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The effect of acetylcholine on twitches of the locust leg
Comp. Biochem. Physiol., 1963, Vol. 10, pp. 203 to 208. Pergamon Press Ltd. Printed in Great Britain
THE EFFECT OF ACETYLCHOLINE ON T W I T C H E S OF THE LOCUST LEG R. B. H I L L Department of Zoology, University of Glasgow*
(Received 16 April 1963) A b s t r a c t - - 1 . Concentrations of acetylcholine from 10-~ to 10-3 molar can
increase the amplitude of isotonic twitches of the extensor tibiae of Schistocerca
gregaria.
2. Fully isometric twitches are not affected by the same concentrations of acetylcholine. 3. The lack of action on isometric twitches may explain previous negative results of the application of acetylcholine to insect nerve-muscle preparations. INTRODUCTION THERE is a lack of conclusive evidence for an action of acetylcholine on insect neuromuscular preparations, although it appears possible that acetylcholine may play a role in transmission in insect central nervous systems. Acetylcholine, cholinesterase and choline acetylase are found in high concentrations in the central nervous systems of a number of insects (Prosser, 1961). As pointed out by Roeder (1953), the effects of anticholinesterases on synapfic transmission in insect central nervous systems are in accordance with the concept of chemical mediation but the reported physiological inactivity of exogenous acetylcholine is contradictory. However, Suga & Katsuki (1961) have found for the grasshopper Gampsocleis buergeri that acetylcholine has an excitatory effect on the large auditory fibres lying between the brain and the metathoracic ganglion. Voskressenskaya (1958) states as a generality that the excitatory influence of acetylcholine on insect skeletal muscles may be observed only at a certain stage of ontogenetic development. She found that acetylcholine caused a strong tonic contraction of caterpillar muscles (Voskressenskaya, 1946). The rhythmicity of pupal thoracic muscles was augmented by acetylcholine during the first days of the pupal stage but butterfly muscles did not respond to the application of acetylcholine. The wing muscles of fifth instar locusts (Voskressenskaya, 1958) responded to the application of acetylcholine by tonic contraction only until the sixth or seventh day, and in adult locusts there was no motor reaction to acetylcholine. Harlow (1958) found that acetylcholine could produce a tetanic extension when perfused through the isolated hind leg of Lo~sta migratoria. However, in studies of * Present address: Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire. 203
204
R.B. HILL .
"fast" twitches of the metathoracic extensor tibiae of the closely related locust, Schistocerca gregaria, Hill & Usherwood (1961) found injected acetylcholine to have no effect on isometric tension development as recorded with an R C A 5734 mechanoelectronic transducer. Subsequently, it seemed desirable to see if acetylcholine should have an action on isotonic twitches, since the fibres might be in a physiological state more comparable to that of the preparations studied by Vosskressenskaya and by Harlow. METHODS "Fast" twitches of the Schistocerca gregaria metathoracic extensor tibiae in situ were elicited by repetitive maximal stimulation of the crural nerve trunk with square pulses of variable strength delay, and duration delivered through chlorided silver electrodes. Isotonic records were made on the smoked drum of a kymograph, using a counterbalanced lever. A stop was placed so that the tibia came to rest at right angles to the femur. Records of isometric twitches were made with an RCA 5734 mechano-electronic transducer, attached directly to the tibia. Semi-isometric recordings were made by attaching the transducer indirectly to the tibia with a length of thread. Both the latter were thus free-loaded rather than after-loaded. Acetyleholine iodide (Eastman Kodak) in solution in locust saline (Hoyle, 1955b) was applied directly to the exposed ventral surface of the extensor tibiae and allowed to remain in the bath formed by the dorsal and lateral exoskeleton of the leg until an effect was seen. Simultaneous recordings of isometric twitch tension and of intracellular potentials were made as described previously (Hill & Usherwood, 1961). Calculations of the effect of acetylcholine on isotonic twitches were made on the basis of the percentage increase in the height of rise from the resting base line, measured on the kymograph records. RESULTS Acetylcholine was found to increase the amplitude of isotonic twitches due to repetitive stimulation, but not to cause contraction of the resting extensor tibiae. Ordinarily the increase was gradual (Fig. 1) and subsequent bathing of the continuously twitching leg in locust saline, for ½-1 hr, resulted in a return to twitches of the original size or smaller. Occasionally there would be an abrupt transition, following addition of acetylcholine, to responses of an amplitude comparable to a maximum tetanus. Concentrations of acetylcholine from 10 -2 M to 10 -7 M were found effective (Fig. 2) on the first trial. In preparations from fourteen different locusts where the isotonic twitch was potentiated by acetylcholine, four of these responded to 10 -6 M. Preparations previously exposed to acetylcholine showed a smaller increase of amplitude on a second application and the response to a third application was usually so small as to be of dubious significance. Mytolon (2, 5-bis (3-diethylaminopropylamino) benzoquinone bis-benzyl chloride), a potent antagonist of acetylcholine for the Venus heart (Luduena & Brown, 1952), did not block the acetylcholine effect but appeared to abolish the tachyphylaxis or desensitization, so that repeated applications of the same concentration of acetylcholine were equally effective. A possible effect of the anticholinesterase eserine was obscured because it proved to be toxic. At a concentration of 10 .4 M eserine caused a gradual diminution in the amplitude of twitches. Nevertheless, subsequent to eserinization,
THE EFFECT OF A C E T Y L C H O L I N E O N T W I T C H E S OF THE LOCUST L E G
205
application of acetylcholine at a given concentration could elicit twitches of as great an amplitude as before eserinization. T h u s the percentage increase in amplitude sometimes appeared greater after eserinization. Acetylcholine iodide failed to affect the amplitude of isometric twitches over a range of concentrations from 10 -7 M to 10 -2 M, even after prolonged treatment with high concentrations of cetyltrimethylammonium bromide which was applied to the exposed extensor tibiae in the hope that it might lower permeability barriers (Hoyle, 1953) by chemodissection (Walsh & Deal, 1959). Measurement of isometric records did not reveal any change in duration of the rising or of the falling phase of twitch tension after application of acetylcholine.
FIc. 1. Kymograph record of extensor tibiae twitches, elicited by repetitive stimulation. At the first arrow the bathing saline was replaced by saline containing 10 -6 molar acetylcholine. At the second arrow the acetylcholine was washed off with locust saline. The drum was then stopped for ½ hr. Time signal, every 10 sec. Use of semi-isometric conditions (flee-loaded) made it possible to record an increase in twitch tension after application of 10 -7 M acetylcholine, which became greater as conditions were made less isometric. T h a t is, use of a long thread running around a pulley between the tibia and the shaft of the transducer made it possible to record an increase in tension when acetylcholine was applied to the extensor tibiae, presumably by allowing some shortening. In insect leg muscle the electrically recorded membrane response to stimulation of the "fast" axon is a large "all-or-nothing" event (Hoyle, 1955a) which is believed to be due to a simultaneous depolarization at the numerous nerve endings
206
R.B. HTLL
on the surface of each muscle fibre (Hoyle, 1957a; H~imori, 1961), setting up junctional potentials and the resulting local spike responses simultaneously throughout the fibre (Hoyle, 1957b). Except at the highest concentration used (10 -2 M), acetylcholine had no effect on the duration or magnitude of the "fast" electrical response observed intracellularly during isometric twitches of the extensor tibiae. 300
250
200
~
J50
100 @
l
I
50 ¸
o
; i0-z
]
10-6 Molar
10 -5 concentration
10 -4 of
10-3
iO -z
ACh
FIG. 2. Relation of molar concentration of acetylcholine to per cent increase twitch amplitude. Each point represents a trial on a fresh preparation. DISCUSSION Acetylcholine iodide appears to increase the degree of shortening during twitches of the extensor tibiae of Schistocercagregaria but not to increase the force developed unless some shortening is permitted. As added iodide is ineffective at the lower concentrations used (i.e. 10 -3 M-10 -7 M), the effect noted must be due to acetylcholine. Harlow (1958) has previously found that 10 -1 M to 10 -5 M acetylcholine perfused through the isolated metathoracic leg of Locusta migratoria would sometimes produce a large kick extra to the repetitive stimulation and sometimes cause a tetanus which would relax during 2-3 rain. The "extra large kicks" in her figure look very much like the sudden transition to large contractile responses which I occasionally found. However, I never found acetylcholine to produce a tetanus unless the occasional large responses were brief tetani. Harlow did not report the gradual increase in size of repetitive twitches which I regularly saw. Instead, the response to stimulation continued, superimposed on a tetanus. We both found desensitization to result from repeated doses of acetylcholine.
T H E EFFECT OF A C E T Y L C H O L I N E ON T W I T C H E S OF T H E LOCUST LEG
207
The unexpected finding that Mytolon prevented desensitization but did not block the action of acetylcholine might be interpreted as indicating a reversible competition for receptor sites, in which acetylcholine could displace Mytolon but only in a dynamic relation which would prevent acetylcholine tachyphylaxis. If the toxic effect of eserine is due to anticholinesterase activity, it is difficult to reconcile with the idea that acetylcholine might be the transmitter at motor endplates of the extensor tibiae. Although applied acetylcholine reinforces twitches of the extensor tibiae, the curious effects of Mytolon and eserine do not justify the suggestion that acetylcholine might be the chemical transmitter for neuromuscular junctions of insect striated muscle. One question raised by the results reported here concerns the mode of action of acetylcholine. It might be expected that acetylcholine would exert an influence on a cell at the membrane. If the mechanism of the positive effect of acetylcholine on isotonic twitches were through changing the permeability of the cell membrane to an ion, e.g. calcium, then acetylcholine should markedly affect the magnitude of the "fast" membrane response, as does calcium (Hoyle, 1955b). In fact, acetylcholine at very high concentrations (10 -2 M) does somewhat increase the size of the "fast" response but this does not solve the problem of its mode of action at very low concentrations where no effect on the action potential is seen. However, the results reported above do appear to rule out the possibility that a prolongation of the action potential might be caused by acetylcholine. The second question arises from the observation that acetylcholine had no effect on twitches recorded isometrically, but a clearcut effect on twitches recorded isotonically, and a lesser effect on twitches recorded semi-isometrically. One possible explanation of an apparent increase in isotonic shortening, caused by a drug which does not increase isometric tension, might be that the duration of the shortening phase of the twitch was increased. That could appear, through the inertial distortion of the isotonic tracing lever system, as an increase in amplitude. If that were the explanation, the difference between isotonic and isometric effects of acetylcholine would be merely apparent, and the isometric records should reveal a change in duration of the period of development of tension. Since they did not, it seems that acetylcholine increases the amount of shortening without increasing the duration of the shortening phase. This could mean increase in the velocity of shortening, or it could mean an increase in the stiffness of the series elastic element (Hill, A. V., 1950). The observation that acetylcholine has an augmenting effect on twitches of the extensor tibiae is congruent with the observation by Hill & Usherwood (1961) that 5-hydroxytryptamine, which often has an opposite effect to that of acetylcholine, blocks neuromuscular transmission in the locust leg. The failure of acetylcholine to affect the preparation used by Hill & Usherwood (1961) is explained, since acetylcholine does not affect isometric twitches of the Schistocercagregaria extensor tibiae.
208
R . B . HILL SUMMARY
1. Results of a p p l y i n g acetylcholine to insect n e r v e - m u s c l e p r e p a r a t i o n s are often i n c o n c l u s i v e . Hill & U s h e r w o o d (1961) r e p o r t e d t h a t i n j e c t e d acetylcholine h a d n o effect o n i s o m e t r i c twitches of the locust leg. 2. A c e t y l c h o l i n e iodide i n s o l u t i o n i n locust saline was a p p l i e d to the exposed m e t a t h o r a c i c e x t e n s o r tibialis of Schfstocerca gregaria while m a x i m a l twitches were b e i n g elicited b y s t i m u l a t i o n of the n e r v e t r u n k c o n t a i n i n g the " f a s t " axon. 3. C o n c e n t r a t i o n s of acetylcholine f r o m 10 7 M to 10 -2 M were f o u n d to e n h a n c e the a m p l i t u d e of isotonic twitches b u t did n o t e n h a n c e t e n s i o n of isometric twitches.
Acknowledgements--I wish to acknowledge my indebtedness to Professor C. M. Yonge for making available the facilities of the Department of Zoology and to Dr. Graham Hoyle for his advice. This investigation was carried out during the tenure of a post-doctoral fellowship, HF-5211-C2, from the National Heart Institute, United States Public Health Service. REFERENCES H.~.MORI J. (1961) Innervation of insect leg muscle. Acta Biol. Acad. Sci. Hung. 12, 219-230. HARLOW P. ANNE (1958) The action of drugs on the nervous system of the locust (Locusta migratoria). Ann. Appl. Biol. 46, 55-73. HILL A. V. (1950) The series elastic component of muscle. Proc. Roy. Soc. B 137, 273-280. HILL R. B. & USHERWOODP. N. R. (1961) The action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust Schistocerca gregaria. J. Physiol. 157, 393401. HOVLE G. (1953) Potassium ions and insect nerve muscle. J. Exp. Biol. 30, 121-135. HOVLE G. (1955a) Neuromuscular mechanisms of a locust skeletal muscle. Proc. Roy. Soc. B 143, 343-367. HOYLE G. (1955b) The effects of some common cations on neuromuscular transmission in insects. J. Physiol. 127, 90-103. HOYLE G. (1957a) Nervous control of insect muscle. In Recent Advances in Invertebrate Physiology. Univ. of Oregon Publications. HOYLE G. (1957b) Comparative Physiology of the Nervous Control of Muscular Contraction. Cambridge University Press. LUDUENA F. P. & BROWN T. G. (1952) Mytolon and related compounds as antagonists of acetylcholine on the heart of Venus mercenaria. J. Pharmacot. 105, 232-239. PROSSER C. L. (1961) Nervous systems. In Comparative Animal Physiology, 2nd ed. (Edited by PROSSERC. L. & BROWN F.A.). Saunders, Philadelphia. ROEDER K. D. (1953) Electric activity in nerves and ganglia. In Insect Physiology (Edited by ROEDERK. D.). Chapman & Hal], London ; John Wiley, New York. SUGA N. & KATSUKIY (1961) Pharmacological studies on the auditory synapses in a grasshopper. J. Exp. Biol. 38, 759-770. VOSKRESSENSKAYAA. K. (1946) Study of the reaction of muscles of insects in the process of metamorphosis. Izv. Ahad. Nauk., Sect. Biol. 1946, 1, 163-170. VOSKRESSENSKAYAA. K (1958) Data about the functional evolution of the neuromuscular apparatus. In Problems of Evolution of Physiological Functions. USSR Acad. Sci. WALSH R. R. & DEAL S. E. (1959) Reversible conduction block produced by lipid-insoluble quaternary ammonium ions in cetyltrimethylammonium bromide-treated nerves. Amer. J. Physiol. 197, 547-550.
THE EFFECT OF ACETYLCHOLINE ON T W I T C H E S OF THE LOCUST LEG R. B. H I L L Department of Zoology, University of Glasgow*
(Received 16 April 1963) A b s t r a c t - - 1 . Concentrations of acetylcholine from 10-~ to 10-3 molar can
increase the amplitude of isotonic twitches of the extensor tibiae of Schistocerca
gregaria.
2. Fully isometric twitches are not affected by the same concentrations of acetylcholine. 3. The lack of action on isometric twitches may explain previous negative results of the application of acetylcholine to insect nerve-muscle preparations. INTRODUCTION THERE is a lack of conclusive evidence for an action of acetylcholine on insect neuromuscular preparations, although it appears possible that acetylcholine may play a role in transmission in insect central nervous systems. Acetylcholine, cholinesterase and choline acetylase are found in high concentrations in the central nervous systems of a number of insects (Prosser, 1961). As pointed out by Roeder (1953), the effects of anticholinesterases on synapfic transmission in insect central nervous systems are in accordance with the concept of chemical mediation but the reported physiological inactivity of exogenous acetylcholine is contradictory. However, Suga & Katsuki (1961) have found for the grasshopper Gampsocleis buergeri that acetylcholine has an excitatory effect on the large auditory fibres lying between the brain and the metathoracic ganglion. Voskressenskaya (1958) states as a generality that the excitatory influence of acetylcholine on insect skeletal muscles may be observed only at a certain stage of ontogenetic development. She found that acetylcholine caused a strong tonic contraction of caterpillar muscles (Voskressenskaya, 1946). The rhythmicity of pupal thoracic muscles was augmented by acetylcholine during the first days of the pupal stage but butterfly muscles did not respond to the application of acetylcholine. The wing muscles of fifth instar locusts (Voskressenskaya, 1958) responded to the application of acetylcholine by tonic contraction only until the sixth or seventh day, and in adult locusts there was no motor reaction to acetylcholine. Harlow (1958) found that acetylcholine could produce a tetanic extension when perfused through the isolated hind leg of Lo~sta migratoria. However, in studies of * Present address: Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire. 203
204
R.B. HILL .
"fast" twitches of the metathoracic extensor tibiae of the closely related locust, Schistocerca gregaria, Hill & Usherwood (1961) found injected acetylcholine to have no effect on isometric tension development as recorded with an R C A 5734 mechanoelectronic transducer. Subsequently, it seemed desirable to see if acetylcholine should have an action on isotonic twitches, since the fibres might be in a physiological state more comparable to that of the preparations studied by Vosskressenskaya and by Harlow. METHODS "Fast" twitches of the Schistocerca gregaria metathoracic extensor tibiae in situ were elicited by repetitive maximal stimulation of the crural nerve trunk with square pulses of variable strength delay, and duration delivered through chlorided silver electrodes. Isotonic records were made on the smoked drum of a kymograph, using a counterbalanced lever. A stop was placed so that the tibia came to rest at right angles to the femur. Records of isometric twitches were made with an RCA 5734 mechano-electronic transducer, attached directly to the tibia. Semi-isometric recordings were made by attaching the transducer indirectly to the tibia with a length of thread. Both the latter were thus free-loaded rather than after-loaded. Acetyleholine iodide (Eastman Kodak) in solution in locust saline (Hoyle, 1955b) was applied directly to the exposed ventral surface of the extensor tibiae and allowed to remain in the bath formed by the dorsal and lateral exoskeleton of the leg until an effect was seen. Simultaneous recordings of isometric twitch tension and of intracellular potentials were made as described previously (Hill & Usherwood, 1961). Calculations of the effect of acetylcholine on isotonic twitches were made on the basis of the percentage increase in the height of rise from the resting base line, measured on the kymograph records. RESULTS Acetylcholine was found to increase the amplitude of isotonic twitches due to repetitive stimulation, but not to cause contraction of the resting extensor tibiae. Ordinarily the increase was gradual (Fig. 1) and subsequent bathing of the continuously twitching leg in locust saline, for ½-1 hr, resulted in a return to twitches of the original size or smaller. Occasionally there would be an abrupt transition, following addition of acetylcholine, to responses of an amplitude comparable to a maximum tetanus. Concentrations of acetylcholine from 10 -2 M to 10 -7 M were found effective (Fig. 2) on the first trial. In preparations from fourteen different locusts where the isotonic twitch was potentiated by acetylcholine, four of these responded to 10 -6 M. Preparations previously exposed to acetylcholine showed a smaller increase of amplitude on a second application and the response to a third application was usually so small as to be of dubious significance. Mytolon (2, 5-bis (3-diethylaminopropylamino) benzoquinone bis-benzyl chloride), a potent antagonist of acetylcholine for the Venus heart (Luduena & Brown, 1952), did not block the acetylcholine effect but appeared to abolish the tachyphylaxis or desensitization, so that repeated applications of the same concentration of acetylcholine were equally effective. A possible effect of the anticholinesterase eserine was obscured because it proved to be toxic. At a concentration of 10 .4 M eserine caused a gradual diminution in the amplitude of twitches. Nevertheless, subsequent to eserinization,
THE EFFECT OF A C E T Y L C H O L I N E O N T W I T C H E S OF THE LOCUST L E G
205
application of acetylcholine at a given concentration could elicit twitches of as great an amplitude as before eserinization. T h u s the percentage increase in amplitude sometimes appeared greater after eserinization. Acetylcholine iodide failed to affect the amplitude of isometric twitches over a range of concentrations from 10 -7 M to 10 -2 M, even after prolonged treatment with high concentrations of cetyltrimethylammonium bromide which was applied to the exposed extensor tibiae in the hope that it might lower permeability barriers (Hoyle, 1953) by chemodissection (Walsh & Deal, 1959). Measurement of isometric records did not reveal any change in duration of the rising or of the falling phase of twitch tension after application of acetylcholine.
FIc. 1. Kymograph record of extensor tibiae twitches, elicited by repetitive stimulation. At the first arrow the bathing saline was replaced by saline containing 10 -6 molar acetylcholine. At the second arrow the acetylcholine was washed off with locust saline. The drum was then stopped for ½ hr. Time signal, every 10 sec. Use of semi-isometric conditions (flee-loaded) made it possible to record an increase in twitch tension after application of 10 -7 M acetylcholine, which became greater as conditions were made less isometric. T h a t is, use of a long thread running around a pulley between the tibia and the shaft of the transducer made it possible to record an increase in tension when acetylcholine was applied to the extensor tibiae, presumably by allowing some shortening. In insect leg muscle the electrically recorded membrane response to stimulation of the "fast" axon is a large "all-or-nothing" event (Hoyle, 1955a) which is believed to be due to a simultaneous depolarization at the numerous nerve endings
206
R.B. HTLL
on the surface of each muscle fibre (Hoyle, 1957a; H~imori, 1961), setting up junctional potentials and the resulting local spike responses simultaneously throughout the fibre (Hoyle, 1957b). Except at the highest concentration used (10 -2 M), acetylcholine had no effect on the duration or magnitude of the "fast" electrical response observed intracellularly during isometric twitches of the extensor tibiae. 300
250
200
~
J50
100 @
l
I
50 ¸
o
; i0-z
]
10-6 Molar
10 -5 concentration
10 -4 of
10-3
iO -z
ACh
FIG. 2. Relation of molar concentration of acetylcholine to per cent increase twitch amplitude. Each point represents a trial on a fresh preparation. DISCUSSION Acetylcholine iodide appears to increase the degree of shortening during twitches of the extensor tibiae of Schistocercagregaria but not to increase the force developed unless some shortening is permitted. As added iodide is ineffective at the lower concentrations used (i.e. 10 -3 M-10 -7 M), the effect noted must be due to acetylcholine. Harlow (1958) has previously found that 10 -1 M to 10 -5 M acetylcholine perfused through the isolated metathoracic leg of Locusta migratoria would sometimes produce a large kick extra to the repetitive stimulation and sometimes cause a tetanus which would relax during 2-3 rain. The "extra large kicks" in her figure look very much like the sudden transition to large contractile responses which I occasionally found. However, I never found acetylcholine to produce a tetanus unless the occasional large responses were brief tetani. Harlow did not report the gradual increase in size of repetitive twitches which I regularly saw. Instead, the response to stimulation continued, superimposed on a tetanus. We both found desensitization to result from repeated doses of acetylcholine.
T H E EFFECT OF A C E T Y L C H O L I N E ON T W I T C H E S OF T H E LOCUST LEG
207
The unexpected finding that Mytolon prevented desensitization but did not block the action of acetylcholine might be interpreted as indicating a reversible competition for receptor sites, in which acetylcholine could displace Mytolon but only in a dynamic relation which would prevent acetylcholine tachyphylaxis. If the toxic effect of eserine is due to anticholinesterase activity, it is difficult to reconcile with the idea that acetylcholine might be the transmitter at motor endplates of the extensor tibiae. Although applied acetylcholine reinforces twitches of the extensor tibiae, the curious effects of Mytolon and eserine do not justify the suggestion that acetylcholine might be the chemical transmitter for neuromuscular junctions of insect striated muscle. One question raised by the results reported here concerns the mode of action of acetylcholine. It might be expected that acetylcholine would exert an influence on a cell at the membrane. If the mechanism of the positive effect of acetylcholine on isotonic twitches were through changing the permeability of the cell membrane to an ion, e.g. calcium, then acetylcholine should markedly affect the magnitude of the "fast" membrane response, as does calcium (Hoyle, 1955b). In fact, acetylcholine at very high concentrations (10 -2 M) does somewhat increase the size of the "fast" response but this does not solve the problem of its mode of action at very low concentrations where no effect on the action potential is seen. However, the results reported above do appear to rule out the possibility that a prolongation of the action potential might be caused by acetylcholine. The second question arises from the observation that acetylcholine had no effect on twitches recorded isometrically, but a clearcut effect on twitches recorded isotonically, and a lesser effect on twitches recorded semi-isometrically. One possible explanation of an apparent increase in isotonic shortening, caused by a drug which does not increase isometric tension, might be that the duration of the shortening phase of the twitch was increased. That could appear, through the inertial distortion of the isotonic tracing lever system, as an increase in amplitude. If that were the explanation, the difference between isotonic and isometric effects of acetylcholine would be merely apparent, and the isometric records should reveal a change in duration of the period of development of tension. Since they did not, it seems that acetylcholine increases the amount of shortening without increasing the duration of the shortening phase. This could mean increase in the velocity of shortening, or it could mean an increase in the stiffness of the series elastic element (Hill, A. V., 1950). The observation that acetylcholine has an augmenting effect on twitches of the extensor tibiae is congruent with the observation by Hill & Usherwood (1961) that 5-hydroxytryptamine, which often has an opposite effect to that of acetylcholine, blocks neuromuscular transmission in the locust leg. The failure of acetylcholine to affect the preparation used by Hill & Usherwood (1961) is explained, since acetylcholine does not affect isometric twitches of the Schistocercagregaria extensor tibiae.
208
R . B . HILL SUMMARY
1. Results of a p p l y i n g acetylcholine to insect n e r v e - m u s c l e p r e p a r a t i o n s are often i n c o n c l u s i v e . Hill & U s h e r w o o d (1961) r e p o r t e d t h a t i n j e c t e d acetylcholine h a d n o effect o n i s o m e t r i c twitches of the locust leg. 2. A c e t y l c h o l i n e iodide i n s o l u t i o n i n locust saline was a p p l i e d to the exposed m e t a t h o r a c i c e x t e n s o r tibialis of Schfstocerca gregaria while m a x i m a l twitches were b e i n g elicited b y s t i m u l a t i o n of the n e r v e t r u n k c o n t a i n i n g the " f a s t " axon. 3. C o n c e n t r a t i o n s of acetylcholine f r o m 10 7 M to 10 -2 M were f o u n d to e n h a n c e the a m p l i t u d e of isotonic twitches b u t did n o t e n h a n c e t e n s i o n of isometric twitches.
Acknowledgements--I wish to acknowledge my indebtedness to Professor C. M. Yonge for making available the facilities of the Department of Zoology and to Dr. Graham Hoyle for his advice. This investigation was carried out during the tenure of a post-doctoral fellowship, HF-5211-C2, from the National Heart Institute, United States Public Health Service. REFERENCES H.~.MORI J. (1961) Innervation of insect leg muscle. Acta Biol. Acad. Sci. Hung. 12, 219-230. HARLOW P. ANNE (1958) The action of drugs on the nervous system of the locust (Locusta migratoria). Ann. Appl. Biol. 46, 55-73. HILL A. V. (1950) The series elastic component of muscle. Proc. Roy. Soc. B 137, 273-280. HILL R. B. & USHERWOODP. N. R. (1961) The action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust Schistocerca gregaria. J. Physiol. 157, 393401. HOVLE G. (1953) Potassium ions and insect nerve muscle. J. Exp. Biol. 30, 121-135. HOVLE G. (1955a) Neuromuscular mechanisms of a locust skeletal muscle. Proc. Roy. Soc. B 143, 343-367. HOYLE G. (1955b) The effects of some common cations on neuromuscular transmission in insects. J. Physiol. 127, 90-103. HOYLE G. (1957a) Nervous control of insect muscle. In Recent Advances in Invertebrate Physiology. Univ. of Oregon Publications. HOYLE G. (1957b) Comparative Physiology of the Nervous Control of Muscular Contraction. Cambridge University Press. LUDUENA F. P. & BROWN T. G. (1952) Mytolon and related compounds as antagonists of acetylcholine on the heart of Venus mercenaria. J. Pharmacot. 105, 232-239. PROSSER C. L. (1961) Nervous systems. In Comparative Animal Physiology, 2nd ed. (Edited by PROSSERC. L. & BROWN F.A.). Saunders, Philadelphia. ROEDER K. D. (1953) Electric activity in nerves and ganglia. In Insect Physiology (Edited by ROEDERK. D.). Chapman & Hal], London ; John Wiley, New York. SUGA N. & KATSUKIY (1961) Pharmacological studies on the auditory synapses in a grasshopper. J. Exp. Biol. 38, 759-770. VOSKRESSENSKAYAA. K. (1946) Study of the reaction of muscles of insects in the process of metamorphosis. Izv. Ahad. Nauk., Sect. Biol. 1946, 1, 163-170. VOSKRESSENSKAYAA. K (1958) Data about the functional evolution of the neuromuscular apparatus. In Problems of Evolution of Physiological Functions. USSR Acad. Sci. WALSH R. R. & DEAL S. E. (1959) Reversible conduction block produced by lipid-insoluble quaternary ammonium ions in cetyltrimethylammonium bromide-treated nerves. Amer. J. Physiol. 197, 547-550.