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Post-tetanic potentiation at the neuromuscular junction of the frog in the presence of tetrodotoxin
SHORT COMMUNICATIONS
527
Post-tetanic potentiation at the neuromuscular junction of the frog in the presence of tetrodotoxin Following repetitive stimulation of motor nerves, neuromuscular transmission is potentiated2, 4. This potentiation can be separated into two components3: (1) 'primary potentiation', i.e., facilitation lasting up to several hundred milliseconds following one or a few stimuli, and (2) 'secondary or post-tetanic potentiation' (PTP) following a tetanic stimulus and having a minimum duration of several seconds. Katz and Miledi a have demonstrated that facilitation following a single stimulus is not prevented by the presence of tetrodotoxin (TTX). Since T T X is known to block Na + influx associated with action potentials in nerve and muscle s, it seems clear that facilitation does not require Na ÷ entry into the nerve terminal. However, a question remains as to the role played by Na + in PTP. Birks 1 suggested that the accumulation of intracellular Na + in nerve endings during a train of action potentials is essential for the generation of PTP. Such Na + accumulation may enhance the entrance of Ca z+ which would in turn increase the amount of acetylcholine release in response to subsequent nerve impulses 8. The present experiments were designed to determine if Na ÷ entry and possibly intracellular accumulation of Na + during a train of stimuli to the motor nerve are necessary for PTP in the frog neuromuscular junction. The extensor digitorum muscle of the frog (Rana pipiens) was placed in a recording chamber which contained a solution of the following composition: l 15.6 m M NaCI, 3.5 m M KCI, 1.8 m M CaClz, 10 -6 g/ml neostigmine methylsulfate, and 10 -7 g/ml TTX. Intracellularly recorded end-plate potentials (EPPs) were evoked by
A
B
___,/.,..._
-
"'%'--c.
J2mV ~_.~sec __
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!
L._-----
J2mV
,':! 50msec ~'il
,
Fig. I. EPPs evoked by depolarizing pulses in a TTX-treated preparation. A, The effect of varying current intensities on the EPP amplitude; strength of pulse was progressively increased from a (10/zA) to c (18/~A). B, Potentiation during and after a short train of pulses at 75/sec in another experiment. Test responses were evoked at varying intervals after the end of the train. In both A and B the temperature was maintained at 17°C; pulse durations were 0.9 and 0.5 msec, respectively. Current intensity in B was 40/~A.
Brain Research, 17 (1970) 527-529
528
SHORT COMMUNICATIONS
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Fig. 2. The effect of a train of 500 depolarizing pulses at 50:see on the EPP amplitude and MEPP frequency in a preparation treated with TTX. A. Evoked EPPs at indicated intervals (see) following the train; some traces were retouched with dots. B. Graph illustrating the time course of the EPP amplitude (@) and MEPP frequency ( ~ ) after the tetanus ('hatched bar). Test pulses, 0.9 msec and 11 t~A, were applied at 2-see intervals before and after the train. Ordinates are the ratio of MEPP frequency, f, to control frequency, fc, and EPP amplitude in mV. Abscissa is the time (secJ after the tetanus. Temperature was maintained at 25°C.
electrotonic d e p o l a r i z a t i o n o f the m o t o r nerve endings 6. S p o n t a n e o u s miniature endplate p o t e n t i a l s ( M E P P s ) were also m o n i t o r e d . Fig. 1A illustrates the effects o f d e p o l a r i z i n g pulses o f varying intensities applied to a nerve terminal in the presence o f T T X . In accord with the results o f K a t z and Miledi 6, EPPs e v o k e d by d e p o l a r i z a t i o n o f the nerve t e r m i n a l occur in q u a n t a l steps. a n d the average a m p l i t u d e d e p e n d s on the s t r e n g t h o f the a p p l i e d current pulse. R e c o r d s in Fig. IB d e m o n s t r a t e that p o t e n t i a t i o n following a s h o r t train (q/~ M a l l a r t a n d M a r t i n 7) is not i m p a i r e d by the presence o f T T X . In the e x p e r i m e n t shown in Fig. 2. EPPs were o b t a i n e d at 2-see intervals with c o n s t a n t d e p o l a r i z i n g pulses applied to the m o t o r nerve t e r m i n a l before and after tetanic s t i m u l a t i o n by 500 pulses at 50/see in the presence o f T T X . S a m p l e r e c o r d s in Fig. 2A depict the changes in EPPs at v a r y i n g intervals following the t r a i n o f stimuli. A s shown in Fig. 2B (filled circles), the E P P showed a significant e n h a n c e m e n t in a m p l i t u d e after the tetanic stimuli. In a d d i t i o n , there was an increase in the frequency o f s p o n t a n e o u s M E P P s ~open circles in Fig. 2B). It can be seen t h a t the E P P r e t u r n s to the c o n t r o l level in a p p r o x i m a t e l y 60 sec. O n the o t h e r hand, p o t e n t i a t i o n o f M E P P frequency is greater in the percentile c h a n g e but has a shorter time course t h a n that for the e v o k e d s y n a p t i c response. These dissimilarities in the time course a n d in the m a g n i t u d e o f p o t e n t i a t i o n between M E P P and EPP have previously been n o t e d following a train o f nerve a c t i o n potentials a. F r o m the e x p e r i m e n t s described above, it is clear t h a t P T P o f n e u r o m u s c u l a r t r a n s m i s s i o n is not e l i m i n a t e d in the presence o f T T X . Since T T X blocks t h e i n w a r d s o d i u m currents associated with impulse g e n e r a t i o n 5. it is c o n c l u d e d that N a + entry into the nerve t e r m i n a l or the resultant intracellular N a + a c c u m u l a t i o n d u r i n g a train Brain Research. 17 0970) 527-529
SHORT COMMUNICATIONS
529
o f nerve stimuli is n o t i m m e d i a t e l y required for the g e n e r a t i o n o f PTP. F u r t h e r m o r e , the test d e p o l a r i z i n g pulses used in the present study before and after the tetanic stimulation were identical in m a g n i t u d e and d u r a t i o n . This eliminates the possibility that P T P m a y be due entirely to changes in configuration o f action potentials o f the presynaptic t e r m i n a l following a tetanic stimulus. The a u t h o r is indebted to Dr. M. K u n o for his advice and criticism in p r e p a r i n g the manuscript. The a u t h o r is a p r e d o c t o r a l trainee under U.S. Public H e a l t h Service P h a r m a cology Research T r a i n i n g G r a n t 5 T 1 - G M 153-11. Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah 84112 (U.S.A.)
DANIEL WEINREICH
1 BIRKS, R. 1., The role of sodium ions in the metabolism of acetylcholine, Canad. J. Biochem. Physiol., 41 (1963) 2573-2597. 2 FENG,T. P., Studies on the neuromuscular junction. XXXI. The changes of the end-plate potential during and after prolonged stimulation, Chin. J. Physiol., 16 (1941) 341-472. 3 HUBBARD,J. I., Repetitive stimulation at the mammalian neuromuscular junction and the mobilization of transmitter, J. Physiol. (Lond.), 169 (1963) 641-662. 4 HUGHES,J. R., Post-tetanic potentiation, Physiol. Rev., 38 (1958) 91-113. 5 KAO, C. Y., Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena, Pharmacol. Rev., 18 (1966)997-1049. 6 KATZ, B., AND MILEDI, R., Tetrodotoxin and neuromuscular transmission, Proc. roy. Soc. B, 167 (1967) 8-22. 7 MALLART, A., ANt) MARTtN, A. R., An analysis of facilitation of transmitter release at the neuromuscular junction of the frog, J. Physiol. (Lond.), 193 (1967) 679-694. 8 ROSENTHAL,J., Post-tetanic potentiation at the neuromuscular junction of the frog, J. Physiol. (Lond.), 203 (1969) 121-133. (Accepted November 20th, 1969)
Brain Research, 17 (1970) 527-529
527
Post-tetanic potentiation at the neuromuscular junction of the frog in the presence of tetrodotoxin Following repetitive stimulation of motor nerves, neuromuscular transmission is potentiated2, 4. This potentiation can be separated into two components3: (1) 'primary potentiation', i.e., facilitation lasting up to several hundred milliseconds following one or a few stimuli, and (2) 'secondary or post-tetanic potentiation' (PTP) following a tetanic stimulus and having a minimum duration of several seconds. Katz and Miledi a have demonstrated that facilitation following a single stimulus is not prevented by the presence of tetrodotoxin (TTX). Since T T X is known to block Na + influx associated with action potentials in nerve and muscle s, it seems clear that facilitation does not require Na ÷ entry into the nerve terminal. However, a question remains as to the role played by Na + in PTP. Birks 1 suggested that the accumulation of intracellular Na + in nerve endings during a train of action potentials is essential for the generation of PTP. Such Na + accumulation may enhance the entrance of Ca z+ which would in turn increase the amount of acetylcholine release in response to subsequent nerve impulses 8. The present experiments were designed to determine if Na ÷ entry and possibly intracellular accumulation of Na + during a train of stimuli to the motor nerve are necessary for PTP in the frog neuromuscular junction. The extensor digitorum muscle of the frog (Rana pipiens) was placed in a recording chamber which contained a solution of the following composition: l 15.6 m M NaCI, 3.5 m M KCI, 1.8 m M CaClz, 10 -6 g/ml neostigmine methylsulfate, and 10 -7 g/ml TTX. Intracellularly recorded end-plate potentials (EPPs) were evoked by
A
B
___,/.,..._
-
"'%'--c.
J2mV ~_.~sec __
'_ktll. ~
!
L._-----
J2mV
,':! 50msec ~'il
,
Fig. I. EPPs evoked by depolarizing pulses in a TTX-treated preparation. A, The effect of varying current intensities on the EPP amplitude; strength of pulse was progressively increased from a (10/zA) to c (18/~A). B, Potentiation during and after a short train of pulses at 75/sec in another experiment. Test responses were evoked at varying intervals after the end of the train. In both A and B the temperature was maintained at 17°C; pulse durations were 0.9 and 0.5 msec, respectively. Current intensity in B was 40/~A.
Brain Research, 17 (1970) 527-529
528
SHORT COMMUNICATIONS
A 10, -
,T,
" con
4
°'~ °M
2,----. 41 ~
"----_
° ° o
-
...................
0
, 3 2 ..,12~,v __ ~ . ~ c
]6
1:iiiii -~_. . . .
(~
"" "
tif
•
| ,,~
° • "°. °.. 11" " ................
fO 2"0-3"0~'114"0 5"0
6"0
7"0
TIME-sec
Fig. 2. The effect of a train of 500 depolarizing pulses at 50:see on the EPP amplitude and MEPP frequency in a preparation treated with TTX. A. Evoked EPPs at indicated intervals (see) following the train; some traces were retouched with dots. B. Graph illustrating the time course of the EPP amplitude (@) and MEPP frequency ( ~ ) after the tetanus ('hatched bar). Test pulses, 0.9 msec and 11 t~A, were applied at 2-see intervals before and after the train. Ordinates are the ratio of MEPP frequency, f, to control frequency, fc, and EPP amplitude in mV. Abscissa is the time (secJ after the tetanus. Temperature was maintained at 25°C.
electrotonic d e p o l a r i z a t i o n o f the m o t o r nerve endings 6. S p o n t a n e o u s miniature endplate p o t e n t i a l s ( M E P P s ) were also m o n i t o r e d . Fig. 1A illustrates the effects o f d e p o l a r i z i n g pulses o f varying intensities applied to a nerve terminal in the presence o f T T X . In accord with the results o f K a t z and Miledi 6, EPPs e v o k e d by d e p o l a r i z a t i o n o f the nerve t e r m i n a l occur in q u a n t a l steps. a n d the average a m p l i t u d e d e p e n d s on the s t r e n g t h o f the a p p l i e d current pulse. R e c o r d s in Fig. IB d e m o n s t r a t e that p o t e n t i a t i o n following a s h o r t train (q/~ M a l l a r t a n d M a r t i n 7) is not i m p a i r e d by the presence o f T T X . In the e x p e r i m e n t shown in Fig. 2. EPPs were o b t a i n e d at 2-see intervals with c o n s t a n t d e p o l a r i z i n g pulses applied to the m o t o r nerve t e r m i n a l before and after tetanic s t i m u l a t i o n by 500 pulses at 50/see in the presence o f T T X . S a m p l e r e c o r d s in Fig. 2A depict the changes in EPPs at v a r y i n g intervals following the t r a i n o f stimuli. A s shown in Fig. 2B (filled circles), the E P P showed a significant e n h a n c e m e n t in a m p l i t u d e after the tetanic stimuli. In a d d i t i o n , there was an increase in the frequency o f s p o n t a n e o u s M E P P s ~open circles in Fig. 2B). It can be seen t h a t the E P P r e t u r n s to the c o n t r o l level in a p p r o x i m a t e l y 60 sec. O n the o t h e r hand, p o t e n t i a t i o n o f M E P P frequency is greater in the percentile c h a n g e but has a shorter time course t h a n that for the e v o k e d s y n a p t i c response. These dissimilarities in the time course a n d in the m a g n i t u d e o f p o t e n t i a t i o n between M E P P and EPP have previously been n o t e d following a train o f nerve a c t i o n potentials a. F r o m the e x p e r i m e n t s described above, it is clear t h a t P T P o f n e u r o m u s c u l a r t r a n s m i s s i o n is not e l i m i n a t e d in the presence o f T T X . Since T T X blocks t h e i n w a r d s o d i u m currents associated with impulse g e n e r a t i o n 5. it is c o n c l u d e d that N a + entry into the nerve t e r m i n a l or the resultant intracellular N a + a c c u m u l a t i o n d u r i n g a train Brain Research. 17 0970) 527-529
SHORT COMMUNICATIONS
529
o f nerve stimuli is n o t i m m e d i a t e l y required for the g e n e r a t i o n o f PTP. F u r t h e r m o r e , the test d e p o l a r i z i n g pulses used in the present study before and after the tetanic stimulation were identical in m a g n i t u d e and d u r a t i o n . This eliminates the possibility that P T P m a y be due entirely to changes in configuration o f action potentials o f the presynaptic t e r m i n a l following a tetanic stimulus. The a u t h o r is indebted to Dr. M. K u n o for his advice and criticism in p r e p a r i n g the manuscript. The a u t h o r is a p r e d o c t o r a l trainee under U.S. Public H e a l t h Service P h a r m a cology Research T r a i n i n g G r a n t 5 T 1 - G M 153-11. Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah 84112 (U.S.A.)
DANIEL WEINREICH
1 BIRKS, R. 1., The role of sodium ions in the metabolism of acetylcholine, Canad. J. Biochem. Physiol., 41 (1963) 2573-2597. 2 FENG,T. P., Studies on the neuromuscular junction. XXXI. The changes of the end-plate potential during and after prolonged stimulation, Chin. J. Physiol., 16 (1941) 341-472. 3 HUBBARD,J. I., Repetitive stimulation at the mammalian neuromuscular junction and the mobilization of transmitter, J. Physiol. (Lond.), 169 (1963) 641-662. 4 HUGHES,J. R., Post-tetanic potentiation, Physiol. Rev., 38 (1958) 91-113. 5 KAO, C. Y., Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena, Pharmacol. Rev., 18 (1966)997-1049. 6 KATZ, B., AND MILEDI, R., Tetrodotoxin and neuromuscular transmission, Proc. roy. Soc. B, 167 (1967) 8-22. 7 MALLART, A., ANt) MARTtN, A. R., An analysis of facilitation of transmitter release at the neuromuscular junction of the frog, J. Physiol. (Lond.), 193 (1967) 679-694. 8 ROSENTHAL,J., Post-tetanic potentiation at the neuromuscular junction of the frog, J. Physiol. (Lond.), 203 (1969) 121-133. (Accepted November 20th, 1969)
Brain Research, 17 (1970) 527-529