Effect of Phasic Activation on Endplate Potential in Rat Diaphragm

Michelle Moyer1,2 and Erik van Lunteren1,3

Departments of  1Medicine,  2Biology, and  3Neurosciences, Case Western Reserve University and Cleveland Veterans Administration Medical Center, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Moyer, Michelle and Erik van Lunteren. Effect of Phasic Activation on Endplate Potential in Rat Diaphragm. J. Neurophysiol. 82: 3030-3040, 1999. 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 µ-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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Peripheral fatigue of skeletal muscle may result from neurotransmission failure, muscular failure, or combinations thereof. Neurotransmission failure may in turn result from three processes: failure of action potential conduction along the axon, diminished release of acetylcholine from the presynaptic terminal, and reduced excitability of the endplate due to acetylcholine receptor desensitization. Depending on the pattern and intensity of neuromuscular activation, neurotransmission fatigue can account for as low as 15% to as high as 75% or more of peripheral fatigue (Aldrich et al. 1986; Kelsen and Nochomovitz 1982; Kuei et al. 1990; van Lunteren and Moyer 1996). Furthermore, declines in neurotransmission can occur rapidly [e.g., evoked endplate potential (EPP) amplitude decreases by 25-75% over the course of <= 10 stimuli (Bazzy 1994; Fournier et al. 1991; Hong and Chang 1989; Hubbard and Wilson 1973)], and the degree of decline heightens with increasing frequency of stimulation (Fournier et al. 1991; Hubbard and Wilson 1973; Krnjevic and Miledi 1958).

Previous estimates of the susceptibility of neurotransmission to failure are based almost entirely on paradigms in which motor nerves are stimulated continuously without interruption (e.g., Bazzy 1994; Fournier et al. 1991; Giniatullin et al. 1989; Hatt and Smith 1976; Hong and Chang 1989; Hubbard and Wilson 1973; Katz and Thesleff 1957; Krnjevic and Miledi 1958; Magleby and Pallotta 1981; Sandercock et al. 1985; Smith 1980; Wilson 1979). For some muscles, especially those used for postural tasks, this is a physiologically relevant activation pattern. However, many muscles (in particular thoracic muscles used for breathing) are activated intermittently, with a duty cycle as short as ~0.25-0.35 (Kong and Berger 1986; St. John and Bartlett 1979). This pattern of activation potentially allows decrements in neurotransmission to recover between contractions, but only if the mechanism(s) that led to failure allow for a sufficiently fast rate of recovery.

The overall hypothesis of the present study is that there is a rapid recovery from the decline in neurotransmission that occurs during repetitive stimulation, thereby substantially preserving neurotransmission during intermittent compared with continuous stimulation. Furthermore, the rapid onset of and recovery from neurotransmission failure from train to train during intermittent stimulation are due to rapid changes in the magnitude of neurotransmitter release rather than to axonal block or postsynaptic receptor desensitization, because these latter processes diminish in importance as mechanisms of neurotransmission failure when stimulation is intermittent.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Male Sprague-Dawley rats (250-350 g, n = 40) were anesthetized with urethan (initial dose 1g/kg ip, with supplemental doses of 0.1-0.2 g/kg ip as needed to ensure deep anesthesia). All experiments were performed in accordance with the animal care and welfare guidelines of the National Institutes of Health. The diaphragm was removed surgically. The muscle was left intact to the ribs and the central tendon, and both phrenic nerves were isolated and removed with the diaphragm. The phrenic nerve hemi-diaphragm preparation was stretched out and pinned in a silicone-elastomer (Sylgard)-lined 35 mm Petri dish. The muscle was continuously bathed in Krebs solution composed of (in mM) 135 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 1 NaH2PO4, 15 NaHCO3, and 11 glucose, with pH adjusted to 7.25-7.35, bubbled continuously with 95% O2-5% CO2 at 20-22°C. The oxygenated Krebs solution flowed into the Petri dish to superfuse the muscle-nerve preparation, and the overflow was evacuated by suction. The solution was not bubbled directly in the Petri dish to minimize vibration during electrophysiological recording. This system was verified to be well-oxygenated using a dissolved oxygen meter (World Precision Instruments, Sarasota, FL).

Intracellular membrane potentials, EPPs, and miniature EPPs (MEPPs) of muscle fibers were recorded using intracellular glass microelectrodes fabricated with a Flaming Brown micropipette puller (Sutter Instruments, Novato, CA; resistance 5-15 MOmega when filled with 3 M KCl). Microelectrodes were lowered slowly by a micropositioner (David Kopf Instruments, Tujunga, CA) to impale the muscle fiber. The presence of MEPPs verified that the muscle fibers were impaled in the region of the endplate. The phrenic nerve was drawn into a suction electrode (A-M Systems, Everett, WA). Muscle action potentials were inhibited after 15 min equilibration with µ-conotoxin (Bachem, King of Prussia, PA) (Bazzy 1994; Breugelmans and Bazzy 1997; Hong and Chang 1989, 1991), which preferentially blocks muscle over nerve sodium channels (Cruz et al. 1985; Prior et al. 1993). In preliminary studies, a conotoxin concentration of 2.5 µM was found to allow recording EPPs of a size comparable with that reported in previous studies (Breugelmans and Bazzy 1997; Hong and Chang 1989, 1991), and this concentration was used for the data reported here. All chemicals other than conotoxin were obtained from Sigma Chemical (St. Louis, MO).

EPPs were evoked using supramaximal stimuli applied to the phrenic nerve (pulse width 0.2 ms). Muscle fibers underwent one of four protocols: 1) continuous pattern of stimulation for 2 min, 2) intermittent pattern of stimulation for 2 min, 3) continuous stimulation for 30 s followed by single EPPs at 1, 2, 5, and 10 min after the cessation of repetitive stimulation, or 4) intermittent stimulation for 30 s followed by single EPPs at 1, 2, 5, and 10 min after the cessation of repetitive stimulation. Single EPPs at 1, 2, 5, and 10 min after the cessation of repetitive stimulation were evoked in the latter two protocols to examine the recovery of the EPP. Protocols 1 and 2 examined stimulation frequencies of 5, 20, 50, and 100 Hz. A wide range of stimulation frequencies was used in this study because in the rat, phrenic motoneurons have discharge rates between 34 and 76 Hz with a mean of 56 Hz during eupnea (Kong and Berger 1986), and motor units of limb muscles have firing frequencies between 18 and 91 Hz (Hennig and Lomo 1985). Protocols 3 and 4 examined only 20 and 100 Hz and a shorter period of time, these frequencies and duration having been found to be sufficient to produce substantial EPP rundown in the studies examining longer durations of stimulation in the first two protocols. During continuous stimulation, pulses were applied for the entire duration of stimulation thus allowing no recovery time. During intermittent stimulation, trains of pulses with a train duration of 0.33 s were delivered once per second, thereby allowing the neuromuscular junction a 0.67-s recovery period before the onset of the subsequent train. Thus each train consisted of 2 pulses for 5 Hz, 7 pulses for 20 Hz, 17 pulses for 50 Hz, and 34 pulses for 100-Hz stimulation. MEPP data were recorded before and after the 30-s period of repetitive stimulation (protocols 3 and 4). Sample sizes for each experimental arm ranged from four to nine and are indicated in the figure legends. Each hemidiaphragm underwent only a single stimulation paradigm at a single frequency. Potentials were recorded with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA), digitized, collected on-line (Axotape software, Axon Instruments, Foster City, CA), and stored on the hard drive of a computer for future analysis. EPPs and MEPPs were analyzed using manually controlled cursors to measure the peak height of each potential.

EPPs were analyzed on the basis of both time and pulse number, the latter to correct for the variations in pulse number per unit of time with the different stimulation protocols. To compare rate of EPP rundown among the various stimulation paradigms, each EPP was normalized to the amplitude of the first EPP in the stimulation protocol. Decay constants were calculated for intra-train EPP amplitude declines during intermittent stimulation using the equation f(x) = Ae-bx starting at f(0) = A and decaying with time constant 1/b. Cumulative EPP amplitude curves were calculated from the normalized EPP amplitudes added together consecutively and plotted as a function of pulse number. EPP amplitude at the onset of repetitive stimulation was given a value of 1 unit, and the amplitude of all subsequent EPPs were normalized relative to this value. Normalization was done to factor out the effects of small variability in absolute EPP size (in mV) among the neuromuscular junctions, because a slight difference in absolute EPP amplitude at the onset of stimulation would have a large cumulative effect over time. EPP generation failures were counted in a train every 10 s during intermittent stimulation. During continuous stimulation, failures were counted using the corresponding number pulses. The number of failures per number of stimuli in a train at the specific frequency was expressed as a function of time and pulse number. MEPP amplitudes were measured with two manually controlled cursors to determine the voltage difference (one placed on the average baseline value, the other on the maximum peak) using Axoscope software (Axon Instruments, Foster City, CA). MEPPs were analyzed on the basis of amplitude for a period of 6 s before and after stimulation. All values presented are means ± SE. Statistical analysis of EPP amplitude in each train and percent occurrence of EPP generation failures was done with repeated measures two-way ANOVA followed by the Newman-Keuls test when the ANOVA indicated statistical significance. Decay constants were analyzed using a one-way ANOVA. Statistical comparison of MEPP amplitude before and after stimulation was done using the paired t-test. A P value of <0.05 (2-tailed) indicated significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mean EPP size at the onset of stimulation was 21.5 ± 1.2 (SE) mV and was similar among stimulation frequencies (P = 0.96). The mean resting membrane potential at the onset of stimulation was -71.4 ± 0.7 mV and also was similar among stimulation frequencies (P = 0.74). Single EPPs evoked at 0, 1, 2, 5, and 10 min in otherwise quiescent control strips declined in amplitude by <1% per minute (at the end of 10 min EPP size declined to 92.3 ± 4.8% of initial values, n = 3).

EPP rundown during continuous stimulation

EPP amplitude during continuous stimulation depended on stimulation frequency, pulse number, and time (Fig. 1). At all frequencies there was an initial rapid decrease (Fig. 1, left panel) followed by a slower rate of decline and/or a plateau over an expanded timeframe (Fig. 1, right panel). The initial rate of decline was greater at high than low stimulation frequencies, whether analyzed as a function of time or pulse number. Furthermore, the magnitude of the declines was large, with EPP size decreasing by >50% over <100 pulses at the higher frequencies. EPP amplitudes during 100- and 50-Hz continuous stimulation were significantly smaller than those during 5- and 20-Hz stimulation when compared over time and pulse number (Fig. 1). These differences were significant after the 6th pulse and 7th pulse for 100 and 50 Hz, respectively.



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Fig. 1. Endplate potential (EPP) amplitude as a function of time (A) and pulse number (B) during continuous 5-, 20-, 50-, and 100-Hz stimulation, expressed as a percent of initial values (mean ± SE). Left panels: data during the initial portion of stimulation (400 ms). Right panels: data over a expanded time frame. *Significant difference from 5 Hz. **Significant difference from 5 and 20 Hz. ***Significant difference from 5, 20, and 50 Hz. #Significant difference from 50 Hz. ##Significant difference from 20 Hz. Only 50 vs. 100 Hz values were compared statistically in the left panel of A due to differences in time frames. n = 5 for all experimental arms.

EPP rundown during intermittent stimulation

An example of EPP rundown at the onset of intermittent 20- and 50-Hz stimulation is shown in Fig. 2. There was considerable rundown during each of the 10 trains depicted (the 1st and last of which are amplified in the bottom panel), whereas there was near complete recovery by the beginning of the subsequent train. Although intra-train rundown (EPP amplitude decline within a train) was clearly evident at all frequencies, inter-train rundown (EPP amplitude decline from one train to the next) was substantially less. Average values for EPP rundown during intermittent stimulation are depicted as a function of time and pulse number in Fig. 3, A and B. During 5-Hz intermittent stimulation, EPP amplitude showed only modest intra-train and inter-train decline. During 20-, 50-, and 100-Hz intermittent stimulation, there was a progressive increase in the degree of intra-train rundown of EPP amplitude as stimulation frequency increased. For example, the amplitude of the last EPP of the first train declined to ~80% of initial during 20-Hz stimulation, ~60% of initial during 50-Hz stimulation, and ~40% of initial during 100-Hz stimulation. On the other hand, recovery of EPP amplitude between trains was considerable at all frequencies, so that inter-train EPP rundown was affected much less by stimulation frequency.



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Fig. 2. Example of EPPs recorded from the diaphragm during intermittent 20-Hz (A) and 50-Hz (B) stimulation (train duration 333 ms, train interval 1 s). Top panels in each illustrate EPPs during each of the 1st 10 trains, and bottom panels illustrate a time-expanded view of EPPs during the 1st and 10th train. Stimulus artifacts have been removed from the traces to improve clarity. EPP size decreased during the course of each train but recovered substantially from the end of one train to the onset of the subsequent train. Calibration: membrane potential 5 mV, time 500 ms (top) and 50 ms (bottom).



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Fig. 3. EPP amplitude as a function of time (A) and pulse number (B) during intermittent 5-, 20-, 50-, and 100-Hz stimulation, expressed as a percent of initial values (mean ± SE). Trains 1-5 and train 30 are depicted for 5, 20, and 50 Hz, and trains 1, 2, and 30 are depicted for 100 Hz. n = 5 for all experimental arms. C: changes in EPP size during 50-Hz intermittent stimulation based on measured EPP size compared with changes based on EPP size after correction for nonlinear summation (50-Hz data same as in A and B).

To better quantify the rate of EPP rundown during the course of each train, exponential decay time constants of the EPP amplitude rundown were calculated and are presented in Table 1 (the rate constant during 5-Hz stimulation could not be calculated due to the fact that there were only 2 EPPs per train). The rate constants shortened as stimulation frequency increased, with those during 20-Hz stimulation being significantly different from those during 50- and 100-Hz stimulation.


                              
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Table 1. Exponential decay time constants for the intra-train rate of decline in EPP amplitude for the 1st, 2nd, and 30th train during intermittent stimulation at 20, 50, and 100 Hz

The maintenance of EPP amplitude over time during the 1st, 7th, and 17th pulse in each train as a function of stimulation frequency is illustrated in Fig. 4. (The 7th pulse was the last EPP in a 20-Hz train, and the 17th pulse was the last EPP in a 50-Hz train.) The size of the first EPP in each train was similar for all frequencies, with the magnitude of the dimunition being frequency independent (P = 0.96). However, there was more depression within each train as stimulation frequency increased. At the seventh pulse in each train, the EPP amplitudes during 50- and 100-Hz stimulation were significantly lower than the EPP amplitudes during 20-Hz stimulation, starting after 50 s of stimulation. However, the EPP amplitudes during 50- and 100-Hz stimulation were not significantly different from each other. By the 17th pulse in each train, the EPP amplitudes during 50 and 100 Hz were significantly different from each other, even initially.



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Fig. 4. Changes over time in EPP amplitude within each train are compared for intermittent 20-, 50-, and 100-Hz stimulation, expressed as a percent of initial values (mean ± SE). Panels depict values for the 1st EPP of each train, the 7th EPP of each train, and the 17th EPP of each train. The 7th EPP of the train corresponds to the last EPP of the train during 20-Hz stimulation, and the 17th EPP of each train corresponds to the last EPP of the train during 50-Hz stimulation. *Significant difference from 5 Hz. **Significant difference from 20 Hz. ***Significant difference from 50 Hz. n = 5 for all experimental arms.

Comparison of EPP rundown during continuous and intermittent stimulation

EPP amplitudes of intermittently stimulated neuromuscular junctions are compared with EPP amplitudes of continuously stimulated neuromuscular junctions with respect to time and pulse number in Fig. 5. During 5-Hz stimulation, continuous and intermittent stimulation caused similar reductions in EPP rundown. During 20-, 50-, and 100-Hz stimulation, EPP amplitude during continuous stimulation decreased significantly faster than intermittent stimulation when compared with respect to the amplitude of the first EPP in each train but not when compared with respect to the amplitude of the last EPP in each train. As expected, during intermittent stimulation, the amplitude of the last EPP of each train was significantly lower than that of the first EPP of each train, especially during 50- and 100-Hz stimulation.



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Fig. 5. Amplitude of EPPs during continuous vs. intermittent stimulation over time (A) and pulse number (B), expressed as a percent of the initial EPP amplitude (mean ± SE). Train stimulation is indicated by solid symbols and continuous stimulation by open symbols. EPP amplitudes for train values are the 1st EPP in the train (---) or the last EPP in the same train (· · ·). Continuous data were measured at the corresponding time to the 1st EPP in the train during intermittent stimulation. Data for 100 Hz is restricted to 1st 10 s in A due to EPP drop-out during continuous stimulation. In B the 1st and last EPP in each specified train (1-5, 10, 20, 30, and 40) were measured in addition to the corresponding EPP during the continuous stimulation protocol. *Significant difference between EPP amplitudes during continuous stimulation and the 1st EPP during intermittent stimulation. **Significant difference between the last EPP and the 1st EPP in the train during intermittent stimulation. n = 5 for all experimental arms.

Cumulative EPP amplitude values were calculated by successively adding the amplitudes of each EPP during the stimulation period to provide an estimate of the total neurotransmitter release in the neuromuscular junction. The cumulative EPP amplitudes over 600 pulses during continuous and intermittent stimulation, respectively, were 353 ± 27 and 394 ± 21 for 20 Hz, 245 ± 20 and 308 ± 18 for 50 Hz, and 137 ± 13 and 203 ± 19 for 100-Hz stimulation. The cumulative EPP amplitude was significantly greater for intermittent than continuous stimulation at frequencies of both 50 and 100 Hz (P < 0.05 and P < 0.02, respectively).

Effects of correcting EPP size for nonlinear summation

It is possible that not correcting EPP sizes for nonlinear summation underestimated the decline in neurotransmitter release in the present study. The 50-Hz data of Fig. 3 were therefore recalculated based on corrected EPP sizes, using the formula of McLachlan and Martin (1981), a reversal potential of 0 mV and a value for f (correction factor) of 0.8. EPP rundown based on corrected values was up to 10% greater than that based on measured values (Fig. 3C). However, the overall pattern of fast intra-train rundown, fast recovery from the end of one train to the onset of the next train, and slow inter-train rundown was apparent with both corrected and measured EPP data. Use of corrected EPP sizes also did not qualitatively alter the relationships between different stimulation frequencies or between intermittent and continuous stimulation (data not shown), as would be expected given that EPP size and resting membrane potential were similar at the onset of all stimulation paradigms (so that degree of correction was similar among groups as well).

Recovery protocol

EPP amplitude following the cessation of repetitive stimulation was >95% initial after 1 min of recovery time. EPP amplitude remained stable thereafter for the 10 min over which this was tested. The amplitudes of single EPPs evoked at 1, 2, 5, and 10 min after the completion of repetitive stimulation were not significantly different from initial EPP amplitudes during either continuous stimulation (P = 0.86 and 0.59 for 20 and 100 Hz, respectively) or intermittent stimulation (P = 0.81 and 0.86 for 20 and 100 Hz, respectively).

Axonal conduction block

EPPs were evoked without fail during both continuous and intermittent stimulation at 5, 20, and 50 Hz. Continuous 100-Hz stimulation led to the failure of EPP generation, starting on average 6.3 s (~630 pulses) after the onset of stimulation. After 10 s of continuous 100-Hz stimulation, EPPs were evoked during 69% ± 10 of the stimulation attempts, and this declined to as low as 60% ± 7 as stimulation continued. In contrast, EPP generation failure did not occur during intermittent stimulation at 100 Hz, even after 4,000 pulses (P < 0.0005 vs. continuous stimulation).

Postsynaptic receptor desensitization

MEPP amplitudes were measured for 6 s before and after stimulation to determine the pre- or postsynaptic contribution to EPP amplitude rundown following stimulation. The number of MEPPs analyzed averaged 104 per diaphragm sample. The mean MEPP size for all fibers before stimulation was 0.16 ± 0.01 mV, a size that has been recorded by others (Hatt and Smith 1976; Plomp et al. 1995; Wilson 1979; Wilson and Cardaman 1984). MEPP size was not significantly different before compared with after 30 s of repetitive stimulation (for continuous stimulation P = 0.65 and 0.18 for 20 and 100 Hz, respectively; for intermittent stimulation P = 0.16 and 0.28 for 20 and 100 Hz, respectively). Any trends toward declines in MEPP amplitude (average of 5%) were substantially smaller than the corresponding declines in EPP amplitude (average of 64%) in these same neuromuscular junctions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EPP rundown during intermittent stimulation

The present data provide direct evidence for the protective effects of intermittent compared with continuous stimulation. The better preservation of overall EPP size during intermittent relative to continuous stimulation is due to the repetitive inter-train recovery of EPP size, rather than to a slower overall rate of EPP decline. That is, the first EPP of each train was considerably larger than the corresponding EPP size during continuous stimulation, whereas the last EPP of each train was similar in size to that of the corresponding EPP during continuous stimulation. Furthermore, intra-train reductions in EPP amplitude are stimulation frequency dependent, based both on the total decline and on the rate constant of EPP decline. This is similar to that seen during the early portion of continuous stimulation. In addition, the degree of recovery of EPP size from train to train is relatively independent of stimulation frequency, based on findings that the decline of the first EPP of each train over time was similar at all stimulation frequencies.

One trivial explanation for these findings is that the intra-train EPP rundown and subsequent recovery in between trains result from transmitter depletion and repletion, and that the inter-train EPP rundown is the consequence of deterioration of the experimental preparation. However, this seems unlikely based on the full recovery of EPP size by 1 min after cessation of repetitive stimulation. A second explanation is that intra-train EPP rundown and the recovery from EPP rundown occur over a single-order exponential time course, in which insufficient time for recovery in between trains results in the inter-train rundown. If this were the case, however, the size of the first EPP in each train should decline faster during high compared with low frequency stimulation, which was not seen. (Any small differences noted among stimulation frequencies in the size of the 1st EPP of the train over time in Fig. 4 would disappear were the data to be expressed as a function of pulse number rather than time.) A third explanation is that EPP rundown and recovery occur over a second-order exponential time course, with the relative contribution of the fast and slow phases to the dynamic changes in EPP amplitude varying as a function of stimulation frequency. The effects of the slow phase predominate at low stimulation frequencies (e.g., 5 Hz) and account for the incomplete recovery between trains and hence much of the inter-train EPP rundown; the effects of the fast phase become increasingly apparent with increasing stimulation frequencies and account for the intra-train EPP rundown. This could explain the relative frequency-independent inter-train rundown but highly frequency-dependent intra-train rundown. Whether these two phases represent distinct cellular processes cannot be established from the present data. However, Wu and Betz (1998) have recently proposed a model in which synaptic vesicles reside in three pools: 1) a docked pool, which is located at the membrane and is ready for immediate release, 2) a reserve pool, which resides away from the membrane and replenishes the docked pool, and 3) a fused pool, which consists of vesicles exposed to the extracellular fluid during the time between exocytosis and endocytosis and which replenishes the reserve pool. It is possible that in the present study the fast intra-train decline and near-complete recovery in EPP size reflects depletion and replenishment of the docked pool, the slower inter-train decline in EPP size reflects depletion of the reserve pool, and the recovery of EPP size after 1 min of inactivity reflects repletion of the reserve pool by the docked pool. In some respects this is analogous to the distinctions that are made between "low frequency" and "high frequency" fatigue when describing changes in muscle force over time during repetitive contractions, although the time course and particularly the mechanisms of action differ for muscle force loss and EPP rundown.

There is a paucity of other data addressing alterations in EPP size or other parameters of neurotransmission failure during intermittent stimulation, and the manner in which this relates to continuous stimulation. Bark and Scharf (1986) recorded neuromuscular conduction times and muscle compound action potential areas as indirect measures of neurotransmission failure. They found greater neurotransmission failure during high compared with low frequency stimulation, and greater neurotransmission failure during continuous compared with intermittent stimulation. The limitation of the measures used by Bark and Scharf (1986) is that they reflect not only neural and neuromuscular junction function, but also the action potential properties of the muscle membrane, which can change with fatigue (Grabowski et al. 1972; Hanson 1974; Lannergren and Westerblad 1987; Metzger and Fitts 1986). More recently, Correia-de-Sa et al. (1996) compared 3H-acetylcholine release from rat phrenic nerve terminals during continuous 50-Hz stimulation (500 pulses) with that during intermittent 50-Hz stimulation (5 times 100 pulses, with 20-s interburst intervals). Total acetylcholine release during the 500 pulses was almost 3 times greater during intermittent compared with continuous stimulation (63 × 103 vs. 23 × 103 dpm/g). Although the interpulse intervals used for the intermittent 50-Hz stimulation were longer than what would normally occur during rhythmic activities, these data suggest that recovery between trains can be sufficient to substantially improve neuromuscular junction function. The studies of Bark and Scharf (1986) and Correia-de-Sa et al. (1996) are therefore consistent with the present findings.

Axonal conduction block

Studies using continuous stimulation paradigms have also found that axonal conduction block may occur concomitantly with reductions in EPP size, particularly during prolonged stimulation. One of the earlier descriptions of this did not find a consistent relationship between stimulation frequency and extent of axonal conduction block (Krnjevic and Miledi 1958). More recently, Sandercock et al. (1985) found that axonal conduction block occurred frequently during prolonged continuous stimulation at 80 Hz but was less common during 20-Hz stimulation. In addition, Fournier et al. (1991) found that during 10- to 40-Hz stimulation, propagation failure rate was very low (0-4%), whereas during 75-Hz stimulation the propagation failure rate was much higher (26%) despite the decrement in EPP amplitude being no greater than during 40-Hz stimulation. The present study is in agreement with the latter two studies, in that EPP dropout was only evident at the highest stimulation frequency and only during continuous stimulation.

Axonal conduction block occurs predominantly at axonal branch points (Smith 1980). This has been attributed to K+ efflux and Na+ influx altering transmembrane ion gradients, thereby producing a depolarizing block (Hatt and Smith 1976; Smith 1980). During continuous stimulation, the onset of axonal conduction block is slow (Hatt and Smith 1976; Smith 1980), whereas the recovery occurs rapidly, being complete after 1-2 s (Smith 1980). The disconcordancy between the rate of onset and recovery of axonal conduction block may explain the protective effect of intermittent relevant to continuous stimulation.

Postsynaptic receptor desensitization

Reduction of acetylcholine release from the presynaptic terminal is a more important mechanism than postsynaptic receptor desensitization in producing reductions in EPP size during continuous stimulation (Giniatullin et al. 1989; Katz and Thesleff 1957; Magleby and Pallotta 1981). Receptor desensitization can clearly occur in response to prolonged or repeated exogenous acetylcholine application (Katz and Thesleff 1957; Magazanik and Vyskocil 1970). However, most studies have found minimal or no desensitization when acetylcholine is released from nerve terminals, except during very high-frequency or closely paired stimulation, or in the presence of acetylcholinesterase inhibitors and/or drugs such as proadifen (Giniatullin et al. 1989; Katz and Thesleff 1957; Magleby and Pallotta 1981). Both the onset and recovery from desensitization are slow processes, generally occurring over the course of many seconds to a few minutes (Giniatullin et al. 1989; Katz and Thesleff 1957; Magleby and Pallotta 1981). The present study found no significant change in MEPP size during either continuous or intermittent stimulation, consistent with previous studies.

Influence of correcting EPP size for nonlinear summation

Several methods have been established for the correction of EPP sizes for nonlinear summation, most notably that of McLachlan and Martin (1981). In mouse diaphragm the discrepancy between measured and corrected size is negligible at EPP sizes below 5% of resting potential (McLachlan and Martin 1981). However, the discrepancy increases progressively with increasing EPP size, so that there is an ~18% difference between uncorrected and corrected values at a measured EPP size of 20% of resting potential. Correction for nonlinear summation results in EPP size more accurately reflecting the amount of neurotransmitter released by the presynaptic terminal. On the other hand, the integrity of neuromuscular transmission is ultimately dependent on whether EPP size exceeds the threshold for action potential generation postsynaptically, which is a function of actual rather than corrected EPP size. In the present study, the extent to which not correcting for nonlinear summation underestimated EPP rundown was similar for all groups. Therefore the magnitude of the protective effect of intermittent compared with continuous stimulation was not affected by analyzing measured EPP amplitudes.

Possible temperature effects

The experiments in the present study were performed at 20-22°C to assure that the phrenic nerve-diaphragm preparation remained stable throughout the setup (which included a 15-min incubation period with conotoxin) and experimental (which for selected studies included a 10-min recovery) period. It was critical that there be no deterioration of the preparation because this could have contributed to reductions in EPP size over time. The complete recovery of EPP size poststimulation indicates that the preparation did remain viable throughout the duration of the experiment. Several other earlier studies using mammalian muscle also utilized a cooler temperature (Bazzy 1994; Fournier et al. 1991; Gertler and Robbins 1978; Hubbard and Wilson 1973; McLachlan and Martin 1981; Wilson 1979; Wood and Slater 1997), which therefore enabled the present results to be directly compared with previous data. The present study used a wider range of stimulation frequencies (5-100 Hz) than has been reported for phrenic nerve firing frequencies in vivo at 37°C (34-76 Hz) (Kong and Berger 1986). This ensures that the conclusions derived from the present study are not uniquely dependent on a single stimulation frequency.

Possible influence of fiber type heterogeneity

Previous studies have suggested heightened susceptibility of fast (in particular IIb) fibers to neurotransmission failure based on extracellularly recording muscle fiber action potential properties in response to nerve stimulation (Clamann and Robinson 1985; Pagala et al. 1984; Sandercock et al. 1985) or examining glycogen depletion during muscle versus nerve stimulation (Johnson and Sieck 1993). The former approach is affected by factors distal to the neuromuscular junction. In particular, depolarization of resting membrane potential due to K+ efflux and Na+ influx may inactivate Na+ channels (Ruff and Whittlesey 1992), thereby reducing the height of action potentials (and eventually preventing action potential initiation if membrane depolarization is sufficiently severe). Changes in muscle action potential occur not only when muscle is activated by the nerve but also when muscle is stimulated directly (Lannergren and Westerblad 1987). Rate of K+ efflux during repetitive contractions is higher for fast than slow fibers (Juel 1986; Lindinger et al. 1987), and Na+ channels of fast fibers are more susceptible than those of slow fibers to inactivation in response to membrane depolarization (Ruff and Whittlesey 1992). Hence reductions in action potential size and force due to membranous ionic shifts may be greater in fast than slow fibers. The approach of examining glycogen depletion circumvents this problem. However, Johnson and Sieck (1993) examined glycogen depletion only after 8 min of stimulation, so it is not known how soon after the onset of repetitive stimulation fiber differences become manifest.

Both of the above approaches assess the point at which overt failure of neuromuscular transmission occurs at a given neuromuscular junction. This point is dependent on many factors, including the magnitude of the decline in EPP amplitude (which in turn reflects the integrity of axonal conduction, the amount of neurotransmitter release, and the extent of postsynaptic receptor desensitization), the relationship between amount of neurotransmitter released and EPP size, the safety factor of neuromuscular transmission, the extent of membrane depolarization (which as noted above differs among fiber subtypes), and the effects of resting membrane depolarization on action potential threshold. Which of these factors accounts for the greater vulnerability of fast and particularly type IIb fibers to overt neurotransmission failure is not clear from previous studies. The present study recorded changes in endplate potential size over time, and hence focused on presynaptic factors (because there was little evidence for postsynaptic receptor desensitization).

In the present study there was an inherent bias toward sampling large over small fibers and hence type IIb and IIx fibers over other fiber types. In this and other studies using conotoxin to record full-sized action potentials, there was little variability from fiber to fiber in the rate and extent of EPP amplitude reductions for a given stimulation paradigm (Bazzy 1994; Hong and Chang 1989). The low degree of variability suggests that the data were sampled from a homogeneous population of fiber types, or variability among fibers in the rate of EPP reduction is not the major factor that accounts for variability among fiber types in overt transmission failure. It is not possible to distinguish between these explanations, and hence the present data may apply mainly to fast fibers. Future studies are needed comparing slow with fast twitch muscles to more definitively address the issue of fiber type heterogeneity in the extent to which neurotransmitter release declines during repetitive stimulation.

Conclusions

The major finding of the present study was that there was better preservation of neurotransmission during intermittent compared with continuous stimulation, as assessed both as a function of time and pulse number. Specifically, the periods of quiescence in between trains during intermittent stimulation allowed for the near complete recovery of neurotransmitter release from the decline that had occurred during the previous train. The result was only modest inter-train reductions despite substantial intra-train reductions of neurotransmitter release. As a consequence, the cumulative EPP amplitude was greater during intermittent than continuous stimulation. Thus the numerous previous investigations concerning EPP amplitude rundown during continuous stimulation considerably overestimate the extent of rundown that occurs during intermittent activation.

In addition to allowing recovery from rundown of transmitter release, intermittent stimulation protected against the development of axonal conduction block during high-frequency stimulation. Receptor desensitization was not noted during continuous stimulation, and hence intermittent stimulation did not provide any protection in this regard. Therefore out of the three potential mechanisms for the decrements and recovery of EPP size during intermittent stimulation (reduction of acetylcholine release, postsynaptic desensitization, and axonal conduction block), the majority of the rapid decline and recovery of EPP size from one train to the next can be attributed to alterations in acetylcholine release.

Neurotransmission at the neuromuscular junction is an all-or-none phenomenon, in which EPP size is either sufficient or insufficient to generate an action potential. Under normal circumstances, substantially more neurotransmitter is released, and hence a larger EPP is generated than that needed to generate an action potential, the so-called safety factor for neuromuscular transmission. The safety factor ensures that neurotransmission is preserved even when EPP size decreases during repetitive stimulation. The magnitude of the safety factor in mammalian neuromuscular junctions has been estimated at 1.8-3.7 by Gertler and Robbins (1978) and 3.5-5.0 by Wood and Slater (1997). Based on these values, neurotransmission will fail when EPP height decreases by 44-83%. During continuous stimulation, once EPP size falls below the threshold value, it remains so for the subsequent duration of stimulation. Neurotransmission thereby fails completely at that neuromuscular junction, resulting in the total absence of muscle contraction. In contrast, during intermittent stimulation, the repeated good recovery of acetylcholine release during the periods of quiescence results in EPP size during the early portion of the trains remaining above threshold. As a consequence, the integrity of neurotransmission may be preserved during the early portion of the train, allowing partial muscle contraction. This may be an especially important consideration in neuromuscular junctions with a low safety factor of neurotransmission, as well as for neuromuscular junctions in which the safety factor is reduced as a result of disease (e.g., myasthenia gravis).


    ACKNOWLEDGMENTS

This study was supported in part by the Veterans Administration Medical Research Service and by Specialized Center of Research Grant HL-42215 from the National Heart Lung and Blood Institute.


    FOOTNOTES

Address for reprint requests: E. van Lunteren, Pulmonary Section, 111J(W), Cleveland VA Medical Center, 10701 East Boulevard, Cleveland, OH 44106.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 February 1999; accepted in final form 20 August 1999.


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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society