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Post-tetanic potentiation of miniature end-plate potential frequency at neuromuscular junction of the rat soleus muscle
amp. ~ioehem. Physiol. Vol. 8X4, No. 3, pp. 737444, 1987
0~9629/87 63.00+ 0.00 @ 1987PergamonJoumaIs Ltd
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POST-TETANIC POTENTIATION OF MINIATURE END-PLATE POTENTIAL FREQUENCY AT NEUROMUSCULAR JUNCTION OF THE RAT SOLEUS MUSCLE TD~IIIHIKo YAMASHITA*and ISAO OOTA Department of Physiology and *Department of Orthopedic Surgery, Sapporo Medical College, Sapporo 060, Japan (Received 6 October 1986)
Abstract-l.
Changes in miniature end-plate potential (m.e.p.p.) frequency by repetitive nerve stimulation were examined in the rat soleus muscle. 2. The increase of m.e.p.p. frequency was induced by repetitive stimulation and persisted for several minutes after the tetanus. That is, post-tetanic potentiation (PTP) of neuromuscular transmission was first demonstrated here in the rat soleus muscle. 3. The time course of the decay of m.e.p.p. frequency after the tetanus showed a double exponential curve which consisted of a fast decaying component (augmentation) and a slow decaying component (potentiation). 4. The magnitude of PTP depended on the stimulation frequency and its duration. It increased with the increase of duration and was at its maximum at a frequency of 100 Hz. 5. No PTP was elicited by repetitive stjm~ation under conditions in which end-plate potential (e.p.p.) was compietely suppressed, and, moreover, m.e.p.p. frequency tended to decrease after the tetanus.
INTRODUCTION Following repetitive stimulation of the presynaptic nerve, transmitter release increases for some time. This phenomenon is known as post-tetanic potentiation (PTP) (Hughes, 1958). PTP, which shows synaptic plasticity, has been observed in a variety of synapses (Bliss and Lomo, 1973; Castelluci and Kandel, 1976; Tsukahara, 1981; Brown and McAfee, 1982; Koyano et al., 1985). In neuromuscular junctions, the PTP of neuromuscular transmission has been studied under conditions of reduced quanta1 content by many investigators (Feng, 1941; Liley and North, 1953; de1 Castillo and Katz, 1954; Miledi and Thies, 1971; Magleby and Zengel, 1976; Erulkar and Rahamimoff, 1978). PTP can be observed as an increase in the spon~neous occurrence of miniature end-plate potential (m.e.p.p.), or as an increase in the amplitude of end-plate potential (e.p.p.) evoked by nerve stimulation. These two types of PTPs have similar time courses of decay (Zengel and Magleby, 1981). PTP of neuromuscular transmission has been investigated in frog sartorius muscles (de1 Castillo and Katz, 1954; Erulkar and Rahamimoff, 1978; Magleby and Zengel, 1976; Misler and Huribut, 1983) and rat diaphragms (Liley and North, 1953; Gage and Hubbard, 1966; Wilson and Skirboll, 1974). Especially in frog sartorius muscles, the kinetic properties of PTP, such as the time course of decay and the parameters of magnitude, have been analyzed in detail. However, there has been no report showing the existence of PTP in the muscles of mammalian limbs, or analyzing its kinetic properties. In this study, we examined changes in m.e.p.p, frequency by repetitive stimulation of the motor
nerve and demonstrated the existence of PTP in a rat tibia1 nerve-soteus muscle preparation. Furthermore, its basic kinetic properties, such as the time course of the decay of m.e.p.p. frequency, and the effects of changes in stimulation conditions were analyzed. MATERIALSAND METHODS Preparation and solution
Experiments were performed on a tibia1 nerve-soleus muscle preparation from male Wistar rats (200-2508 in weight). Under i.p. anesthesia using sodium pentobarbital (50 mg/kg body wt), preparations were dissected in modified Krebs solution. The dissected nerve-muscle preparations were mounted at resting length in a 30-ml organ chamber. Modified Krebs solution, bubbled with 95% O,-5% CO,, continuously circulated in the organ chamber at a rate of 2-5 mljmin. The composition of the modified Krebs solution was (mM): NaCl, 135; KCI, 5; NaHCO,, 15; Na,HP04, 1; glucose 1I. To suppress the action potential of the muscle fiber. 8-12 mM MeCl, and 1.8-2.0 mM CaCl, were added to this solution. ?he*pH was adjusted to 7.2 before use. Experiments were carried out at room temperature (Zo-25°C). A glass suction electrode was used as a stimulating electrode, and the tibia1 nerve was drawn into it. Tetanic trains of square pulses (supramaximal voltage was 5-10 V, and duration, 0.05 msec) were delivered to the nerve by an electric stimulator (Nihon Kohden, SEN-7103). The frequency and duration of repetitive stimulations were varied over the ranges of 255OOHz and l-5Ose.c, respectively.
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Recording
Figure 1 shows a block diagram of the system used for the recording and analysis of experimental data. Miniature
TOSHIHIKO YAMASHITA
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ISAO OOTA RESULTS
Changes in m.e.p.p. frequency stimulation
Date recorder
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Fig. 1. Block diagram of the system for recording and analysis of experimental data.
end-plate potentials (m.e.p.p.s) were recorded from endplate regions using an intracellular micro-electrode technique. Glass micro-electrodes were filled with 3 M KC1 and
had tip resistances of IO-15 Ma. Signals from recording electrodes were amplified by a micro-electrode amplifier (Nihon Kohden, MEZ-8201) and a high-gain pre-amplifier (Nihon Kohden, AVH-IO), and then recorded with an FM
after repetitiue nerve
Following repetitive stimulation of the motor nerve, m.e.p.p. frequency markedly increased, and then decayed gradually with time, as seen in Fig. 2. Figure 3 shows a typical time course of changes in m.e.p.p. frequency before and after repetitive stimulation. The average frequency of m.e.p.p. before the stimulation (control frequency) was 1.7 Hz. Repetitive nerve stimulation at the rate of 100 Hz for 30 set produced a large increase in m.e.p.p. frequency, and the average frequency in the 5 set immediately after the stimulation was 29.0 Hz. After that, the m.e.p.p. frequency declined rapidly to 6.6 Hz at 50 set after the tetanus. Following this fast decline, a small transient increase in m.e.p.p. frequency appeared (arrow in Fig. 3A), and then the decay changed into a slow decline phase. At 300 set after the tetanus, m.e.p.p. frequency was 3.2 Hz, which was an obvious increase over the control frequency. Miniature end plate potential frequencies after the tetanus were plotted semilogarithmically against time as shown in Fig. 3B. The two lines in this figure represent the two different exponential decays of m.e.p.p. frequency. Thus, the time course of the decay of m.e.p.p. frequency after repetitive stimulation represents a double exponential curve which consists of two components; a fast component of decay immediately after the tetanus, and the subsequent slow component of
A
tape recorder (SONY, KS-609W), while being sent to a CRT display (Nihon Kohden, VC-10) for monitoring. Miniature end-plate potentials which occurred for 100 set before, and 500 set after, the repetitive stimulation, were recorded in each trial. If any stimulus in the repetitive stimulation failed to evoke an end-plate potential (e.p.p.), the trial was discarded. Intervals between trials were at least 20 min, to allow the increased m.e.p.p. frequency to return to the control level. Analysis The data recorded on video tapes were analyzed by a data analyzer (Nihon Kohden, ATAC-450). Miniature end-plate potential frequency was measured by a pulse count program and represented in histograms in real time. The control frequency was defined as the average of m.e.p.p. frequencies for 1OOsec before the repetitive stimulation. Since it was impossible to identify and count m.e.p.p.s during the tetanus because of the overlap of e.p.p.s and stimulation pulses, the data during the tetanus were excluded from analysis. To present a detailed picture of the time course of the decay of m.e.p.p. frequency after the tetanus, the pulse count program was modified by a ‘moving-bin technique’ (Rahamimoff and Yaari, 1973). The m.e.p.p. frequency was calculated for a block of time (bin size), and then the block was advanced in time by steps (A bin) smaller than the bin size. Therefore, the block moved (bin/A bin - 1) times. The m.e.p.p. frequency was plotted at the center of the time bin for each successive position. In this study, bin size was 25 set and A bin size was S sec. The histograms were drawn by an X-Y plotter and the values of m.e.p.p. frequencies were printed by a printer. The time constant of the decay of m.e.p.p. frequency was calculated by the analysis of exponential regression using a personal computer (NEC, PC-9801).
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lOmst3c Fig. 2. Post-tetanic potentiation of miniature end-plate potential frequency due to repetitive stimulation of motor nerve at 1OOHz for 30 sec. A shows m.e.p.p. frequency before the tetanic stimulation. B shows m.e.p.p. frequency immediately after the tetanic stimulation. C, D and E show m.e.p.p. frequency at 30, 60 and 120 set, respectively after the tetanic stimulation. Intracellular recording was done in a modified Krebs solution containing 2.0 mM CaCl,, and 10 mM MgCl,. Calibration: vertical bar, 0.5 mV; horizontal bar, 10 msec.
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Fig. 3. Effect of tetanic stimulation (100 Hz, 30 see) on m.e.p.p. frequency. (A) Time course of changes in m.e.p.p. frequency before and after tetanic stimulation. A moving bin display was used, with a bin size of 25 set and A bin size of 5 sec. The m.e.p.p. frequency before the tetanic stimulation (control frequency) was 1.7 Hz. The m.e.p.p. frequency was 29.0 Hz immediately after the tetanic stimulation and 3.2 Hz at 3OOsec after the stimulation. The arrow indicates a rebound of m.e.p.p. frequency after a fast decline. The medium contained 2.0 mM CaCl,, IO mM MgC12modified Krebs solution. (B) Semilogarithmic plot of post-tetanic changes in m.e.p.p. frequency shown in A. The two straight fines indicate the two different phases of decay, au~en~tion and potentiation.
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Fig. 4. Post-tetanic potentiation of m.e.p.p. frequency due to stimulation at various rates (denoted by each set of points). The frequency of m.e.p.p. was normalized by dividing by the control frequency. The number of stimuli was constant at 2000. Moving bins: bin = 25 set, A bin = 5 sec.
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decay. Figure 3B shows the re-increase of m.e.p.p. frequency which appeared at the intersection of the two lines. This indicates that the small re-increase (rebound) of m.e.p.p. frequency seen in Fig. 3A occurred during the transition phase between the two components. The time constant of the fast decaying component (augmentation) was 31.2 set and that of the slow decaying component (potentiation) was 163.9sec. M.e.p.p. frequency should return to the control level at about 10 min after the tetanus according to the analysis of exponential regression; that is to say the PTP should persist for about 10 min.
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ISAO OOTA
tetanus in two preparations, so that it was impossible to calculate the time constants in these trials. Effect of repetitive stimulation on m.e.p.p. frequency when e.p.p. was suppressed
End-plate potential (e.p.p.) can be observed in low-Ca*+, high-Mg2+ solutions, since quanta1 contents are reduced enough to suppress the action potential of muscle fiber. However, when quanta1 contents are more strongly reduced, even e.p.p. is suppressed. The effect of repetitive stimulation on m.e.p.p. frequency under such conditions was examined. In many trials using a solution containing
Effect of the frequency of stimulation on PTP
The correlation between the magnitude of PTP and the stimulation frequency was examined by changing the frequency step-by-step while maintaining a constant number of stimuli (2000). As shown in Fig. 4, the magnitude of PTP varied with the changes in the frequency. The magnitude of PTP was estimated by calculating the following parameters in each trial: (1) the ratio of m.e.p.p. frequency in the 5 set immediately after the tetanus/the control frequency (normalized initial post-tetanic frequency, that isf); (2) the time constant of the augmentation phase (ra) and (3) the time constant of the potentiation phase (rp). The results from two different preparations are shown in Fig. 5. The PTP series was obtained from a single junction in each preparation. The stimulation frequency was varied from 25 to 500 Hz. The values off increased with the increase of stimulation frequency from 25 to 100 Hz, and decreased with the increase of frequency from 100 to 500Hz (Fig. SA). Similarly, the values of r, and tp increased with each stimulation frequency up to 100 Hz, and then decreased with successive frequencies above 100 Hz (Fig. 5B and C). Thus, the parameters depended on the stimulation frequency and their maximum values were at the frequency of 100 Hz. The potentiation phase was not clear in two preparations at 25 Hz, and in one preparation at 500 Hz, so that it was impossible to calculate the time constants in these trials. Efect
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The correlation between the magnitude of PTP and the duration of the stimulation was examined by changing the duration step-by-step, while maintaining a constant frequency of 100 Hz. As shown in Fig. 6, the magnitude of PTP varied with the changes in the duration of stimulation. The PTP magnitude was estimated by calculating the above-mentioned parameters. The results from three different preparations are shown in Fig. 7. The PTP series was obtained from a single junction in each preparation. The duration of the stimulation varied from 1 to 50 sec. The value of fincreased with the increases in the stimulation duration in all preparations (Fig. 7A). The values of r, and 7p also increased with the increases in the duration of the stimulation (Fig. 7B and C). The augmentation phase and potentiation phase did not appear at I-set tetanus in any preparations, and the potent&ion phase drd not appear at 5-set
Fig. 5. Effect of the stimulation frequency on the parameters of the magnitude of PTP. A: The no~ali~d initial posttetanic frequency (f ). B: The time constant of augmentation (ra). C: The time constant of potentiation (T,). The results obtained from two different preparations are shown. The number of stimuli . .was . constant at 2000. The values of the parameters reached their maxima at a stimulation rate of 100 Hz in both preparations.
PTP at the rat neuromuscular jun~ion
Time
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Fig. 6. Post-tetanic potentiation of m,e.p.p. frequency due to stimulation of various durations (denoted by each set of points). The stimulation frequency was constant at 100 Hz. Moving bins: bin = 25 set, A bin = 5 sec. The frequency of m.e.p.p. was normalized by dividing by the control frequency.
1.8 mM CaCl, and 12 mM MgCI,, failures of e.p.p. occurred during repetitive stimulation. Under such conditions, experiments were performed on preparations in which all stimuli during the tetanus failed to evoke e.p.p. The duration of the stimulation varied from 5 to 40 set, with a constant frequency of 100 Hz. Miniature end-plate potential frequency was not increased by repetitive stimulation, as can be seen in Fig. 8. The correlation between the value offand the duration of the stimulation is shown in Fig. 9. The value off remained at about 1.0 in spite of increases in the duration of the stimulation. Thus, repetitive nerve stimulation under these conditions did not produce PTP. Furthermore, the correlation between the value off’, the ratio expressed by the average m.e.p.p. frequency for 1OOsec after the tetanus/the control m.e.p.p. frequency, and the duration of the stimulation was examined (Fig. 9). The values off’ were Iess than 1.O at every stimulation duration, and tended to decrease with the increase of the duration of the stimulation. Thus, m.e.p.p. frequency after the tetanus showed a tendency to be more and more depressed with the increases in the stimulation duration. DlscussIoN The present study shows that repetitive nerve stimulation causes a remarkable increase in m.e.p.p. frequency, and that the increase persists for several minutes after the tetanus in rat tibia1 nerve-soleus muscle preparations. Thus, post-tetanic potentiation (PTP) of neuromuscular t~smission was demonstrated in the rat soleus muscle. According to previous studies on the PTP of neuromuscular transmission in the frog sartorius muscle (Magleby and Zengel, 1976; Zengel and Magleby, 1981), the decay of m.e.p.p. frequency is composed of four ex-
ponentially decaying components; the first facilitation, the second facilitation, augmentation and potentiation. The time constants of each component were reported as follows: the first facilitation, 47 + 9 msec (mean t_ SE); the second facilitation, 472 + 108 msec; augmentation, 6.9 & 1.6 set; and potentiation, 82 + 25 set for a tetanus with 100-200 stimuli (Zengel and Magleby, 1981). Another report on the frog revealed that, when the changes in m,e.p.p. frequency were recorded per second for several minutes, its time course represented a double exponential curve composed of two components; the fast decaying phase (au~en~tion), and the following slow decaying phase (potentiation). Since facilitation is a very short phase which appears immediately, within 1 set after the tetanus, it cannot be detected by observing changes per second (Lev-Tov and Rahamimoff, 1980). Considering these findings, and the results of this experiment, such as the time course and the PTP time constants of decay, the fast and slow components of PTP found in this experiment correspond to augmentation and potentiation, respectively. Therefore, t, and rp represent the time constants of augmentation and potentiation, respectively. As mentioned above, it was not possible to measure facilitation in this experiment. Lev-Tov and Rahamimoff (1980) have reported on the kinetic properties of the PTP of neuromuscular transmission in the frog sartorius muscle. In their report, the value off (the normalized initial posttetanic frequency) was 18.36 + 8.89 (mean + SE) for a tetanus of 100 Hz for 20 set, and 48.45 f 21.59 for a tetanus of 100 Hz for 40 sec. In contrast, under the same conditions, the respective values off in our experiment using the rat soleus muscle were 7.31 + 0.40 and 14.20 f 2.43. These values are smaller than those of the frog sartorius muscle. The time constants of augmentation (7,) for 100-HZ
142
TOSHIHIKO
YAMASHITA and ISAOOQTA
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stimulation in the frog were 5.32 + 2.01 set for the 20-set tetanus, and 9.29 + 2.05 set for the 40-set tetanus, whereas in our expe~ment the figures were 27.10 i_ 9.39 and 45.50 rfl 5.50 set, respectively. These results indicate that the augmentation phase in the rat soleus muscle persists longer than that in the frog sartorius muscle. In contrast, the time constants of the potentiation phase (rr) were 132.72 + 19.90 set for the lOO-Hz-20-set tetanus, and 163.05 + 31.74 see for the lOO-Hz-40-set tetanus in the frog, whereas, in our experiment, the respective zp for the rat were 85.33 f. 20.96 and 141.14 f 27.54 sec. Thus, it is clear that the potentiation phase in the rat soleus muscle is shorter than that in the frog sartorius muscle. The parameters of the magnitude of PTP increased depending on the duration of the repetitive stimulation (Fig. 7). They also depended on the stimulation
frequency, and reached their maximum at a frequency of 100 Hz (Fig. 5). These results in the rat soleus muscle show the same kinetic properties as in the frog neuromuscular transmission, in that the magnitude of PTP depends on the duration and frequency of the repetitive stimulation (Lev-Tov and Rahamimoff, 1980). As the above results demonstrate, the kinetic properties of PTP of neuromuscular transmission in the rat soleus muscle are similar to those in the frog sartorius muscle in terms of the time course of the decay and the dependence on the stimulation frequency and duration, though there is a certain difference in their ma~itudes. Previous works on the frog neuromuscular junction have shown that Ca2+ plays an important role in the PTP mechanism, and the parameters of the magnitude of PTP give some indication of the kinetics of Caz+-metaboIism in the nerve terminal (LevTov and Rahamimoff, 1980; Zengel and Magleby, 1981; Pawson and Grinnel, 1984). An action potential at the motor nerve terminal induced by a nerve stimulus causes a voltage-dependent Caz+ influx into the terminal, and provokes the quanta1 release of the transmitter (Katz, 1969). Therefore, high frequency stimulation of the motor nerve produces a large increase in the concentration of intracellular Ca2+ ([Ca*‘]i”) and leads to an increase in the probability of transmitter release, which appears as an increase of m.e.p.p. frequency (tetanic potentiation). Therefore, if m.e.p.p. frequency immediately after the tetanus is divided by the control frequency, the resulting ratio (f) would reflect the amount of Ca2+ influx induced by repetitive stimulation. The augmentation phase represents the processes responsible for reducing the amount of intracellular Ca2+ which entered the nerve terminal during the tetanus (Lev-Tov and Rahamimoff, 1980), so that the time constant of augmentation reflects the rate of Ca*+ extrusion from the nerve terminal. The potentiation phase is prolonged by processes maintaining a high basal level of [Ca’+],, such as the following: (1) the effects of Ca’+ which entered during the tetanus and was not completely extruded (Erulkar and Rahamimoff, 1978), (2) Ca2+ entry through a Ca’+-Na+ exchange (Misler and Hurlbut, 1983), (3) mobili~tion of Ca*+ from internal stores (Zengel and Magleby, 1981) and (4) increased permeability of the terminal membrane for Ca*+ (Zengel and Magleby, 1981). As mentioned earlier, this study shows thatfand zp in the rat soleus muscle were smaller than those in the frog sartorius muscle, while 7, in the rat was larger than that in the frog. These results indicate that, in the rat soleus muscle, the amount of Ca2+ influx during the tetanus is smaller, that its extrusion is slower, and that the subsquent entry or mobilization of Ca* + in the slowly decaying component is smaller than that in the frog sartorius muscle. Thus, the nerve terminal in the rat soleus muscle appears to have a smaller Ca’+ conductance than that in the frog sartorius muscle. In the present experiment, it was revealed that the magnitude of PTP depended on the duration and frequency of repetitive stimulation in the rat soleus muscle. When the duration of the tetanus was prolonged, the amount of Ca’+ influx might increase because of the increase in the number of action
PTP at the rat neuromuscular junction
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potentials, and consequently, the magnitude of PTP increased with the increase in the duration of the stimulation. However, the magnitude of PTP reached its maximum at a frequency of 100 Hz, and decreased gradually with both frequency increases and decreases. In studies on the giant synapse of the squid (Llinas et al., 1981; Llinas, 1982), the duration of Ca* + current, induced by a presynaptic action potential, was about 1.5msec In contrast, the interval between stimuli is 40 msec at a frequency of 25 Hz. Thus, it appears that Ca*+ efflux occurs between voltage-dependent Ca2+ influxes, and the accumulation of CaZ+ in the nerve terminal is reduced. The amount of Ca*+ ef?lux is gradually reduced with the increase of the stimulation frequency to 50 and 100 Hz because of the decrease of the interval time to 20 and IOmsec, so that the magnitude of PTP
increases with frequency up to 100 Hz. In contrast, at the frequency of 200 Hz, the interval between stimuli is 5 msec, and little Ca*+ is extruded, so that CaZC largely a~umulates in the the terminal. Consequently, by a mechanism similar to the Ca-induced Ca inactivation which has been observed in the neurons of molluscs (Brehm and Eckert, 1978; Fckert and Tillotson, 1981; Plant and Standen, 1981; Chad et al., 1984), Ca*+ influx could be suppressed, and the magnitude of PTP is reduced. The repetitive stimulation of 500 Hz presumably exceeds the maximal frequency which can be physiologically followed, so not all stimuli would be effective. For the reasons mentioned above, repetitive stimulation of around 100 Hz appears to be most effective for a~umulating Ca2+ in the nerve terminal Under conditions in which e.p.p. fails to be evoked by a nerve stimulation in a low-Ca*+, high-Mg*+ solution, Ca*+ influx into the terminal is strongly reduced because Ca’+ channels in the presynaptic membrane are blocked by Mg2+ (Katz and Miledi, 1969; Ross and Stuart, 1978; Akaike et al., 1978; Hagiwara and Byerly, 1981). In this experiment, PTP was not elicited under conditions in which no e.p.p. was induced by repetitive stimulation. This result would indicate that Ca2+ influx into the terminal is essential to induce PTP. Lev-Tov and Rahamimoff (1980) have also reported that the magnitude of PTP was remarkably reduced in Ca*+-free solution in which no Ca2+ entered the terminal during the tetanus. In their report, however, the values off were 14.76 f 4.25 and 24.3 2 5.97 for lOO-Hz tetanuscs of 20 and 40 set, respectively. They speculated that these increases could account for the mobilization of Ca*+ from internal stores such as mitochondria, vesicles, membranes, etc. In contrast, the value off in this study was about 1.0, irrespective of the duration of the stimulation, and, moreover, the average m.e.p.p. frequency for 100 set after the tetanus tended to
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T~~HIHIKO YAMASHITA and ISAOOOTA
744
decrease compared with the average frequency before the tetanus. These results suggest that there is little Ca2+ mobilization from internal stores in the rat soleus muscle. The depression after the tetanus could be caused by the interruption of Ca*+ influx through Ca channels which supply Ca2+ for decrease during the tetanus. The re-increase (rebound) of m.e.p.p. frequency following the fast decaying component seen in Fig. 3 was observed in many other trials, and all of them appeared at the transition phase between augmentation and potentiation. This phenomenon could be caused by a transient increase of [Ca2+h due to an overlap of Ca2+ which entered during the tetanus and was not completely extruded, and increased Ca2+ induced by the mechanisms such as Ca2+-Na+ exchange. As in an earlier report on frog neuromuscular transmission (Pawson and Grinnel, 1984), the parameters of the magnitude of PTP give an indication of the kinetics of Ca2+ metabolism in the presynaptic terminal in the rat soleus muscle. Since Ca2+ is closely related to the probability of transmitter release, the study of the kinetics of Ca2+ metabolism in the nerve terminal could give clues for the estimation of the activity of transmitter release.
Feng T. P. (1941) Studies of neuromuscular junction. XXVI. The changes in the end-plate potential during and after prolonged stimulation. Clin. J. Physiol. 16, 341-372. Gage P. W. and Hubbard J. I. (1966) An investigation of the post-tetanic potentiation of the end-plate potentials at a mammalian neuromuscular junction. J. Physiol. 184, 353-375. Hagiwara S. and Byerly L. (1981) Calcium channel. A. Rev. Neurosci. 4, 69-125. Hughes J. R. (1958) Post-tetanic potentiation. Physiol. Rev. 38, 91-113.
Katz B. (1969) The Release of Neural Transmitter Substances, pp. 5-39. Liverpool University Press, Liverpool. Katz B. and Miledi R. (1969) Tetrodotoxin-resistance electrical activity in presynaptic terminals, J. Physiol. 203, 459487.
Koyano K., Kuba K. and Minota S. (1985) Long-term potentiation of transmitter release induced by repetitive presynaptic activities in bullfrog sympatlretic ganglia. J. Physiol. 359, 219-233.
Lev-Tov A. and Rahamimoff R. (1980) A study of tetanic and post-tetanic potentiation of miniature end-plate potentials at the frog neuromuscular junction. J. Physiol. 309, 247-273.
Liley A. W. and North K. A. K. (1953) An electrical investigation of effects of repetitive stimulation on mammalian neuromuscular junction. J. Neurophysiol. 16, 509-527.
Llinas R. R. (1982) Calcium in synaptic transmission. Scient. Am. 241, 38-47.
Acknowledgements-The
authors thank Professor Hideyo Yabu, Department of Physiology, and Professor Seiichi Ishii, Department of Orthopedic Surgery, Sapporo Medical College, for their critical reviewing and improving this manuscript, and Dr Genichirou Katahira, Department of Orthopedic Surgery, Sapparo Medical College, for his valuable advice.
REFERENCES Akaike N., Lee K. S. and Brown A. M. (1978) The calcium current of Helix neuron. J. gen. Physiol.11, 509-531. Bliss T. V. P. and Lsmo T. (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J. Physiol. 232, 331-356. Brehm P. and Eckert R. (1978) Calcium entry leads to inactivation of calcium channel in Paramecium. Science 202, 1203-1206.
Brown T. H. and McAfee D. A. (1982) Long-term synaptic potentiation in the superior cervical ganglion. Science 215, 1411-1413. Castelluci V. and Kandel E. R. (1976) Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia. Science 194, 11761178. Chad J., Eckert R. and Ewald D. (1984) Kinetics of calcium-dependent inactivation of calcium current in voltage-clamped neurones of Aplysia californica. J. Physiol. 347. 279-300.
de1 Castilio J. and Katz B. (1954) Quanta1 components of the end-plate notential. J. Phvsiol. 124. 560-573. Eckert R. Hnd Tillotson D. (1981) Calcium-mediated inactivation of the calcium conductance in caecium-loaded grant neurones of Aplysia calijonica. J. Physiol. 314, 265-280.
Erulkar S. D. and Rahamimoff R. (1978) The role of calcium ions in tetanic and post-tetanic increase of miniature end-plate potential frequency. J. Physiol. 278, 501-511.
Llinas R. R., Steinberg I. 2. and Walton K. (1981) Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse. Biophys. J. 33, 323-351.
Magleby K. L. and Zengel J. E. (1976) Long term changes in augmentation, potentiation, and depression of transmitter release as a function of repeated synaptic activity at the frog neuromuscular junction. J. Physiol. 257, 471494.
Miledi R. and Thies R. (1971) Tetanic and post tetanic rise in frequency of miniature end plate potentials in low calcium solutions. J. Physiol. 212, 245-257. Misler S. and Hurlbut W. P. (1983) Post-tetanic potentiation of acetylcholine release at the frog neuromuscular junction develops after stimulation in Car+-free solutions. Proc. Nain. Acad. Sci. U.S.A. 80, 315-319.
Pawson P. A. and Grinnell A. D. (1984) Post-tetanic potentiation in strong and weak neuromuscular junctions: physiological differences caused by a differential Cai+-in&x. Brain Res. 323, 311-315. Plant T. D. and Standen N. B. (1981) Calcium current inactivation in identified neuron& of Helix aspersa. J. Physiol. 321, 273-285.
Rahamimoff R. and Yaari Y. (1973) Delaved release of transmitter at the frog neuromuscular junction. J. Physiol. 228, 241-257.
Ross W. N. and Stuart A. E. (1978) Voltage sensitive calcium channels in the presynaptic terminals of a decrementally conducting photoreceptor. J. Physiol. 274, 173-191.
Tsukahara N. (1981) Synaptic plasticity in the mammalian central nervous system. 2. I&v. Neurosci. 4, 351-379. Wilson D. F. and Skirboll L. R. (1974) Basis for nost-tetanic potentiation at the mammal&n neuromuscular junction. Am. J. Physiol. 227, 92-95.
Zengel J. E. and Magleby K. L. (1981) Changes in miniature endplate potential frequency during repetitive nerve stimulation in the presence of Ca*+, Ba*+ and Sr2+ at the frog neuromuscular junction. J. gen. Phys;ol. 77,503-529.
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POST-TETANIC POTENTIATION OF MINIATURE END-PLATE POTENTIAL FREQUENCY AT NEUROMUSCULAR JUNCTION OF THE RAT SOLEUS MUSCLE TD~IIIHIKo YAMASHITA*and ISAO OOTA Department of Physiology and *Department of Orthopedic Surgery, Sapporo Medical College, Sapporo 060, Japan (Received 6 October 1986)
Abstract-l.
Changes in miniature end-plate potential (m.e.p.p.) frequency by repetitive nerve stimulation were examined in the rat soleus muscle. 2. The increase of m.e.p.p. frequency was induced by repetitive stimulation and persisted for several minutes after the tetanus. That is, post-tetanic potentiation (PTP) of neuromuscular transmission was first demonstrated here in the rat soleus muscle. 3. The time course of the decay of m.e.p.p. frequency after the tetanus showed a double exponential curve which consisted of a fast decaying component (augmentation) and a slow decaying component (potentiation). 4. The magnitude of PTP depended on the stimulation frequency and its duration. It increased with the increase of duration and was at its maximum at a frequency of 100 Hz. 5. No PTP was elicited by repetitive stjm~ation under conditions in which end-plate potential (e.p.p.) was compietely suppressed, and, moreover, m.e.p.p. frequency tended to decrease after the tetanus.
INTRODUCTION Following repetitive stimulation of the presynaptic nerve, transmitter release increases for some time. This phenomenon is known as post-tetanic potentiation (PTP) (Hughes, 1958). PTP, which shows synaptic plasticity, has been observed in a variety of synapses (Bliss and Lomo, 1973; Castelluci and Kandel, 1976; Tsukahara, 1981; Brown and McAfee, 1982; Koyano et al., 1985). In neuromuscular junctions, the PTP of neuromuscular transmission has been studied under conditions of reduced quanta1 content by many investigators (Feng, 1941; Liley and North, 1953; de1 Castillo and Katz, 1954; Miledi and Thies, 1971; Magleby and Zengel, 1976; Erulkar and Rahamimoff, 1978). PTP can be observed as an increase in the spon~neous occurrence of miniature end-plate potential (m.e.p.p.), or as an increase in the amplitude of end-plate potential (e.p.p.) evoked by nerve stimulation. These two types of PTPs have similar time courses of decay (Zengel and Magleby, 1981). PTP of neuromuscular transmission has been investigated in frog sartorius muscles (de1 Castillo and Katz, 1954; Erulkar and Rahamimoff, 1978; Magleby and Zengel, 1976; Misler and Huribut, 1983) and rat diaphragms (Liley and North, 1953; Gage and Hubbard, 1966; Wilson and Skirboll, 1974). Especially in frog sartorius muscles, the kinetic properties of PTP, such as the time course of decay and the parameters of magnitude, have been analyzed in detail. However, there has been no report showing the existence of PTP in the muscles of mammalian limbs, or analyzing its kinetic properties. In this study, we examined changes in m.e.p.p, frequency by repetitive stimulation of the motor
nerve and demonstrated the existence of PTP in a rat tibia1 nerve-soteus muscle preparation. Furthermore, its basic kinetic properties, such as the time course of the decay of m.e.p.p. frequency, and the effects of changes in stimulation conditions were analyzed. MATERIALSAND METHODS Preparation and solution
Experiments were performed on a tibia1 nerve-soleus muscle preparation from male Wistar rats (200-2508 in weight). Under i.p. anesthesia using sodium pentobarbital (50 mg/kg body wt), preparations were dissected in modified Krebs solution. The dissected nerve-muscle preparations were mounted at resting length in a 30-ml organ chamber. Modified Krebs solution, bubbled with 95% O,-5% CO,, continuously circulated in the organ chamber at a rate of 2-5 mljmin. The composition of the modified Krebs solution was (mM): NaCl, 135; KCI, 5; NaHCO,, 15; Na,HP04, 1; glucose 1I. To suppress the action potential of the muscle fiber. 8-12 mM MeCl, and 1.8-2.0 mM CaCl, were added to this solution. ?he*pH was adjusted to 7.2 before use. Experiments were carried out at room temperature (Zo-25°C). A glass suction electrode was used as a stimulating electrode, and the tibia1 nerve was drawn into it. Tetanic trains of square pulses (supramaximal voltage was 5-10 V, and duration, 0.05 msec) were delivered to the nerve by an electric stimulator (Nihon Kohden, SEN-7103). The frequency and duration of repetitive stimulations were varied over the ranges of 255OOHz and l-5Ose.c, respectively.
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Figure 1 shows a block diagram of the system used for the recording and analysis of experimental data. Miniature
TOSHIHIKO YAMASHITA
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ISAO OOTA RESULTS
Changes in m.e.p.p. frequency stimulation
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Fig. 1. Block diagram of the system for recording and analysis of experimental data.
end-plate potentials (m.e.p.p.s) were recorded from endplate regions using an intracellular micro-electrode technique. Glass micro-electrodes were filled with 3 M KC1 and
had tip resistances of IO-15 Ma. Signals from recording electrodes were amplified by a micro-electrode amplifier (Nihon Kohden, MEZ-8201) and a high-gain pre-amplifier (Nihon Kohden, AVH-IO), and then recorded with an FM
after repetitiue nerve
Following repetitive stimulation of the motor nerve, m.e.p.p. frequency markedly increased, and then decayed gradually with time, as seen in Fig. 2. Figure 3 shows a typical time course of changes in m.e.p.p. frequency before and after repetitive stimulation. The average frequency of m.e.p.p. before the stimulation (control frequency) was 1.7 Hz. Repetitive nerve stimulation at the rate of 100 Hz for 30 set produced a large increase in m.e.p.p. frequency, and the average frequency in the 5 set immediately after the stimulation was 29.0 Hz. After that, the m.e.p.p. frequency declined rapidly to 6.6 Hz at 50 set after the tetanus. Following this fast decline, a small transient increase in m.e.p.p. frequency appeared (arrow in Fig. 3A), and then the decay changed into a slow decline phase. At 300 set after the tetanus, m.e.p.p. frequency was 3.2 Hz, which was an obvious increase over the control frequency. Miniature end plate potential frequencies after the tetanus were plotted semilogarithmically against time as shown in Fig. 3B. The two lines in this figure represent the two different exponential decays of m.e.p.p. frequency. Thus, the time course of the decay of m.e.p.p. frequency after repetitive stimulation represents a double exponential curve which consists of two components; a fast component of decay immediately after the tetanus, and the subsequent slow component of
A
tape recorder (SONY, KS-609W), while being sent to a CRT display (Nihon Kohden, VC-10) for monitoring. Miniature end-plate potentials which occurred for 100 set before, and 500 set after, the repetitive stimulation, were recorded in each trial. If any stimulus in the repetitive stimulation failed to evoke an end-plate potential (e.p.p.), the trial was discarded. Intervals between trials were at least 20 min, to allow the increased m.e.p.p. frequency to return to the control level. Analysis The data recorded on video tapes were analyzed by a data analyzer (Nihon Kohden, ATAC-450). Miniature end-plate potential frequency was measured by a pulse count program and represented in histograms in real time. The control frequency was defined as the average of m.e.p.p. frequencies for 1OOsec before the repetitive stimulation. Since it was impossible to identify and count m.e.p.p.s during the tetanus because of the overlap of e.p.p.s and stimulation pulses, the data during the tetanus were excluded from analysis. To present a detailed picture of the time course of the decay of m.e.p.p. frequency after the tetanus, the pulse count program was modified by a ‘moving-bin technique’ (Rahamimoff and Yaari, 1973). The m.e.p.p. frequency was calculated for a block of time (bin size), and then the block was advanced in time by steps (A bin) smaller than the bin size. Therefore, the block moved (bin/A bin - 1) times. The m.e.p.p. frequency was plotted at the center of the time bin for each successive position. In this study, bin size was 25 set and A bin size was S sec. The histograms were drawn by an X-Y plotter and the values of m.e.p.p. frequencies were printed by a printer. The time constant of the decay of m.e.p.p. frequency was calculated by the analysis of exponential regression using a personal computer (NEC, PC-9801).
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lOmst3c Fig. 2. Post-tetanic potentiation of miniature end-plate potential frequency due to repetitive stimulation of motor nerve at 1OOHz for 30 sec. A shows m.e.p.p. frequency before the tetanic stimulation. B shows m.e.p.p. frequency immediately after the tetanic stimulation. C, D and E show m.e.p.p. frequency at 30, 60 and 120 set, respectively after the tetanic stimulation. Intracellular recording was done in a modified Krebs solution containing 2.0 mM CaCl,, and 10 mM MgCl,. Calibration: vertical bar, 0.5 mV; horizontal bar, 10 msec.
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Fig. 3. Effect of tetanic stimulation (100 Hz, 30 see) on m.e.p.p. frequency. (A) Time course of changes in m.e.p.p. frequency before and after tetanic stimulation. A moving bin display was used, with a bin size of 25 set and A bin size of 5 sec. The m.e.p.p. frequency before the tetanic stimulation (control frequency) was 1.7 Hz. The m.e.p.p. frequency was 29.0 Hz immediately after the tetanic stimulation and 3.2 Hz at 3OOsec after the stimulation. The arrow indicates a rebound of m.e.p.p. frequency after a fast decline. The medium contained 2.0 mM CaCl,, IO mM MgC12modified Krebs solution. (B) Semilogarithmic plot of post-tetanic changes in m.e.p.p. frequency shown in A. The two straight fines indicate the two different phases of decay, au~en~tion and potentiation.
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Fig. 4. Post-tetanic potentiation of m.e.p.p. frequency due to stimulation at various rates (denoted by each set of points). The frequency of m.e.p.p. was normalized by dividing by the control frequency. The number of stimuli was constant at 2000. Moving bins: bin = 25 set, A bin = 5 sec.
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decay. Figure 3B shows the re-increase of m.e.p.p. frequency which appeared at the intersection of the two lines. This indicates that the small re-increase (rebound) of m.e.p.p. frequency seen in Fig. 3A occurred during the transition phase between the two components. The time constant of the fast decaying component (augmentation) was 31.2 set and that of the slow decaying component (potentiation) was 163.9sec. M.e.p.p. frequency should return to the control level at about 10 min after the tetanus according to the analysis of exponential regression; that is to say the PTP should persist for about 10 min.
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ISAO OOTA
tetanus in two preparations, so that it was impossible to calculate the time constants in these trials. Effect of repetitive stimulation on m.e.p.p. frequency when e.p.p. was suppressed
End-plate potential (e.p.p.) can be observed in low-Ca*+, high-Mg2+ solutions, since quanta1 contents are reduced enough to suppress the action potential of muscle fiber. However, when quanta1 contents are more strongly reduced, even e.p.p. is suppressed. The effect of repetitive stimulation on m.e.p.p. frequency under such conditions was examined. In many trials using a solution containing
Effect of the frequency of stimulation on PTP
The correlation between the magnitude of PTP and the stimulation frequency was examined by changing the frequency step-by-step while maintaining a constant number of stimuli (2000). As shown in Fig. 4, the magnitude of PTP varied with the changes in the frequency. The magnitude of PTP was estimated by calculating the following parameters in each trial: (1) the ratio of m.e.p.p. frequency in the 5 set immediately after the tetanus/the control frequency (normalized initial post-tetanic frequency, that isf); (2) the time constant of the augmentation phase (ra) and (3) the time constant of the potentiation phase (rp). The results from two different preparations are shown in Fig. 5. The PTP series was obtained from a single junction in each preparation. The stimulation frequency was varied from 25 to 500 Hz. The values off increased with the increase of stimulation frequency from 25 to 100 Hz, and decreased with the increase of frequency from 100 to 500Hz (Fig. SA). Similarly, the values of r, and tp increased with each stimulation frequency up to 100 Hz, and then decreased with successive frequencies above 100 Hz (Fig. 5B and C). Thus, the parameters depended on the stimulation frequency and their maximum values were at the frequency of 100 Hz. The potentiation phase was not clear in two preparations at 25 Hz, and in one preparation at 500 Hz, so that it was impossible to calculate the time constants in these trials. Efect
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The correlation between the magnitude of PTP and the duration of the stimulation was examined by changing the duration step-by-step, while maintaining a constant frequency of 100 Hz. As shown in Fig. 6, the magnitude of PTP varied with the changes in the duration of stimulation. The PTP magnitude was estimated by calculating the above-mentioned parameters. The results from three different preparations are shown in Fig. 7. The PTP series was obtained from a single junction in each preparation. The duration of the stimulation varied from 1 to 50 sec. The value of fincreased with the increases in the stimulation duration in all preparations (Fig. 7A). The values of r, and 7p also increased with the increases in the duration of the stimulation (Fig. 7B and C). The augmentation phase and potentiation phase did not appear at I-set tetanus in any preparations, and the potent&ion phase drd not appear at 5-set
Fig. 5. Effect of the stimulation frequency on the parameters of the magnitude of PTP. A: The no~ali~d initial posttetanic frequency (f ). B: The time constant of augmentation (ra). C: The time constant of potentiation (T,). The results obtained from two different preparations are shown. The number of stimuli . .was . constant at 2000. The values of the parameters reached their maxima at a stimulation rate of 100 Hz in both preparations.
PTP at the rat neuromuscular jun~ion
Time
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Fig. 6. Post-tetanic potentiation of m,e.p.p. frequency due to stimulation of various durations (denoted by each set of points). The stimulation frequency was constant at 100 Hz. Moving bins: bin = 25 set, A bin = 5 sec. The frequency of m.e.p.p. was normalized by dividing by the control frequency.
1.8 mM CaCl, and 12 mM MgCI,, failures of e.p.p. occurred during repetitive stimulation. Under such conditions, experiments were performed on preparations in which all stimuli during the tetanus failed to evoke e.p.p. The duration of the stimulation varied from 5 to 40 set, with a constant frequency of 100 Hz. Miniature end-plate potential frequency was not increased by repetitive stimulation, as can be seen in Fig. 8. The correlation between the value offand the duration of the stimulation is shown in Fig. 9. The value off remained at about 1.0 in spite of increases in the duration of the stimulation. Thus, repetitive nerve stimulation under these conditions did not produce PTP. Furthermore, the correlation between the value off’, the ratio expressed by the average m.e.p.p. frequency for 1OOsec after the tetanus/the control m.e.p.p. frequency, and the duration of the stimulation was examined (Fig. 9). The values off’ were Iess than 1.O at every stimulation duration, and tended to decrease with the increase of the duration of the stimulation. Thus, m.e.p.p. frequency after the tetanus showed a tendency to be more and more depressed with the increases in the stimulation duration. DlscussIoN The present study shows that repetitive nerve stimulation causes a remarkable increase in m.e.p.p. frequency, and that the increase persists for several minutes after the tetanus in rat tibia1 nerve-soleus muscle preparations. Thus, post-tetanic potentiation (PTP) of neuromuscular t~smission was demonstrated in the rat soleus muscle. According to previous studies on the PTP of neuromuscular transmission in the frog sartorius muscle (Magleby and Zengel, 1976; Zengel and Magleby, 1981), the decay of m.e.p.p. frequency is composed of four ex-
ponentially decaying components; the first facilitation, the second facilitation, augmentation and potentiation. The time constants of each component were reported as follows: the first facilitation, 47 + 9 msec (mean t_ SE); the second facilitation, 472 + 108 msec; augmentation, 6.9 & 1.6 set; and potentiation, 82 + 25 set for a tetanus with 100-200 stimuli (Zengel and Magleby, 1981). Another report on the frog revealed that, when the changes in m,e.p.p. frequency were recorded per second for several minutes, its time course represented a double exponential curve composed of two components; the fast decaying phase (au~en~tion), and the following slow decaying phase (potentiation). Since facilitation is a very short phase which appears immediately, within 1 set after the tetanus, it cannot be detected by observing changes per second (Lev-Tov and Rahamimoff, 1980). Considering these findings, and the results of this experiment, such as the time course and the PTP time constants of decay, the fast and slow components of PTP found in this experiment correspond to augmentation and potentiation, respectively. Therefore, t, and rp represent the time constants of augmentation and potentiation, respectively. As mentioned above, it was not possible to measure facilitation in this experiment. Lev-Tov and Rahamimoff (1980) have reported on the kinetic properties of the PTP of neuromuscular transmission in the frog sartorius muscle. In their report, the value off (the normalized initial posttetanic frequency) was 18.36 + 8.89 (mean + SE) for a tetanus of 100 Hz for 20 set, and 48.45 f 21.59 for a tetanus of 100 Hz for 40 sec. In contrast, under the same conditions, the respective values off in our experiment using the rat soleus muscle were 7.31 + 0.40 and 14.20 f 2.43. These values are smaller than those of the frog sartorius muscle. The time constants of augmentation (7,) for 100-HZ
142
TOSHIHIKO
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stimulation in the frog were 5.32 + 2.01 set for the 20-set tetanus, and 9.29 + 2.05 set for the 40-set tetanus, whereas in our expe~ment the figures were 27.10 i_ 9.39 and 45.50 rfl 5.50 set, respectively. These results indicate that the augmentation phase in the rat soleus muscle persists longer than that in the frog sartorius muscle. In contrast, the time constants of the potentiation phase (rr) were 132.72 + 19.90 set for the lOO-Hz-20-set tetanus, and 163.05 + 31.74 see for the lOO-Hz-40-set tetanus in the frog, whereas, in our experiment, the respective zp for the rat were 85.33 f. 20.96 and 141.14 f 27.54 sec. Thus, it is clear that the potentiation phase in the rat soleus muscle is shorter than that in the frog sartorius muscle. The parameters of the magnitude of PTP increased depending on the duration of the repetitive stimulation (Fig. 7). They also depended on the stimulation
frequency, and reached their maximum at a frequency of 100 Hz (Fig. 5). These results in the rat soleus muscle show the same kinetic properties as in the frog neuromuscular transmission, in that the magnitude of PTP depends on the duration and frequency of the repetitive stimulation (Lev-Tov and Rahamimoff, 1980). As the above results demonstrate, the kinetic properties of PTP of neuromuscular transmission in the rat soleus muscle are similar to those in the frog sartorius muscle in terms of the time course of the decay and the dependence on the stimulation frequency and duration, though there is a certain difference in their ma~itudes. Previous works on the frog neuromuscular junction have shown that Ca2+ plays an important role in the PTP mechanism, and the parameters of the magnitude of PTP give some indication of the kinetics of Caz+-metaboIism in the nerve terminal (LevTov and Rahamimoff, 1980; Zengel and Magleby, 1981; Pawson and Grinnel, 1984). An action potential at the motor nerve terminal induced by a nerve stimulus causes a voltage-dependent Caz+ influx into the terminal, and provokes the quanta1 release of the transmitter (Katz, 1969). Therefore, high frequency stimulation of the motor nerve produces a large increase in the concentration of intracellular Ca2+ ([Ca*‘]i”) and leads to an increase in the probability of transmitter release, which appears as an increase of m.e.p.p. frequency (tetanic potentiation). Therefore, if m.e.p.p. frequency immediately after the tetanus is divided by the control frequency, the resulting ratio (f) would reflect the amount of Ca2+ influx induced by repetitive stimulation. The augmentation phase represents the processes responsible for reducing the amount of intracellular Ca2+ which entered the nerve terminal during the tetanus (Lev-Tov and Rahamimoff, 1980), so that the time constant of augmentation reflects the rate of Ca*+ extrusion from the nerve terminal. The potentiation phase is prolonged by processes maintaining a high basal level of [Ca’+],, such as the following: (1) the effects of Ca’+ which entered during the tetanus and was not completely extruded (Erulkar and Rahamimoff, 1978), (2) Ca2+ entry through a Ca’+-Na+ exchange (Misler and Hurlbut, 1983), (3) mobili~tion of Ca*+ from internal stores (Zengel and Magleby, 1981) and (4) increased permeability of the terminal membrane for Ca*+ (Zengel and Magleby, 1981). As mentioned earlier, this study shows thatfand zp in the rat soleus muscle were smaller than those in the frog sartorius muscle, while 7, in the rat was larger than that in the frog. These results indicate that, in the rat soleus muscle, the amount of Ca2+ influx during the tetanus is smaller, that its extrusion is slower, and that the subsquent entry or mobilization of Ca* + in the slowly decaying component is smaller than that in the frog sartorius muscle. Thus, the nerve terminal in the rat soleus muscle appears to have a smaller Ca’+ conductance than that in the frog sartorius muscle. In the present experiment, it was revealed that the magnitude of PTP depended on the duration and frequency of repetitive stimulation in the rat soleus muscle. When the duration of the tetanus was prolonged, the amount of Ca’+ influx might increase because of the increase in the number of action
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potentials, and consequently, the magnitude of PTP increased with the increase in the duration of the stimulation. However, the magnitude of PTP reached its maximum at a frequency of 100 Hz, and decreased gradually with both frequency increases and decreases. In studies on the giant synapse of the squid (Llinas et al., 1981; Llinas, 1982), the duration of Ca* + current, induced by a presynaptic action potential, was about 1.5msec In contrast, the interval between stimuli is 40 msec at a frequency of 25 Hz. Thus, it appears that Ca*+ efflux occurs between voltage-dependent Ca2+ influxes, and the accumulation of CaZ+ in the nerve terminal is reduced. The amount of Ca*+ ef?lux is gradually reduced with the increase of the stimulation frequency to 50 and 100 Hz because of the decrease of the interval time to 20 and IOmsec, so that the magnitude of PTP
increases with frequency up to 100 Hz. In contrast, at the frequency of 200 Hz, the interval between stimuli is 5 msec, and little Ca*+ is extruded, so that CaZC largely a~umulates in the the terminal. Consequently, by a mechanism similar to the Ca-induced Ca inactivation which has been observed in the neurons of molluscs (Brehm and Eckert, 1978; Fckert and Tillotson, 1981; Plant and Standen, 1981; Chad et al., 1984), Ca*+ influx could be suppressed, and the magnitude of PTP is reduced. The repetitive stimulation of 500 Hz presumably exceeds the maximal frequency which can be physiologically followed, so not all stimuli would be effective. For the reasons mentioned above, repetitive stimulation of around 100 Hz appears to be most effective for a~umulating Ca2+ in the nerve terminal Under conditions in which e.p.p. fails to be evoked by a nerve stimulation in a low-Ca*+, high-Mg*+ solution, Ca*+ influx into the terminal is strongly reduced because Ca’+ channels in the presynaptic membrane are blocked by Mg2+ (Katz and Miledi, 1969; Ross and Stuart, 1978; Akaike et al., 1978; Hagiwara and Byerly, 1981). In this experiment, PTP was not elicited under conditions in which no e.p.p. was induced by repetitive stimulation. This result would indicate that Ca2+ influx into the terminal is essential to induce PTP. Lev-Tov and Rahamimoff (1980) have also reported that the magnitude of PTP was remarkably reduced in Ca*+-free solution in which no Ca2+ entered the terminal during the tetanus. In their report, however, the values off were 14.76 f 4.25 and 24.3 2 5.97 for lOO-Hz tetanuscs of 20 and 40 set, respectively. They speculated that these increases could account for the mobilization of Ca*+ from internal stores such as mitochondria, vesicles, membranes, etc. In contrast, the value off in this study was about 1.0, irrespective of the duration of the stimulation, and, moreover, the average m.e.p.p. frequency for 100 set after the tetanus tended to
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T~~HIHIKO YAMASHITA and ISAOOOTA
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decrease compared with the average frequency before the tetanus. These results suggest that there is little Ca2+ mobilization from internal stores in the rat soleus muscle. The depression after the tetanus could be caused by the interruption of Ca*+ influx through Ca channels which supply Ca2+ for decrease during the tetanus. The re-increase (rebound) of m.e.p.p. frequency following the fast decaying component seen in Fig. 3 was observed in many other trials, and all of them appeared at the transition phase between augmentation and potentiation. This phenomenon could be caused by a transient increase of [Ca2+h due to an overlap of Ca2+ which entered during the tetanus and was not completely extruded, and increased Ca2+ induced by the mechanisms such as Ca2+-Na+ exchange. As in an earlier report on frog neuromuscular transmission (Pawson and Grinnel, 1984), the parameters of the magnitude of PTP give an indication of the kinetics of Ca2+ metabolism in the presynaptic terminal in the rat soleus muscle. Since Ca2+ is closely related to the probability of transmitter release, the study of the kinetics of Ca2+ metabolism in the nerve terminal could give clues for the estimation of the activity of transmitter release.
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Acknowledgements-The
authors thank Professor Hideyo Yabu, Department of Physiology, and Professor Seiichi Ishii, Department of Orthopedic Surgery, Sapporo Medical College, for their critical reviewing and improving this manuscript, and Dr Genichirou Katahira, Department of Orthopedic Surgery, Sapparo Medical College, for his valuable advice.
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