadmium (75 microM). The amplitude of mEPP(C)s was increased by okadaic acid (1 microM), but neither the decay time constant of miniature endplate currents (mEPC) nor the amplitude of endplate currents evoked by iontophoretic application of carbachol was so altered. The activity of electric eel acetylcholinesterase was unchanged by okadaic acid. The present data do not support the concept that the recruitment of normally silent L-type Ca(2+)-channels contributes to activity-dependent increases in acetylcholine release. The results obtained with okadaic acid suggest that protein phosphorylation and dephosphorylation may regulate the activity of L-type channels and the packaging of acetylcholine.

Effects of Muscle Electrical Activity on the Transmission of Developing Neuromuscular Junction

Miniature endplate potentials (MEPPS) caused by the spontaneous release of ACh from the growth cone of cholinergic neurons, are recorded by the whole-cell patch-clamp technique on a large number of 1-day cultured myoballs which have contact neurites of co-cultured neurons. Both muscle cell and neuron are dissociated from the 1-day-old (about stage 20) Xenopus embryo. Frequency and/or amplitude of MEPPs can obviously increase after the repetitive high-level depolarization caused by the stimuli on muscle cells. No detectable changes of single ACh receptor channel property are observed by using the single-channel recording technique. These results suggest that the mechanism of the increase of MEPPs after electrical activity of postsynaptic muscle cells probably involve some alteration of presynaptic membrane.

Differential effects of age on neuromuscular transmission in partially denervated mouse muscle

The response of the neuromuscular junction to expansion of the motor unit after partial denervation (section of L5 root) was compared in soleus muscles from young (5-8 month) and old (25-30 month) mice. The object was to determine the relative capacity of young and old motor neurons to adapt to an enlarged functional field of innervation, and to delineate physiological parameters that are compromised under these conditions. Neuromuscular function was studied at 30, 60, and 120 d after partial denervation. The initial (18-23) and postoperative number (5-8) of motor units was the same in both age groups. Twitch strength declined in proportion to loss of motor units at 30 d but returned completely (young) or nearly completely (old) by 60 d. In old but not young muscle, the safety factor (assayed by twitch depression in low calcium) was decreased even before functional sprouting had occurred, indicating a reduced safety factor in nondenervated junctions. The proportion of fibers with "long" latencies (delay between stimulation and endplate potential) increased transiently (at 30 d) in young muscle but persisted without recovery at 120 d in regenerated junctions in old muscle. After partial denervation, decline in miniature endplate potential (mepp) amplitude, in mepp frequency, and in estimated quantal content of evoked release was relatively more pronounced in old than in young mice, and in the case of mepp amplitude and frequency, more persistent. Mepp amplitude was also decreased in presumed nondenervated junctions of old muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

On the development of behavioral tolerance to organophosphates II: Neurophysiological aspects

In a recent study (9) it was found in rats that chronic treatment with the irreversible cholinesterase inhibitors DFP or soman led to behavioral tolerance in the case of DFP, but not in the case of soman. Biochemically, no explanation was found for this difference between these two inhibitors. Notably, chronic administration of each of these inhibitors did not affect ohe availability of the nicotinic receptors at the motor endplate, in spite of very low cholinesterase activity. In an attempt to explain the different effects of these inhibitors a neurophysiological approach seemed appropriate. The spontaneous quantal release of acetylcholine from diaphragm muscles in vitro from animals chronically treated with each inhibitor showed a similar trend; compared with controls the MEPP frequency was decreased, which was significant for DFP, and the MEPP amplitude was increased, which was significant for soman. Neuromuscular function of muscle strips obtained from both DFP- or soman-treated animals appeared significantly more sensitive to additional inhibitor added in vitro. This could simply be explained by the high preexisting level of cholinesterase inhibition, but seems in contrast with the phenomenon of tolerance.

A re-examination of the effects of lanthanum on the frog neuromuscular junction.

Lanthanum (La3+, 0.1-2mM) was applied to frog cutaneous pectoris muscles at 20-25 degrees C, or at 3-5 degrees C, and the mean amplitude and rate of occurrence, mean value of r, of the miniature endplate potentials (mepps) were measured as functions of time at single neuromuscular junctions. Some muscles were fixed at 3-5 degrees C and their nerve terminals examined in the electron microscope. When 1 or 2 mM La3+ was applied at room temperature, mean value of r rose to peak values of 0.8-3.4 x 10(3) and then declined to less than 100/s after 30-60 min. When the results are corrected for the dispersion in mepp amplitudes, we estimate that approximately 1.8 x 10(6) mepps occurred in this time. If 0.1 mM La3+ was applied, or if 1 mM La3+ was removed when mean value of r was near its peak, then mean value of r remained high for at least 1 h and approximately 4 x 10(6) mepps occurred. All these mepp counts exceed the 0.7 x 10(6) quanta stored in resting nerve terminals. When 1 or 2 mM La3+ was applied at 3-5 degrees C, mean value of r rose to peak values of 50-700/s and then fell to 20-200/s after 20-30 min. If the La3+ was then removed, mean value of r declined approximately 50% over the next hour; approximately 0.7 x 10(6) mepps occurred at the junctions treated with 1 mM La3+, and their terminals still contained about 69% of their vesicles. Thus, vesicles can be recycled at 3-5 degrees C. Millimolar concentrations of La3+ reduced the mepp amplitude by 70-80% at both temperatures and abolished almost completely the depolarization produced by bath applied acetylcholine or carbachol. These effects were reversible.

Effects of an inhibitor of the synaptic vesicle acetylcholine transport system on quantal neurotransmitter release: an electrophysiological study

The drug 2-(4-phenylpiperidino)cyclohexanol (AH5183), which potently inhibits the active transport of acethylcholine (ACh) into synaptic vesicles, was used as pharmacological tool to study the functional role of synaptic vesicles in quantal transmitter release. Using microelectrode recording techniques, miniature endplate potentials (meps) and nerve-evoked endplate potentials (epps) were recorded from cutaneous pectoris neuromuscular junctions in low Ca 2+/high Mg 2+ Ringer solution, and in normal Ringer with added d-tubocurraine ( d-TC). Stimulation in the presence of AH5183 caused a 40% reduction in quantal size (mepp amplitude), depressed tetanic potentiation, and decreased the number of quanta released with each nerve impulse in the presence of d-TC. All of these effects prepared gradually and only after extended stimulation of the nerve, during which several hundred thousand quanta were released. Consequently, these findings suggest a serial one-time usage of vesicles, with little or no re-entry of recycled vesicles until after a large fraction of the original vesicles has been exhausted. The results primarily show that filling of synaptic vesicles with ACh is crucial for sustaining synaptic transmission, and gives further evidence that the ACh released by nerve impulses originates from these organelles.

Tools to differentiate immunologic and non-immunologic myasthenia gravis in infancy.

Twelve patients between 15 months and 13 years of age with clinical and pharmacological features of myasthenia gravis were studied. Repetitive nerve stimulation did not offer valuable information; the patients demonstrated either inspecific muscular decremental response or had normal behavior. Two clear groups of patients were identified after measurements of acetylcholine receptor (AChR) antibodies and MEPP amplitude recorded in the diaphragm of mice injected with sera from those patients. The first group included patients with positive AChR antibodies titers and decreased MEPP amplitude. The second one had negative AChR antibodies titers and MEPPs with normal amplitude. These data strongly suggest immunologic and non-immunologic mechanisms for the former and later respectively.

Reactivating and cholinolytic action of trimedoxime bromide at the neuromuscular junction of warm-blooded animals

The reactivating and cholinolytic action of trimedoxime bromide was evaluated during experiments on rat soleus and diaphragm muscles accoridng to the amplitude and time course of miniature end-plate potentials and currents (MEPP and MEPC respectively). This agent reactivates acetylcholinesterase (AChE) phosphorylation. The effects of trimedoxime bromide at concentrations of 5·10−6–5·10−4 M following AChE inhibition on the amplitude and duration of MEPP arises from complex interaction between the reactivating and cholinolytic effects. A separate evaluation of the reactivating effect (once the reactivating agent had been removed) revealed that this action increases throughout the entire range of trimedoxime bromide concentration: complete reactivation of AChE phosphorylation was observed under the action of 2–5·10−4 M trimedoxime bromide. Examination of the cholinolytic effect in isolation (with voltage-clamping at the muscle and intact AChE) showed that blockade of open end-plate ionic channels underlies this effect. Reduction in MEPC amplitude together with retarded (but still exponential) decay of signals were distinguishing features of this blockade, confirming that trimedoxime bromide acts as a “very fast blocker”.

Miniature endplate potentials as a tool in neurotoxicology

The toxic effect of thallium added to the bath solution was studied with intra- and extracellular recordings from mammalian nerve-muscle preparations. To elucidate the target region, 3 different functional parameters were studied: 1. (1) Post-synaptic endplate potentials (EPPs) resulting from evoked transmitter release; 2. (2) Post-synaptic miniature endplate potentials (MEPPSs) resulting from spontaneous transmitter release; and 3. (3) Presynaptic ion currents at the nerve terminal. At a concentration of 0.5 mM/l thallium acetate, EPP amplitudes were irreversibly decreased while MEPP amplitudes remained unaffected. MEPP frequencies were reversibly increased, indicating a presynaptic rather than a post-synaptic target site of thallium toxicity. The subpopulation of small MEPPs (sub-MEPPs) behaved like the MEPP population, except that upon addition of 4-AP, the sub-MEPP population was augmented at the cost of the MEPP population. In view of the slow time course of the toxic effects (30 min for a 10-fold increase of MEPP frequency, 100–180 min for a 50% reduction of EPP amplitudes), it is concluded that thallium needs to be transported across the cell membrane before it finally interferes with release mechanisms. It is hypothesised that thallium reduces the number of active sites recruited by one action potential (reduced EPP amplitude), while at the same time the probability of transmitter liberation is enhanced (increased MEPP frequency). The rather indirect mode of action of thallium was also found when presynaptic ion currents were recorded using extracellular electrodes. In proportion to the decrease of the EPP amplitudes, a reduction of all inward and outward currents was observed. This effect was also irreversible. It is concluded that in spite of some similarities, thallium behaves quite differently from bivalent heavy metals like cobalt and cadmium, which act as competitive calcium antagonists at the presynaptic nerve terminal. In these toxic substances, the time course of intoxication is much faster, the required concentration is much lower, and the inhibition of the slow calcium current is reversible.

A myasthenic syndrome with congenital paucity of secondary synaptic clefts: CPSC syndrome.

Two cases of a newly recognized myasthenic syndrome were investigated (CPSC syndrome). The course of the disease was observed for periods of 6 and 3 years. In infancy, exacerbations of the symptoms occurred during febrile illness, but thereafter the clinical course was stable and the children appeared to be only slightly handicapped. Biopsies were taken from the intercostal muscle in both patients. Microelectrode studies revealed small Mepp amplitudes. Light microscopy demonstrated predominance of type I fibers and focal type-grouping. There was a lowered cholinesterase activity and frequent branching of preterminal axons. Electron microscopy revealed that there were few, if any, folds of the postsynaptic membrane and that there were no signs of degeneration. Methods for localization of acetylcholine receptors (AChR) revealed a deficiency and altered distribution of AChRs at these postsynaptic membranes and the occurrence of extrajunctional AChRs in some muscle fibers. It is concluded that the syndrome is a clinicopathological entity, characterized morphologically by a congenital paucity of secondary synaptic clefts (CPSC syndrome).

Effects of hyaluronidase on miniature endplate potentials and currents at the frog neuromuscular junction

The effects of 0.1% testicular hyaluronidase on miniature endplate potentials and currents (MEPP and MEPC) were investigated in frog pectorocutaneous muscle. The action of hyaluronidase on preparations with armine-induced blockade of acetylcholinesterase was associated with decreased amplitude and duration of MEPP and MEPC half-decay time and rising phase. The correlation between amplitude and half-decay time of MEPP and MEPC declined at the same time, while MEPC decay remained exponential. Treating preparations having intact acetylcholinesterase with hyaluronidase increased the length of MEPC halfdecay, with duration of the rising phase and amplitude remaining constant. It is suggested that enzymatic breakdown of a proportion of the glycocalix of cells forming the neuromuscular junction and a portion of the extracellular matrix at the synaptic cleft leads to attenuation of nonspectific acetylcholine binding, thus facilitating acetylcholine diffusion into the synaptic cleft.

Acetylcholine receptors at mature and aged mouse neuromuscular junctions

The density and distribution of junctional and perijunctional ACh receptors (AChR) were studied in young (8-12 months) and old (24-25 months) C57 mice to determine: (1) if increased amplitude of spontaneous postsynaptic potentials previously reported in old C57 muscle was due to increased junctional AChR; (2) if increased extrajunctional AChR would be found in association with previously reported nerve terminal complexity; and (3) if extrajunctional AChR was present as in disused or denervated muscle. Microdissection of individual muscle fibers combined with I125-alpha-bungarotoxin labeling, gamma counting, measurement of surface area, cholinesterase stains, and autoradiography were used to obtain the results. In both young and old mice there was a sharp gradient in AChR between the end-plate and the perijunctional region. End-plate AChR densities and total AChR per end-plate were the same at old and young end-plates, as were perijunctional values. Thus, neither end-plate nor extrajunctional AChR density changes with age. An increased mepp amplitude reported previously in old CB57 animals must be due to other factors. The perijunctional AChR in old mice show no changes characteristic of disuse or denervation, or those which might give rise to the observed nerve terminal complexity.

A Congenital Myasthenic Disorder with Paucity of Secondary Synaptic Clefts: Deficiency and Altered Distribution of Acetylcholine Receptorsa

Congenital myasthenia (CM) constitutes a heterogeneous group of disorders with different underlying defects. The authors investigated a case of CM, presenting with congenital contractures. Endplate studies in the first year of life showed a developmental disorder of postsynaptic membranes. Clinical follow-up demonstrated a beneficial effect of pyridostigmine, resulting in normal motor development. Results of a second biopsy at age 4 are reported in this paper. Microelectrode study showed small Mepp amplitudes, which returned to nearly normal in the presence of neostigmine. In the electronmicroscope the postsynaptic membranes showed a paucity of infoldings, as in the first biopsy. These membranes showed only scanty, patchy enhancement with two different methods for localization of AChR. The extrajunctional membranes showed evidence of local presence of AChR. Our results show a developmental disorder of postsynaptic membranes with a deficiency and altered distribution of AChRs.

Postsynaptic inhibition of the frog neuromuscular transmission by prostaglandin E 1

Intracellular recordings were made from the frog sartorius muscle end plate. Prostaglandin (PG) E 1 (100 nM–10 μM) decreased the amplitude of the end plate potential (EPP). PGE 1 decreased the quantal size of EPPs, while it rather increased the quantum content. The frequency of miniature (m) EPPs was not affected by PGE 1. PGE 1 depressed the acetylcholine (ACh)-induced depolarization, as well as the amplitude of mEPPs. These results suggest that PGE 1 decreases the sensitivity of nicotinic ACh receptors at the end plate membrane, resulting in postsynaptic depressions of neuromuscular transmission.

Acute in vivo exposure to ethylcholine aziridinium (AF64A) depresses the secretion of quanta from motor nerve terminals

Quantal release of acetylcholine was evaluated for soleus nerve-muscle preparations removed from mice treated with the cholinergic neurotoxin AF64A. Treatment with two and three times the LD 50 (i.p.) of AF64A caused a marked reduction of the frequency of miniature end-plate potentials (mepps) and the amplitude of end-plate potentials (epps). Since the amplitude of mepps was not altered, the reduction of epps was not due to reduction in the number of molecules of acetylcholine (ACh) per quantum or the end-plate sensitivity to ACh, but to a reduction of the number of quanta released in response to a nerve action potential. While this effect was not reversed when preparations were washed for 3 h, exposure to the potassium channel blocker, 3,4-diaminopyridine returned the mean quantal content of epps to normal. These data further support a presynaptic site of action for AF64A, and suggest that it may acutely disrupt the ionic processes underlying transmitter release.

Postsynaptic inhibitory effects of phenothiazines at cholinergic synapses may not involve calmodulin.

Treatment of frog cutaneous-pectoris nerve-muscle preparations with calmidazolium (R 24571), a calmodulin-inhibitor, at concentrations of 2 X 10(-7) mol/l and 5 X 10(-7) mol/l had no discernable effect on MEPP amplitude. 10(-6) mol/l calmidazolium caused a small (10-35%) increase in MEPP amplitude in most preparations. The phenothiazine calmodulin-inhibitor chlorpromazine (5 X 10(-6) mol/l) caused a clear reduction in MEPP amplitude (20%) after 30 min treatment. Similar experiments carried out with chlorpromazine sulphoxide (a derivative of chlorpromazine that is 60 X less potent in inhibition of calmodulin-activated enzymes) produced data that were very similar to those obtained with chlorpromazine. It is concluded that the postsynaptic inhibitory effect of phenothiazines at cholinergic synapses is unlikely to involve calmodulin.

The action of thallium acetate on phasic transmitter release in the mouse neuromuscular junction

Endplate potentials (EPP's) and miniature endplate potentials (MEPP's) were recorded from neuromuscular junctions of the mouse phrenic nerve-diaphragm preparation, blocked by high Mg++ (12 X 10(-3) mol/l)-Ringer. Superfusion of the preparations with Mg++-Ringer solutions containing thallium acetate (5 X 10(-4) mol/l Tlac) decreased phasic transmitter release as judged by EPP amplitudes as well as average quantal content, until total synaptic blockade (within about 300 min) occurred. Simultaneously MEPP amplitudes remained unchanged, whereas the frequency of MEPP's increased. When EPP amplitudes and/or quantal content were reduced by 50% (usually within about 180 min), superfusion with Mg++-Ringer solution without Tlac did not restore phasic transmitter release. However, the increase in spontaneous transmitter release was reversible, as MEPP frequencies returned to normal values. 4-Aminopyridine (5 X 10(-4) mol/l 4-AP) as added to the bath solution in the state of 50%-reduced phasic release temporarily restored EPP amplitudes and average quantal content, whereas MEPP amplitudes remained unchanged. It is concluded that thallium irreversibly blocks phasic transmitter release, whereas spontaneous transmitter release is reversibly enhanced.