Mepp Frequency Research Articles

Novel effects of guanidine on the neuromuscular junction

1. 1. The effects of guanidine on the isolated mouse phrenic nerve diaphragm (MPND) and chick biventer cervicis (CBC) neuromuscular preparations were determined by myographic and electrophysiological methods. 2. 2. Guanidine at concentrations of 5–10 mM induced an initial facilitation followed by neuromuscular blockade in both preparations. In the isolated MPND such blockade was associated with the abolition of miniature end-plate potentials (MEPPs), but in the CBC the acetylcholine-induced contracture remained unimpaired. After guanidine removal, a heretofore undescribed pronounced facilitation of neuromuscular transmission associated with an increase in MEPP frequency was observed. Simultaneously, the muscular contractions exhibited delayed relaxation and aftercontractions. 3. 3. The K + channel opener, cromakalim (100–200 μM) inhibited both the well-described initial and the novel postremoval facilitatory effects of guanidine in a concentration-dependent manner. These findings are consistent with the proposal that guanidine blocks K + channels in motor nerve endings. 4. 4. The guanidine-induced NMB was reverted by increasing the Ca 2+ concentration (1.8–5 mM) in the nutritive solution. 5. 5. Tetrodotoxin (TTX, 1.56 μM) did not influence the increase in MEPPS frequency induced by guanidine (10 mM) but did reduce the rise in MEPPS frequency observed after guanidine removal. 6. 6. The present findings indicate that the effects of guanidine on the neuromuscular junction are more complex than currently described because they include a neuromuscular blockade and a postremoval facilitation previously unreported in the literature.

Kinetics, Ca2+Dependence, and Biophysical Properties of Integrin-Mediated Mechanical Modulation of Transmitter Release from Frog Motor Nerve Terminals

Neurotransmitter release from frog motor nerve terminals is strongly modulated by change in muscle length. Over the physiological range, there is an approximately 10% increase in spontaneous and evoked release per 1% muscle stretch. Because many muscle fibers do not receive suprathreshold synaptic inputs at rest length, this stretch-induced enhancement of release constitutes a strong peripheral amplifier of the spinal stretch reflex. The stretch modulation of release is inhibited by peptides that block integrin binding of natural ligands. The modulation varies linearly with length, with a delay of no more than approximately 1-2 msec and is maintained constant at the new length. Moreover, the stretch modulation persists in a zero Ca2+ Ringer and, hence, is not dependent on Ca2+ influx through stretch activated channels. Eliminating transmembrane Ca2+ gradients and buffering intraterminal Ca2+ to approximately normal resting levels does not eliminate the modulation, suggesting that it is not the result of release of Ca2+ from internal stores. Finally, changes in temperature have no detectable effect on the kinetics of stretch-induced changes in endplate potential (EPP) amplitude or miniature EPP (mEPP) frequency. We conclude, therefore, that stretch does not act via second messenger pathways or a chemical modification of molecules involved in the release pathway. Instead, there is direct mechanical modulation of release. We postulate that tension on integrins in the presynaptic membrane is transduced mechanically into changes in the position or conformation of one or more molecules involved in neurotransmitter release, altering sensitivity to Ca2+ or the equilibrium for a critical reaction leading to vesicle fusion.

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.

Effect of thiamine on the processes responsible for acetylcholine secretion in the frog neuromuscular synapses

The effect of vitamin B1 (thiamine, 10−10–10−3 M) on direct (transmitter secretion) and recurrent (resynthesis of the transmitter and its storage in synaptic vesicles) processes of acetylcholine (ACh) secretion was studied in the frog neuromuscular junction. In Ca2+-containing extracellular medium, the facilitatory effects of thiamine and α-latrotoxin (an increase in the frequency of miniature end-plate potentials, MEPP) were additive, regardless of the sequence of their application. After partial exhaustion of the synaptic vesicle stores caused by α-latrotoxin (2 nM) in Ca2+-free extracellular medium, thiamine accelerated the Ca2+-induced recovery of the ACh secretion. In the presence of thiamine, there were two phases in the dependence of quantum content of an end-plate potential (EPP) on stimulation frequency, which are typical of the effects of Sr2+ and Ba2+ on the ACh secretion. Under conditions of depression and postdepression recovery, the effect of thiamine on the time course of the changes in EPP amplitude was similar to that produced by Ba2+. Possible mechanisms of the effects of vitamin B1 on the processes responsible for the ACh secretion and the dependence of the MEPP frequency on the concentrations of thiamine and thiamine diphosphate are discussed in light of the above results.

Prejunctional regulatory actions of androgens on a hormone sensitive muscle.

The influence of androgens and time course of effects induced by hormone deprivation were examined on the spontaneous transmitter release in the levator ani (LA) muscle of 30-180-day-old male rats. The resting membrane potential (RMP) and miniature endplate potentials (mepps) were recorded intracellularly from LA muscle fibers of intact animals or gonadectomized at different ages. In intact animals, the frequency of mepps increased proportionately to the muscle fiber growth up to 60 days, stabilizing thereafter. Gonadectomy at any age did not affect the RMP, but increased the frequency of mepps by 65% to 140%. The effect was detected after 15 days and was unrelated to the degree of muscle atrophy. Independently of the age of gonadectomy control values of mepp frequency were restored after 90 days, while the accompanying postjunctional changes persisted. These results indicate that androgens exert a prejunctional inhibitory influence on the spontaneous transmitter release in the rat LA muscle. The transient nature of the prejunctional effect induced by hormone deprivation indicates an adjustment of nerve terminals to persistent postjunctional alterations.

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 actions of brevetoxin on phrenic nerve and diaphragm muscle in the rat

The mechanism of inhibition of skeletal muscle function by brevetoxin (PbTX-3) was examined in vitro in the rat phrenic nerve-diaphragm preparation. PbTX-3 in low concentrations (< 0.06 μM) preferentially blocked conduction in the phrenic nerve without altering the resting membrane potential of the muscle fibers. Endplate potential failure occurred in an all-or-none fashion in the presence of PbTX-3 (> 0.06 μM). An increase in the frequency of miniature endplate potentials resulting from nerve terminal depolarization was observed only after endplate potential failure. Higher concentrations of toxin (> 0.3 μM) depressed directly-elicited muscle twitches and produced significant muscle membrane depolarization. Tetrodotoxin was effective in reversing membrane depolarization and alterations in MEPP frequency caused by PbTX-3. These findings suggest that diaphragmatic failure in PbTX-3 is primarily caused by a block of impulse conduction in the phrenic nerve due to a higher sensitivity of nerve than muscle membrane to the toxin.

Age-related changes in maturation of regenerated motor innervation

The dependence of spontaneous transmitter release by regenerated nerve endings on age was studied in rat extensor digitorum longus muscle after sciatic nerve crush during the first year of life. Intracellular recordings were carried out at different times after denervation in muscles of 1.5-, 3- or 12-month-old animals. The frequency of miniature endplate potentials was detected. In order to determine the percentage of multiple innervation, nerve-evoked endplate potentials were recorded. The time course of the percentage of polyinnervated muscle cells was similar in the three groups of animals, but mepp frequency increased more slowly with increasing age. Moreover, by extrapolating the intercept of linear regression for mepp frequency vs days from denervation, a conventional initial time of reinnervation may be computed: the results are consistent with a decrease in the nerve regeneration rate during the first year of life.

The effects of muscarine and atropine reveal that inhibitory autoreceptors are present on frog motor nerve terminals but are not activated during transmission.

The role of presynaptic muscarinic receptors in modulating neuromuscular transmission was studied in the isolated sartorius muscle of the frog using electrophysiological techniques. In low calcium solutions muscarine reduced mEPP frequency and the quantal of EPPs. In solutions containing the normal calcium concentration the effect of muscarine on quantal content, but not the effect on mEPP frequency, was somewhat attenuated. Muscarine-induced reductions in the parameters of ACh release were prevented by atropine. Irrespective of the calcium concentration, atropine had no effect on mEPP frequency except where fibres were pretreated with glycerol. In experiments where evoked acetylcholine release was maintained at physiologically relevant levels, atropine had no effect on the quantal content of EPPs evoked at low frequency or on the extent of rundown in trains of EPPs evoked at high frequency.

Transduction of the modulatory effect of catecholamines at the mammalian motor neuron terminal

MEPP frequency (f) was measured in mouse phrenic nerve hemidiaphragm preparations during exposure to adrenoceptor agonist and antagonist drugs. Epinephrine, norepinephrine (NE), and phenylephrine caused a concentration-dependent increase in frequency that was blocked by prazosin but not by yohimbine or nadolol. Isoproterenol had no effect on MEPP(f). The response to NE was not affected by prior incubation of the tissues with pertussis toxin. The response was, however, reduced or abolished by prior exposure to drugs, the actions of which include protein kinase inhibition, and also to a calmodulin inhibitory concentration of W-7. H-7, an inhibitor of protein kinase C and of cyclic nucleotide-dependent kinases, was ineffective. The response to NE was enhanced by 10 mM Li+. The data indicate the existence of a presynaptic alpha 1-adrenoceptor in the motor neuron terminal and suggest that modulation of transmitter release might be mediated by inositol triphosphate liberation, Ca2+ release into the cytosol and activation of a calmodulin-dependent system.

Immunoglobulins from animal models of motor neuron disease and from human amyotrophic lateral sclerosis patients passively transfer physiological abnormalities to the neuromuscular junction.

Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons of unknown etiology. In support of the potential role of autoimmunity in ALS, two immune-mediated animal models of motoneuron disease have been developed that resemble ALS with respect to the loss of motoneurons, the presence of IgG within motoneurons and at the neuromuscular junction, and with respect to altered physiology of the motor nerve terminal. To provide direct evidence for the primary role of humoral immunity, passive transfer with immunoglobulins from the two animal models and human ALS was carried out. Mice injected with serum or immunoglobulins from the animal disease models and human ALS but not controls demonstrated IgG in motoneurons and at the neuromuscular junction. The mice also demonstrated an increase in miniature end-plate potential (mepp) frequency, with normal amplitude and time course and normal resting membrane potential, indicating an increased resting quantal release of acetylcholine from the nerve terminal. The ability to transfer motoneuron dysfunction with serum immunoglobulins provides evidence for autoimmune mechanisms in the pathogenesis of both the animal models and human ALS.

Increased MEPP frequency as an early sign of experimental immune‐mediated motoneuron disease

Intracellular recordings of miniature end-plate potentials were performed in extensor digitorum longus muscles from guinea pigs with experimental immune-mediated motoneuron destruction. In the early stages of the disease, the miniature end-plate potential frequency was elevated compared to that in control and normal animals. The amplitude and time course of the miniature end-plate potentials as well as the resting potential of the muscle fibers were not altered, which implies integrity of the postjunctional membrane. The increase in frequency of miniature end-plate potential reflects an increase of basal acetylcholine release and documents dysfunction of the presynaptic terminal of the neuromuscular junction. The increased frequency was associated with high levels of antimotoneuronal IgG in the blood and the presence of IgG at motor end-plates. These data suggest that the presynaptic terminal of the neuromuscular junction may be involved in the immune attack in animal models of motoneuron degeneration.

Ca 2+ role on the effect of phorbol esters on the spontaneous quantal release of neurotransmitter at the mouse neuromuscular junction

The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release process was studied in presence of different extracellular Ca 2+ concentrations, in the mouse phrenic-diaphragm preparation. Hemidiaphragms were incubated for 2 h at room temperature in the presence or absence of TPA. TPA increased the basal frequency of miniature end plate potentials (mepp's) in a dose-dependent manner, resulting in a maximal increase of 280% at a concentration of 0.5 μM. An inverse relationship between extracellular Ca 2+ concentration and TPA effect was observed: at high extracellular concentrations of Ca 2+ the action of TPA decreased significatively, while at low Ca 2+ concentrations the effect of TPA was remarkably augmented. The highest effect of TPA was obtained when tested in a calcium-free medium. TPA also increased mepp frequency stimulated by 10 mM K +. As at basal conditions, the effect of TPA was higher at lower concentrations of extracellular calcium. The results suggest that the effect of stimulation of PKC on neurotransmitter release at the mice neuromuscular junction is not exerted at the level of calcium influx to the nerve terminal. Moreover the action of calcium and TPA seems to be superimposed. The effect of K + on neurotransmitter release could be explained not only by depolarization of the nerve terminal but by increasing the pool of activable PKC.

Physiological differences between strong and weak frog neuromuscular junctions: a study involving tetanic and posttetanic potentiation

This paper describes the extent of release and terminal variability among normal frog sartorius neuromuscular junctions and seeks physiological correlates for these differences. Terminal length varied over approximately a 10-fold range, quantal content and release per unit terminal length ("release efficacy") over much larger ranges. For purposes of comparison of different junctions, release efficacy in a Ringer's containing 0.25 mM Ca2+ was determined in all cases. In a Ringer's containing 0.1 mM Ca2+, tetanic stimulation causes a buildup of evoked release and of miniature endplate potential (mEPP) frequency. The mEPP frequency at the end of the tetanus is proportional to the evoked release level. Following the tetanus, the mEPP frequency declines in a multiexponential fashion, with the 2 longest decay phases, representing augmentation and posttetanic potentiation (PTP), both having time constants that are positively linearly correlated with the synaptic release efficacy. Longer or higher-frequency tetanic stimulation resulted in a longer time course of decay of mEPP frequency. In a Ca2(+)-free/EGTA Ringer's, tetanic stimulation causes no evoked release, but does lead to an increased mEPP frequency, presumably due to a buildup of free Ca2+ displaced from internal stores by the Na+ influx. Following the tetanus, the mEPP frequency declines to resting level with a time constant that is essentially the same for all junctions, regardless of their release efficacy in Ca2(+)-containing Ringer's. These findings indicate that stronger terminals have a greater influx of Ca2+ per unit length during action potential invasion, but that in the absence of external Ca2+, tetanic stimulation results in comparable release of Ca2+ from internal stores in all terminals and comparable accumulation of Ca2+ in some large compartment, the subsequent emptying of which determines the time course of PTP.

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)

Morphological, physiological and biochemical observations on skate electric organ

The electric organs of two species of skate have been examined morphologically, physiologically and biochemically. They can be easily dissociated into innervated or denervated component electrocytes by a Torpedo Ringer's solution containing 1% collagenase. Collagenase treatment did not, however, separate the Schwann cell cover capping the synaptosomes. Isolated electrocytes generate normal MEPP frequencies and show evoked responses for two days in Torpedo Ringer's. The nerve terminals retain excitability and transmitter release properties up to the time of separation. Since isolated terminals and denervated electrocytes show normal ultrastructural characteristics for up to 12 h, the skate electric organ provides several preparations which are not attainable with Torpedo tissue. Acetylcholine (ACh) content of supernatant fractions containing the synaptosomes was comparable to that found in Torpedo (sps.). Collagenase specifically eliminates the basal lamina associated with the synaptic junctional region. Neuronal cell death and synaptic terminal degeneration were also noted in the adult organs of both species. The skate electric organ is ideally suited for the study of cholinergic development and transmission.

Modulation of tension production by octopamine in the metathoracic dorsal longitudinal muscle of the cricket Teleogryllus Oceanicus

ABSTRACT Application of octopamine to the metathoracic dorsal longitudinal muscle (DLM) of the cricket Teleogryllus oceanicus produced dose-dependent increases of twitch amplitude, contraction rate and relaxation rate. The threshold for octopamine effects was between 10−8 and 10−7mol l−1, while maximal effects were seen at approximately 10−5 mol l−1. The octopamine receptors were classified as octopamine2 receptors on the basis of the differential responsiveness of the muscle to the octopamine agonists naphazoline, tolazoline, clonidine and the octopamine antagonists metoclopramide and chlorpromazine. It was not possible to distinguish between octopamine2A or octopamine2B receptors in this preparation. Octopamine had both presynaptic and postsynaptic effects, since it increased both miniature end-plate potential (mEPP) frequency and muscle relaxation rate. Ata calcium concentration of 11 mmol l−1, octopamine did not affect muscle membrane potential, input resistance or EJP amplitude, but the EJP duration at half amplitude (T1/2) was slightly increased. In low-calcium saline (1.8 mmol l−1), octopamine did not affect membrane potential or T1/2, but EJP amplitude was increased. Stimulation of the octopaminergic dorsal unpaired median neuron (DUMDL), which innervates the metathoracic DLM, increased twitch amplitude in about 25% of the preparations. Failure in the other preparations was apparently due to spike conduction failure within the metathoracic ganglion. These results show that octopamine can be an important modulator of metathoracic DLM tension production.

Maturation of the spontaneous transmitter release by regenerated nerve endings in vitamin E-deficient rats

In order to verify the importance of the protection against lipid peroxidation in presynaptic differentiation and maturation, the reappearance and maturation of the spontaneous transmitter release during the extensor digitorum longus muscle reinnervation following a lesion of the sciatic nerve were studied in normal and vitamin E-deficient rats. The study was carried out by intracellular recordings in order to observe the miniature end plate potentials in the reinnervated end plates. In control and vitamin E-deficient rats the first signs of muscle innervation reappeared simultaneously, but in the latter the spontaneous transmitter release mechanism matured more slowly; furthermore, in the long-term, very low mepp frequencies continued to occur. The data suggest a slowing of the transmitter release mechanism maturation and a protracted rearrangement of innervation in the deficient rats.

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.