Effect of phasic activation on endplate potential in rat diaphragm.

Neuromuscular junction endplate potentials (EPPs) decrease quickly and to a large extent during continuous stimulation. The present study examined the hypothesis that EPP rundown recovers rapidly, thereby substantially preserving neurotransmission during intermittent compared with continuous stimulation. Studies were performed in vitro on rat diaphragm, using mu-conotoxin to allow recording of normal-sized EPPs from intact fibers. During continuous 5- to 100-Hz stimulation, EPP amplitude declined with a biphasic time course. The initial fast rate of decline was modulated substantially by stimulation frequency, whereas the subsequent slow rate of decline was relatively frequency independent. During intermittent 5- to 100-Hz stimulation (duty cycle 0.33), EPP amplitude declined rapidly during each train, but recovered substantially by the onset of the following train. The intra-train declines were substantially greater than the inter-train declines in EPP amplitude. Intra-train reductions in EPP amplitude were stimulation frequency dependent, based on both the total decline and rate constant of EPP decline. In contrast, the degree of recovery from train to train was independent of stimulation frequency, indicating low frequency dependence of inter-train rundown. The substantial recovery of EPP amplitude in between trains resulted in greater cumulative EPP size during intermittent compared with continuous stimulation. During continuous stimulation, EPP drop-out was only seen during 100-Hz stimulation; this was completed mitigated during intermittent stimulation. Miniature EPP size was unaffected by either continuous or intermittent stimulation. The pattern of rapid intra-train rundown and slow inter-train rundown of EPP size during intermittent stimulation is therefore due to rapid changes in the magnitude of neurotransmitter release rather than to axonal block or postsynaptic receptor desensitization. These findings indicate considerable rundown of EPP amplitudes within a stimulus train, with near complete recovery by the onset of the next train. This substantially attenuates the decrement in EPP amplitude during intermittent compared with continuous stimulation, thereby preserving the integrity of neurotransmission during phasic activation.

Related papers:

title: Fatigue of isolated rat diaphragm: role of impaired neuromuscular transmission.
abstract: We compared the contributions of impaired neuromuscular transmission (transmission fatigue) and impaired muscle contractility (contractile fatigue) to fatigue of the isolated rat diaphragm. To make this comparison, we measured the differences in active tension elicited by direct muscle stimulation and by indirect (phrenic nerve) stimulation before and after fatigue induced by indirect supramaximal stimulation at varying frequencies and durations. Transmission fatigue was observed after all experimental protocols. Although significant contractile fatigue was not demonstrated after brief periods of low-frequency stimulation (6 min, 15 Hz, 25% duty cycle), it was present after longer or higher frequency stimulation. We repeated the direct stimulation in the presence of neuromuscular blockade with 6 microM d-tubocurarine to demonstrate that a reduced response to stimulation of intramuscular branches of the phrenic nerve during direct stimulation was not responsible for the apparent contractile fatigue. Since we found significant decreases in the response to direct stimulation even after neuromuscular blockade, we could verify the presence of contractile fatigue. We conclude that both contractile and transmission fatigue can occur in the isolated rat diaphragm and that transmission fatigue is a much more important factor after brief periods of fatiguing contractions.

title: Fatigue of the mammalian diaphragm in vitro.
abstract: Diaphragm fatigue was studied in innervated diaphragm strips from 63 Sprague-Dawley rats. The experiments examined 1) the effect on the rate of diaphragmatic fatigue of increases in the diaphragm's duty cycle, i.e., the ratio of the period of diaphragmatic contraction (Ti) to the duration of a cycle of contraction and rest (Ttot) and 2) the possibility that impaired neural transmission contributed to the fatigue process. Alterations in the duty cycle of the diaphragm were simulated by varying the pattern of electrical stimuli applied cyclically to the phrenic nerve. Fatigue was assessed from the rate of fall of isometric tension when the muscle was made to contract 90 times/min. The contribution of neural element fatigue was assessed by comparing the tension during phrenic nerve stimulation to the tension developed when the muscle was stimulated directly. Increasing the duty cycle (Ti/Ttot) from 25 to 50 to 75% increased the rate of diaphragmatic fatigue progressively. Ttot constant at 75%, while varying Ti and Ttot, did not affect the rate of fatigue. Increases in duty cycle appear to increase the rate of fatigue by increasing the number of times the contractile process was activated. In fatigued muscle strips diaphragmatic tension was greater in directly stimulated muscle than in muscle strips activated via the phrenic nerve. The results indicate that 1) when the breathing action of the diaphragm is simulated in vitro, increases in duty cycle accelerate the fatigue process and 2) failure of transmission of phrenic impulses to diaphragmatic muscle cells contributes to the fall in tension during fatigue.

title: Relative contribution of neurotransmission failure to diaphragm fatigue.
abstract: Two procedures were used to estimate the relative contribution of neurotransmission failure (NF) to fatigue of the rat diaphragm at different rates of phrenic nerve stimulation. In one, direct muscle stimulation was intermittently superimposed on neural stimulation of the diaphragm, and the relative contribution of NF was estimated by the difference in generated tension. In a second procedure, diaphragm fatigue was induced by using either direct muscle stimulation (with complete blockade of the neuromuscular junction by d-tubocurare) or phrenic nerve stimulation. The relative contribution of NF to diaphragm fatigue was then estimated by comparing the force loss during these two modes of stimulation. With both procedures, it was observed that 1) the relative contribution of NF to diaphragm fatigue was less than 45% at each frequency of phrenic nerve stimulation; 2) the relative contribution of NF to diaphragm fatigue increased at higher rates of phrenic stimulation, reaching a maximum at 75 pulses/s; and 3) the relative contribution of NF to diaphragm fatigue reached a plateau after 2 min of repetitive stimulation.

title: Effects of DAP on diaphragm force and fatigue, including fatigue due to neurotransmission failure.
abstract: Among the aminopyridines, 3,4-diaminopyridine (DAP) is a more effective K+ channel blocker than is 4-aminopyridine (4-AP), and, furthermore, DAP enhances neuromuscular transmission. Because 4-AP improves muscle contractility, we hypothesized that DAP would also increase force and, in addition, ameliorate fatigue and improve the neurotransmission failure component of fatigue. Rat diaphragm strips were studied in vitro (37 degrees C). In field-stimulated muscle, 0.3 mM DAP significantly increased diaphragm twitch force, prolonged contraction time, and shifted the force-frequency relationship to the left without-altering peak tetanic force, resulting in increased force at stimulation frequencies < or = 50 Hz. During 20-Hz intermittent stimulation, DAP increased diaphragm peak force compared with control during a 150-s fatigue run and, furthermore, significantly improved maintenance of intratrain force. The relative contribution of neurotransmission failure to fatigue was estimated by comparing the force generated by phrenic nerve-stimulated muscles with that generated by curare-treated field-stimulated muscles. DAP significantly increased force in nerve-stimulated muscles and, in addition, reduced the neurotransmission failure contribution to diaphragm fatigue. Thus DAP increases muscle force at low-to-intermediate stimulation frequencies, improves overall force and intratrain fatigue during 20-Hz intermittent stimulation, and reduces neurotransmission failure.

title: Developmental changes in rat diaphragm endplate response to repetitive stimulation.
abstract: Endplate potentials (epps) were recorded intracellularly from single diaphragm fibers, in vitro, of newborn (< or = 10 days, n = 10) and older (18-29 days, n = 5) rats with glass microelectrodes. The muscle was stimulated via the phrenic nerve for 1 s at 10, 20 or 50 Hz. Muscle action potentials were blocked by mu conotoxin GIIIA, a specific muscle Na+ channel blocker. In diaphragms from older animals, epps followed nerve stimulation at the 3 frequencies, with a gradual decrement in amplitude to 70% of the first epp at 50 Hz. The younger age group showed an initial enhancement of epp amplitude, followed by large variability in amplitude. These data suggest that neuromuscular transmission failure in the newborn diaphragm is secondary to variability in neurotransmitter release as compared to the more mature diaphragm.

title: Neuromuscular transmission failure during postnatal development
abstract: Neuromuscular transmission failure in rat diaphragm muscle was examined during the first two weeks of postnatal development, and was found to contribute significantly more to diaphragm fatigue induced by repetitive activation than in adult muscle. Intracellular analysis of evoked end-plate potentials indicated that neuromuscular transmission failure was due to both a block of action potential propagation and reduced synaptic efficacy.

title: Use of geographutoxin II (u‐conotoxin) for the study of neuromuscular transmission in mouse
abstract: 1 Endplate potentials (e.p.ps) were investigated in the presence of geographutoxin II (GTXII) in the mouse phrenic nerve diaphragm preparation. This toxin preferentially blocks muscle Na+ channels which allows the study of e.p.ps in the absence of nicotinic receptor antagonists or substances to depress acetylcholine release. 2 GTXII abolished muscle action potentials and antagonized the depolarization of the muscle membrane produced by the crotamine‐induced opening of Na+ channels. 3 E.p.ps as large as 19–25 mV were observed after 2–4 μg ml−1 GTXII. These concentrations of GTXII did not cause discernible changes of resting membrane potential and frequency and amplitude of miniature e.p.ps. 4 Lower concentrations (1–2 μg ml−1) of GTXII caused incomplete blockade of the muscle Na+ channel resulting in exaggerated ‘e.p.ps’, while higher concentrations of GTXII (8 μg ml−1) abolished e.p.ps by a prejunctional effect. 5 Trains of e.p.ps on repetitive stimulation after GTXII neither ran down, as in tubocurarine‐treated preparations, nor facilitated, as in low Ca2+ and/or high Mg2+‐treated preparations, and were indistinguishable from those of untreated cut muscle preparation. 6 In cut muscle preparations, GTXII did not affect the rise and decay times, amplitude or rundown of e.p.ps. 7 It is concluded that GTXII is a useful agent for studying neuromuscular transmission. This method provides e.p.ps which are neither attenuated nor modified because manipulations that alter transmitter release and postjunctional receptor responses are avoided.

title: Neuromuscular transmission in a mammalian preparation in the absence of blocking drugs and the effect of D‐tubocurarine
abstract: 1. Neuromuscular transmission in the presence and absence of D‐tubocurarine was examined in cut muscle preparations of the rat diaphragm using intracellular recording techniques. The method of localizing the end‐plates is described and evidence is presented indicating that end‐plate potential (e.p.p.) recordings were made close to the end‐plate region and that reliable estimates of the quantum content of e.p.p.s could be made.

title: Failure of neuromuscular propagation in rats
abstract: Many interpretations have been given of the mechanism of muscle fatigue. It is evident that deficient contraction may result from failure at any point in a long chain of events. Various authors disagree as to which link in this chain is the most vulnerable. However, all agree that the nerve trunk is for practical purposes indefatigable, and most authors recognize the occurrence of two main types of peripheral fatigue: fatigue of neuromuscular transmission, and fatigue of the contractile system (del Pozo, 1942; Steiman, 1943; Rosenblueth, 1950; Naess & Storm-Mathisen, 1955). Failure of neuromuscular transmission is commonly ascribed to a deficiency or an excess of transmitter substance (Feng, 1937, 1941; Rosenblueth & Morison, 1937). Other schools of thought may be distinguished. Merton (1954) places all emphasis on fatigue of the contractile mechanism, the occurrence of which, on the other hand, is denied by Ramsey & Street (1941, 1942). Other authors have stressed the importance of fatigue in the central nervous system as the determining factors in fatigue of voluntary contraction (Wailer, 1891; Reid, 1929). In the experiments about to be described, we have studied with intracellular micro-electrodes both in vitro and In situ the mechanism of neuromuscular failure in rats; a number of observations were made which, we believe, may throw some new light on this subject. A preliminary account of some of these experiments has already appeared (Krnjevid & Miledi, 1957).

title: Development of desensitization during repetitive end‐plate activity and single end‐plate currents in frog muscle.
abstract: 1. The amplitudes of end‐plate currents (EPCs) in short trains of fifteen to seventeen EPCs at 10 Hz were depressed in the presence of 10 microM‐proadifen when acetylcholinesterase (AChE) was inhibited. 2. The proadifen‐induced EPC depression was voltage‐dependent and the effect was more pronounced at negative membrane potentials. 3. In the presence of proadifen, the mean amplitude of miniature end‐plate currents (MEPCs) was reduced by 36% 5 s after the EPC train as compared with MEPCs before the train. 4. Without proadifen, but with inhibited AChE, an increase of temperature from 20 to 26 degrees C and elevation of external Ca2+ from 1.8 to 2.5 mM led to EPC amplitude depression in the train, which was also potential‐dependent. 5. After AChE inhibition, proadifen (10 microM) progressively shortened MEPC decay without significant reduction of amplitude up to 40 min of exposition. MEPCs were not affected by proadifen when AChE was active. 6. It is concluded that these postsynaptic effects of proadifen can be explained neither by its action on the resting acetylcholine receptors (AChR) nor on open ion channels but are due to its desensitization‐promoting action.

title: Synaptic depression related to presynaptic axon conduction block.
abstract: 1. The depression of synaptic transmission, which occurs during prolonged repetitive activation, was examined in the opener muscle of the crayfish walking leg. 2. Excitatory post‐synaptic potentials (e.p.s.p.s) initially facilitated but then declined to low amplitudes after about 4000 stimulus pulses had been delivered; this depression is presynaptic in origin; 3. Axon conduction blocks occured at points of bifurcation along the entire length of the presynaptic nerve. This resulted in failure of the nerve impulse to invade some branches of the terminal arborization. 4. Nerve terminal invasion failure caused either intermittent or complete inactiviation of some synaptic release sites; this was associated with depression of the post‐synaptic response. 5. The statistics of transmitter release during prolonged repetitive stimulation were examined by focal extracellular recording methods. Transmitter release could be described by binomial statistics, and depression involved a drop in m, n and p. 6. The rate of spontaneous quantal release did not decrease, however, arguing against transmitter depletion. 7. It is concluded that repetitive stimulation eventually leads to depolarization of the axon membrane. This causes impulse propagation failure which reduces the number of synaptic release sites that are activated and mimics a drop in the effective stimulation rate; both effects cause synaptic depression.

title: A study of desensitization of acetylcholine receptors using nerve‐released transmitter in the frog
abstract: 1. Desensitization of acetylcholine (ACh) receptors was studied at the frog neuromuscular junction under voltage clamp.

title: Depression, facilitation, and mobilization of transmitter at the rat diaphragm neuromuscular junction.
abstract: The characteristics of depression, facilitation, and mobilization of transmitter were examined at the rat diaphragm neuromuscular junction. Intracellular recording techniques were used to monitor end-plate potentials (EPPs), miniature end-plate potentials (MEPPs) and the muscle resting potentials. The cut-muscle technique was used to prevent muscle action potentials. Quantal release was determined by the direct method. The binomial statistical parameters, releasable store (n) and probability of release (p), were examined under various stimulating conditions to determine the basis for depression and facilitation. The present experiments demonstrate that p remains unchanged during repetitive nerve stimulation at low or moderately high frequencies. The experiments demonstrate that depression is due to a decrease in n and facilitation is due to an elevation in n. It is suggested that the increase in n during facilitation is due to a transient recruitment of inactive releasing sites. Substantial replenishment of n by mobilization occurs within a few ms after a stimulus but a slow residual rate of mobilization is needed to replenish n to resting levels.

title: Firing properties and hypercapnic responses of single phrenic motor axons in the rat.
abstract: Inspiratory phase activity was recorded from 33 phrenic motoneuron (PM) axonal fibers in anesthetized, vagotomized, artificially ventilated adult rats. During control conditions (no inspired CO2 added), the population of PM fibers could be separated into early and late onset types based on the time of firing onset relative to the onset of whole phrenic nerve activity. Mean discharge frequencies of both types were not significantly different. Compared with late PM's, early PM's had more spikes per inspiration, fired for a longer period, and the last spike occurred later and during the postinspiratory period. Further, the mean minimal interspike interval was shorter for early PM's than for late PM's. Increasing inspired CO2 to 0.03 and 0.05 resulted in earlier firing onsets and a greater number of spikes per inspiration, particularly for late PM's. Increases in mean firing frequency occurred for both PM types. Mean minimal interspike intervals for both types of PM's showed progressive reductions as CO2 rose. For almost all of the firing properties examined in this study, responses of rat PM axons were similar to those previously reported for the cat.

title: Comparison of phrenic motoneuron responses to hypercapnia and isocapnic hypoxia.
abstract: This study was undertaken to compare phrenic motoneuron responses to hypercapnia and isocapnic hypoxia. Efferent activity of single phrenic nerve fibers was recorded with that of the contralateral phrenic nerve in decerebrate cats which were vagotomized, paralyzed, and artificially ventilated. At normocapnia in hyperoxia, single phrenic fibers were distributed into approximately equal "early" and "late" populations according to their onset of activity relative to the period of the phrenic burst. Elevations of PACO2 or diminutions of PAO2 resulted in progressive increases in the number of spikes per respiratory cycle and decreases in the modal interspike interval for both early and late units. Moreover, either stimulus caused an onset of late unit activity at progressively earlier portions of inspiration. At equivalent levels of peak integrated phrenic discharge achieved at normocapnia or hypercapnia as compared to normoxia or hypoxia, there were no differences in activity patterns for either early or late units. It is concluded that hypoxia-induced alterations in the activity of single phrenic motoneurons are identical to those changes resulting from hypercapnia.

title: Developmental differences in endplate response to P-type calcium channel blockade in the rat diaphragm.
abstract: Endplate potentials (epps) were recorded intracellularly from single diaphragm fibers of newborn (7-10 days, n = 11) and older (24-30 days, n = 11) rats in the presence of 100 nM omega-agatoxin IVA, a P-type Ca2+ channel blocker. The muscle was stimulated via the phrenic nerve for 1 s at 40 Hz. In both age groups epp amplitude decreased with omega-agatoxin, however the decrease was greater in the older group (mean = 60% of control vs. 40% of control in the younger group). A larger number of fibers in the older group (84% vs. 54% in the young) showed a > or = 50% decrease in epp amplitude. These data suggest that although P-type Ca2+ channels are present in the immature presynaptic nerve terminals at the neuromuscular junction, functional maturation of these channels occurs with development. This may contribute to the susceptibility to neuromuscular transmission failure in the newborn diaphragm.

title: Conus geographus toxins that discriminate between neuronal and muscle sodium channels.
abstract: We describe the properties of a family of 22-amino acid peptides, the mu-conotoxins, which are useful probes for investigating voltage-dependent sodium channels of excitable tissues. The mu-conotoxins are present in the venom of the piscivorous marine snail, Conus geographus L. We have purified seven homologs of the mu-conotoxin set and determined their amino acid sequences, as follows, where Hyp = trans-4-hydroxyproline. GIIIA R.D.C.C.T.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro6]GIIIA R.D.C.C. T.P.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro7]GIIIA R.D.C.C.T.Hyp.P.K.K.C.K.D.R.Q.C.R.Hyp.Q.R.C.C.A-NH2 GIIIB R.D.C.C.T.Hyp.M.K.C.C.A-NH2 [Pro6]GIIIB R.D.C.C.T.P.Hyp.R.K.C.K.D.R.R. C.K.Hyp.M.K.C.C.A-NH2 [M.K.C.C.A-NH2 GIIIC R.D.C.C.T.Hyp.K.K.C.K.D.R.R.C.K.Hyp.L.K.C.C.A-NH2. Using the major peptide (GIIIA) in electrophysiological studies on nerve-muscle preparations and in single channel studies using planar lipid bilayers, we have established that the toxin blocks muscle sodium channels, while having no discernible effect on nerve or brain sodium channels. In bilayers the blocking kinetics of GIIIA were derived by statistical analysis of discrete transitions between blocked and unblocked states of batrachotoxin-activated sodium channels from rat muscle. The kinetics conform to a single-site, reversible binding equilibrium with a voltage-dependent binding constant. The measured value of the equilibrium KD for GIIIA is 100 nM at OmV, decreasing e-fold/34 mV of hyperpolarization. This voltage dependence of blocking is similar to that of tetrodotoxin and saxitoxin as measured by the same technique. The tissue specificity and kinetic characteristics suggest that the mu-conotoxins may serve as useful ligands to distinguish sodium channel subtypes in different tissues.

title: Electrophysiological analysis of transmission at the skeletal neuromuscular junction.
abstract: The review is divided into two sections. The first deals with methods and problems associated with performing electrophysiological experimentation on the skeletal muscle neuromuscular junction. The second section concentrates on the computer analysis of electrophysiological data. In the first section, the various techniques available for producing skeletal muscle paralysis are described. These include the use of pharmacological manipulations, such as an excess of magnesium ions or a competitive postjunctional nicotinic acetylcholine antagonist, physiological manipulations, such as cutting the muscle fibers, and the muscle fiber sodium channel toxin, mu-conotoxin. Also, in this section, a comparison is made of the use of voltage- and current-recording techniques, including descriptions of, and solutions to, the problems associated with membrane capacitance, nonlinear summation, membrane space constant, and electrical and mechanical interference. In the second section, details are given of the types of computer system commonly used for the analysis of electrophysiological data and also the requirements of the data analysis software. The use of computer algorithms for signal detection, signal evaluation, signal averaging, and curve fitting are qualitatively described, along with some of the problems and pitfalls often associated with these methods.

title: Non‐linear summation of end‐plate potentials in the frog and mouse.
abstract: 1. End‐plate potentials (e.p.p.s) and end‐plate currents (e.p.c.s) were recorded intracellularly from muscle fibres of frog and mouse at various levels of curarization to determine the relation between the potential change and the underlying synaptic conductance change over a wide range of e.p.p. amplitudes. 2. In frog muscle fibres the e.p.p.‐e.p.c. relation was linear for e.p.p. amplitudes up to about 10 mV, beyond which the rate of increase of e.p.p. amplitude became progressively smaller as the e.p.c. amplitude increased. The equation proposed by Martin (1955) to correct for this non‐linearity consistently over‐corrected the e.p.p. amplitudes. 3. When synaptic potentials and currents of long duration were produced by ionophoresis of ACh onto the end‐plate, the voltage‐current relation showed greater non‐linearity than with nerve‐evoked responses, and correction of the synaptic potential amplitudes resulted in a linear relation. 4. The relation between e.p.p. and e.p.c. amplitudes in mouse muscle showed a greater non‐linearity than in frog muscle and over‐correction by the equation was correspondingly smaller. Theoretical voltage‐current relations were calculated for various membrane models and compared with the relations observed experimentally. The results from mouse muscle agreed with those expected for a point synaptic contact on an infinite cable; those from from muscle were consistent with simple resistive‐capacitative model with no cable extending from the synaptic region. 6. The applicability to the experimental results of several correction factors for non‐linear summation is discussed.

title: Acetylcholine release in myasthenia gravis: Regulation at single end‐plate level
abstract: In myasthenia gravis, loss of acetylcholine receptors at motor end‐plates is induced by antireceptor autoantibodies. At end‐plates in rats in which myasthenia gravis–like symptoms are induced by chronic treatment with α‐bungarotoxin, acetylcholine release is increased. Within muscles from such rats there is a strong correlation between the increase of acetylcholine release at an end‐plate and the loss of postsynaptic acetylcholine receptors, caused by the toxin. The question is whether upregulation of acetylcholine release is a clinically relevant compensatory mechanism in myasthenia gravis or only a feature of the animal model using α‐bungarotoxin. We investigated electrophysiologically the in vitro acetylcholine release at end‐plates of muscles from patients with myasthenia gravis and rats with experimental autoimmune myasthenia gravis where acetylcholine receptor reduction is caused by autoantibody attack. In both human and rat autoimmune myasthenic muscle, the mean quantal content was considerably increased compared with control levels. At each individual myasthenic end‐plate, the increase in quantal content appeared to be correlated with the reduction of the amplitude of the miniature end‐plate potential. This finding suggests the existence of an important compensatory mechanism in myasthenia gravis, in which retrograde acting factors (i.e., from muscle fiber to nerve terminal) upregulate acetylcholine release.

title: Age-associated changes in neuromuscular transmission in the rat.
abstract: The physiological changes in neuromuscular transmission associated with age were examined in rats between the ages of 1 mo (28 days) and 1 yr (364 days). Intracellular recording techniques were used to monitor end-plate potentials and miniature end-plate potentials at the rat diaphragm neuromuscular junction. Muscle action potentials were blocked by cutting the muscle fibers. Neuromuscular transmission was significantly different in 28-day-old rats compared with the older rats (42-364 days). The 28-day-old rats released fewer quanta. The statistical store in the immature rats was also significantly smaller than in the older age groups. The statistical probability of release, however, was not significantly different from the older animals. Examination of the presynaptic parameters in rats between the ages of 42 and 364 days revealed no significant increases in quantal release. It is concluded that the neuromuscular junction matures physiologically by 6 wk of age in rats and remains stable through the 1st yr of life.

title: Kinetics of synaptic depression and vesicle recycling after tetanic stimulation of frog motor nerve terminals.
abstract: We measured the time courses of two key components of the synaptic vesicle cycle during recovery from synaptic depression under different conditions, and used this and other information to create a kinetic model of the vesicle cycle. End plate potential (EPP) amplitudes were used to follow recovery from synaptic depression after different amounts of tetanic stimulation. This provided an estimate of the time course of vesicle mobilization from the reserve pool to the docked (readily releasable) pool. In addition, FM1-43 was used to measure the rate of membrane retrieval after tetanic stimulation, and the amount of membrane transferred to the surface membrane. This provided a measure of the rate of refilling of the reserve pool with recycled vesicles. The time courses of both synaptic depression and endocytosis were slowed by prolonged tetanic stimulation. This behavior could be fitted by a simple model, assuming a first-order kinetics for both vesicle endocytosis and mobilization. The results show that a nearly 20-fold decrease in the rate constant of endocytosis greatly delays refilling of the depleted reserve pool. However, to fully account for the slower recovery of depression, a decrease in the rate constant of vesicle mobilization from the reserve pool of about sixfold is also required.

title: Effect of phrenic nerve stimulation on neural transmission and diaphragmatic force generation in the dog.
abstract: The effect of supramaximal bilateral phrenic nerve stimulation on neural transmission and diaphragmatic force generation was studied in anesthetized dogs. Different combinations of duty cycle and stimulation frequency were examined during intermittent stimulation (pacing) for 15 min. The effect of different stimulation frequencies was also examined during continuous stimulation for 60 sec. Force declined more with increasing stimulation frequency and duty cycle with intermittent stimulation, and with increasing stimulation frequency with continuous stimulation. Neural transmission decreased with increasing stimulation frequencies. The changes were always greater with continuous than with intermittent stimulation. We found no unique relationship between changes in neural transmission and changes in force generation, suggesting that if neural transmission failure is causally responsible for fatigue, it does so by a very complex mechanism.

title: The effect of repetitive stimulation at low frequencies upon the electrical and mechanical activity of single muscle fibres
abstract: Summary1.The effect of repetitive stimulation at low frequencies (0.2–5/sec) upon isometric twitch tension was analysed in isolated muscle fibres of the frog.2.The number of twitches until tension decreased to 10–20% of the original value (fatigue effect) was 5000–10000 in unpoisoned fibres at 20° C, 400–800 in CN-poisoned fibres at 10° C, 1800–2500 in CN-poisoned fibres at 20° C and 100–200 in CN and IAA-poisoned fibres at 1° C.3.In fatigued fibres the overshoot of the action potential was often only a few millivolts lower than in normal fibres, the spike duration was longer, and the after-potential was increased.4.In completely exhausted fibres, which developed no tension when they were activated, the effective memrane resistance was extremely low, the resting potential was high, and early and late after-potentials were absent. Since an exchange of external Cl by SO4 caused no depolarization, it is suggested that the decrease in resistance is due to an increase in potassium conductance.5.A reduction of the external sodium concentration from 115 to 46 mM abolished the overshoot of the action potential, while twitch tension remained unaltered.6.In fatigued fibres normal twitch height could be restored by the addition of caffeine, and maximal contracture tension could be obtained after a complete depolarization in a potassium sulphate solution which contained caffeine.7.From the results quoted under 3, 5, and 6 it is concluded that neither an alteration in the action potential nor an exhaustion of energy reserves or a failure in the contractile mechanism can be regarded as the cause of fatigue.8.In fatigued fibres the S-shaped curve which relates peak tension to membrane potential (threshold curve) was shifted by about 20 mV to more positive potentials. In addition maximal contractile strength was frequently no longer attained, even after complete depolarization.9.The rate by which the contractility of depolarized fibres can be reactivated after a sudden repolarization, and probably also the steady relation between the state of the contractile system and membrane potential, were equal in normal and fatigued fibres.10.Fibres recovered from fatigue within one to three hours.11.From the results quoted under 8 and 9 it is tentatively suggested that a decline of the concentration of an “activator of contraction”—localized in some part of the tubular system—is the cause of the observed deficiency in excitation—contraction coupling.

title: Action potential fatigue in single skeletal muscle fibres of Xenopus.
abstract: Action potential fatigue has been studied in single short toe muscle fibres of Xenopus under three different conditions: in rested fibres which produced maximum tension, in fibres during post-contractile depression (PCD), a state of depressed tension generation but seemingly normal membrane properties, and in fibres de-tubulated by glycerol treatment. The fibres were stimulated continuously at 70 Hz (22.5 degrees C) and membrane potential was measured throughout the stimulation period with an intracellular microelectrode. Rested and PCD fibres exhibited similarities in the development of action potential fatigue during a 30 s stimulation period; the amplitude was reduced by 86 and 70 mV, respectively, and the duration, measured at a level of one-third of the peak amplitude, was increased from 1.1 to 4.2 and 1.3 to 3.7 ms, respectively. De-tubulated fibres were more resistant to action potential fatigue; the amplitude decreased by only 20 and 35 mV during 30 and 60 s of stimulation, respectively, and the duration was increased from 1.1 to 2.7 ms. It is concluded that action potential fatigue in skeletal muscle fibres is primarily caused by failing regenerative activity in the t-tubules, which is reflected in an altered shape of conventionally recorded action potentials.

title: Fatigue from high- and low-frequency muscle stimulation: Role of sarcolemma action potentials
abstract: This study compared the effect of high- (75 Hz, 1 min) and low- (5 Hz, 1.5 min) frequency stimulation on sarcolemmal action potentials of rat phrenic nerve-diaphragm preparations, measured in vitro at 25 degrees C. High-frequency stimulation reduced peak tetanic tension to 21 +/- 1% (means +/- SE) of initial, whereas 5 Hz stimulation produced less of a decline (71 +/- 2% of initial). Despite an initial faster rate of force recovery after 75-Hz stimulation, tetanic tension was still significantly depressed at 0.25 and 1 min relative to the values after 5-Hz stimulation (P less than 0.05). Resting membrane potential, and action potential overshoot and area were not significantly altered by fatigue. Action potential amplitude (AMP) was initially depressed by repetitive stimulation but increased significantly during recovery (P less than 0.05). No significant difference occurred in AMP recovery between the high- vs. low-frequency stimulation groups. The rate of rise and fall of the action potential was reduced after fatiguing stimulation but increased significantly with time (P less than 0.05). Moreover, the time to peak height of the action potential was prolonged by fatigue but significantly declined to resting values with time (P less than 0.05). During recovery, fatigue from high-frequency stimulation was associated with a greater prolongation in duration and time to baseline of the action potential relative to low-frequency stimulation (P less than 0.05). Action potential variables altered by stimulation generally recovered within 1 to 3 min, whereas peak tetanic tension did not completely return to resting values until 10 to 15 min of recovery. We conclude that high- and low-frequency stimulation elicits virtually identical perturbations in sarcolemmal action potentials, and thus changes in surface membrane properties cannot explain the decreased tetanic tension that follows 75-Hz stimulation. It appears that events distal to the sarcolemma are responsible for fatigue from both high- and low-frequency stimulation.

title: Presynaptic A1 inhibitory/A2A facilitatory adenosine receptor activation balance depends on motor nerve stimulation paradigm at the rat hemidiaphragm.
abstract: 1. Adenosine modulates acetylcholine (ACh) release from the rat motor nerve terminals. Tonic activation of presynaptic A1 inhibitory and/or A2A facilitatory adenosine receptors is regulated by the concentration of the nucleoside at the synapse. The parameters (frequency, duration of pulses, train length) of nerve stimulation determine the amount of transmitter and/or modulator released, and have long been proposed as important features of synaptic control. This prompted us to investigate which was the prevailing response to adenosine on evoked [3H]-ACh release from rat phrenic nerve hemidiaphragm preparations in different stimulation conditions. 2. With low-frequency, short-duration pulses (5 Hz, 40 microseconds in duration), the adenosine inhibitory tonus (approximately 30%) predominates. The magnitude of the adenosine tonic inhibition was dependent on the number of pulses (250-750) delivered in each stimulation train, e.g., the facilitatory effect of adenosine deaminase (ADA, 0.5 U/ml) and the inhibitory effect of the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI, 5 microM) reached significance only when > 250 pulses were applied. Facilitation was only observed with high concentrations of either exogenous adenosine (> 100 microM) or NBTI (> 10 microM). 3. When the stimulation pulse duration was increased to 1 ms (5 Hz, 750 pulses), endogenously generated adenosine consistently facilitated evoked [3H]-ACh release. In these conditions, ADA (0.5 U/ml) decreased evoked [3H]-ACh release by 29 +/- 4% (mean +/- SE) (n = 3), and both NBTI (3-30 microM) and adenosine (10-500 microM), which had biphasic effects with pulses of 40 microseconds, facilitated transmitter release. 4. When high-frequency "trains" (50 Hz, 40 microseconds, 500 pulses) were applied, both ADA (0.5 U/ml) and NBTI (5 microM) failed to modify evoked [3H]-ACh release. To bypass putative feedforward inhibition of ecto-5'-nucleotidase induced by released ATP, which might reduce adenosine formation during high-frequency trains, experiments containing a series of five high-frequency "bursts" (50 Hz, 40 microseconds, 100 pulses) with variable interburst intervals (5-20 s) were performed. In such conditions, the prevailing tonic response to adenosine turned out to be facilitatory, because ADA (0.5 U/ml) inhibited and NBTI (5 microM) facilitated evoked [3H]-ACh release. The magnitude of the inhibitory effect of ADA (0.5 U/ml) ranged from -9 +/- 6% (n = 4) to -54 +/- 8% (n = 5) as the interburst interval changed from 5 to 20 s, respectively. 5. Prolongation of individual pulses from 40 microseconds to 1 ms (5 Hz frequency) or increasing the frequency of stimulation (1-50 Hz, 40 microseconds) did not significantly change the excitatory effect of the A2A receptor agonist 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680C). In contrast, the inhibitory effect of the A1 receptor agonist R-N6-phenylisopropyladenosine was significantly attenuated in both stimulation conditions. 6. In conclusion, the results suggest that high-intensity, high-frequency motor nerve stimulation critically influences endogenous adenosine formation and the A1/A2A receptor activation balance, i.e., it potentiates the tonic adenosine A2A-receptor-mediated facilitation of ACh release, whereas activation of the inhibitory A1 receptors becomes less effective. A model is proposed that attempts to further elucidate adenosine's involvement in synaptic transmission adaptation.

title: Dependence of acetylcholine desensitization on the membrane potential of frog muscle fibre and on the ionic changes in the medium
abstract: 1. The rate of desensitization of the post‐synaptic membrane to prolonged action of acetylcholine was investigated during (a) potassium depolarization of the frog muscle fibre, (b) artificial changes of the membrane potential and (c) in the presence of some multivalent cations and of caffeine.

title: The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast‐ and slow‐twitch muscles.
abstract: 1. At the rat neuromuscular junction, the postsynaptic folds and the voltage‐gated sodium channels (VGSCs) within them are thought to amplify the effects of postsynaptic currents. In this study, the contribution of this effect to the safety factor for neuromuscular transmission, the ratio of the normal quantal content to the number of quanta required to reach threshold, has been estimated. 2. Normal quantal content was determined in isolated nerve‐muscle preparations of rat soleus and extensor digitorum longus (EDL) muscles in which muscle action potentials were blocked by mu‐conotoxin. The quantal content estimated from voltage recordings was 61.8 and 79.4 in soleus and EDL, respectively, and from charge measurements derived from current recordings was 46.3 (soleus) and 65.1 (EDL). 3. The threshold for action potential generation in response to nerve stimulation was determined from endplate potentials (EPPs) and endplate currents (EPCs) in preparations partially blocked with d‐tubocurarine. The number of quanta required to reach threshold was estimated from voltage recordings to be 19.7 (soleus) and 23.2 (EDL) and from charge measurements derived from current recordings to be 13.3 (soleus) and 13.0 (EDL). 4. When intracellular electrodes were used to inject current into the muscle fibre, the total charge required to reach threshold was approximately twice that of the nerve‐evoked threshold EPC. 5. The safety factor for nerve‐evoked responses at the junction was 3.5 (soleus) and 5.0 (EDL). In the extrajunctional region the safety factor estimated from injected currents was 1.7 (soleus) and 2.5 (EDL). 6. It is concluded that the effect of the postsynaptic folds and the VGSCs within them is to double the safety factor. At normal frequencies of nerve impulse activity in vivo, this effect is likely to be crucial for ensuring effective neuromuscular transmission.

title: A comparison of electromyographic and mechanical fatigue properties in motor units of the cat hindlimb
abstract: Single motor units were isolated in medial gastrocnemius (MG) or soleus (Sol) muscles of the cat. Single shocks delivered to the motor axon elicited EMG waveforms which were recorded in the muscle. The amplitude of each individual EMG waveform as well as the area under the full-wave rectified waveform were measured. Mechanical properties of the motor units were then measured so that each unit could be classified as FF, FR or S according to the criteria of Burke. Finally, each unit was stimulated continuously at 80 pulses per second (pps), and changes in electrical and mechanical responses were recorded and compared. IEMG was positively correlated with maximum tetanic tension of motor units in MG. The relationship could be fitted with a parabola showing that 'large' motor units produce relatively more electrical activity than do 'small' motor units. Two types of electrical changes were seen during continuous stimulation of motor units at 80 pps: a smooth continuous decline in IEMG was attributed to electrical changes occurring at a site or sites distal to the neuromuscular junction; and random, abrupt changes in unit-EMG waveform and hence IEMG amplitude, were attributed to failure of the action potential to propagate past axonal branch points, resulting in the intermittent failure of groups of muscle fibers to respond to stimuli. The rates of decline of electrical and mechanical activity were compared for motor units. It was found that in fast fatiguing motor units, tetanic tension declined more rapidly than did IEMG, while in slowly fatiguing motor units, IEMG declined more rapidly than did force. We conclude that fatigue of motor units induced by continuous 80 pps stimulation can occur at different sites in the motor unit, and that the site of fatigue depends on motor unit properties or motor unit type.

title: Differential susceptibility of diaphragm muscle fibers to neuromuscular transmission failure.
abstract: The pattern of glycogen utilization was used to determine whether various muscle fiber types in the rat diaphragm are differentially susceptible to neuromuscular transmission failure. Muscle segments from the midcostal region were repetitively stimulated directly or via the phrenic nerve at 10 or 75 Hz. Muscle fiber types were classified histochemically as type I, IIa, or IIb. The amount of muscle fiber glycogen depletion with direct stimulation depended on stimulation rate (75 Hz > 10 Hz) and fiber type (IIb > IIa > I). However, with nerve stimulation, muscle fiber glycogen depletion did not display the same dependency on stimulation rate (10 Hz > 75 Hz), although with stimulation at 10 Hz, the same rank order of fiber depletion was observed (IIb > IIa > I). This rank order of depletion was reversed (I > IIa > IIb) during repetitive stimulation of the nerve at 75 Hz. By intermittently stimulating the muscle directly during continuous nerve stimulation, we determined that neuromuscular transmission failure contributed significantly to the force decline after 2 min of stimulation at 75 Hz but relatively little to the force decline after 2 min of stimulation at 10 Hz. A significantly greater fraction of the force decline could be attributed to neuromuscular transmission failure with repetitive bouts of stimulation at 10 Hz. We conclude that neuromuscular transmission failure causes a significant portion of the force decline after 8 min of stimulation at 10 and 75 Hz, that all diaphragm fiber types are susceptible to neuromuscular transmission failure, but that type IIb fibers are particularly susceptible at higher frequencies of stimulation.

title: Na+ current densities and voltage dependence in human intercostal muscle fibres.
abstract: 1. Voltage‐clamp Na+ currents (INa) were studied in human intercostal muscle fibres using the loose‐patch‐clamp technique. 2. The fibres could be divided into two groups based upon the properties of INa. The two groups of fibres were called type 1 and type 2. 3. Both type 1 and type 2 fibres demonstrated fast and slow inactivation of INa. 4. Type 1 fibres had lower INa on the endplate border and extrajunctional membrane than type 2 fibres and required larger membrane depolarizations to inactivate Na+ channels by fast or slow inactivation of INa. 5. Type 2 fibres had a higher ratio of INa at the endplate border compared to extrajunctional membrane than Type 1 fibres. 6. Measurement of membrane capacitance suggested that the increase in INa at the endplate border was due to increased Na+ channel density. 7. Histochemical staining of some fibres suggested that type 1 fibres were slow twitch and type 2 fibres were fast twitch. 8. Differences in the properties of Na+ channels between fast‐ and slow‐twitch fibres may contribute to the ability of fast‐twitch fibres to operate at high firing frequencies and slow‐twitch fibres to be tonically active.

title: Potassium and sodium shifts during in vitro isometric muscle contraction, and the time course of the ion-gradient recovery
abstract: Intracellular potassium ([K+]i), interstitial potassium ([K+]inter), intracellular sodium ([Na+]i), and resting membrane potential (RMP) were measured before and after repetitive stimulation of mouse soleus and EDL (extensor digitorum longus) muscles. At rest, RMP was −69.8 mV for soleus and −74.9 mV for EDL (37°C). [K+]i was 168 mM and 182 mM, respectively. In soleus, free [Na+]i was 12.7 mM. After repetitive stimulation (960 stimuli) RMP had decreased by 11.9 mV for soleus and by 18.2 mV for EDL. [K+]i was reduced by 32 mM and 48 mM, respectively, whereas [K+]inter was doubled. In soleus [Na+]i had increased by 10.6 mM, demonstrating that the [K+]i-decrease is three times higher than the [Na+]i-increase. It is concluded that this difference reflects different activity induced movements of Na and K, and that the difference is not due to the Na/K pumping ratio. The possible involvement of the potassium loss in muscle fatigue is discussed. After stimulation RMP recovered with a time constant of 0.9 min for soleus and 1.5 min for EDL. Within the first minutes after stimulation the intracellular potassium concentration increased by 20.4 mM/min for soleus and 21.7 mM/min for EDL. Free [Na+]i decreased with less than 10 mM/min. The mechanisms underlying the different rate of changes are discussed.