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The effect of acetylcholine and 5-hydroxy-tryptamine on electrophysiological recordings from muscle fibres of the leech, Hirudo medicinalis
Comp. Biochem. Physiol., 1968, Vol. 24, pp. 987 to 990. Pergamon Press. PHnted in Great Britain
T H E E F F E C T OF A C E T Y L C H O L I N E AND 5-HYDROXYT R Y P T A M I N E ON E L E C T R O P H Y S I O L O G I C A L RECORDINGS FROM MUSCLE FIBRES OF T H E LEECH, HIR UDO M E D I C I N A L I S R. J. WALKER, G. N. W O O D R U F F and G. A. K E R K U T Department of Physiology and Biochemistry, The University of Southampton, Southampton, England (Received 19 August 1967) A b s t r a c t - - 1 . Intracellular recordings from Hirudo muscle fibres indicate
resting potential values of 30-50mV, inside negative; action potentials of 20-50 mV amplitude with a marked after-potential and duration of 20 msec. 2. Iontophoretic application of acetylcholine excites the muscle, producing action potentials; 5-hydroxytryptamine reduces the amplitude of excitatory muscle potentials. INTRODUCTION StNCE the experiments of Fuehner (1918) and Minz (1932) excitation and muscle contraction in the leech has been associated with acetylcholine. The later experiments of Bacq & Coppee (1937) suggested the presence of cholinergic nerves innervating the leech body-wall musculature. The excitatory action of acetylcholine can be antagonized by pretreating the preparation with 5-hydroxytryptamine (Poloni, 1955; Schain, 1961) which itself will relax the muscle. 5-Hydroxytryptamine has been shown to be present in cells in the leech segmental ganglion, including the Retzius cells (Kerkut et al., 1967). These cells send axons down the segmental nerves to innervate the body-wall musculature (Retzius, 1891; Hagiwara & Morita, 1962). The present study was undertaken to investigate the action of acetylcholine and 5-hydroxytryptamine on electrophysiological recordings from the leech body-wall musculature. METHODS
All experiments were performed on the medicinal leech, Hirudo medicinalis. Leeches were pinned on a wax block, dorsal side upwards and a slit made in the lateral body wall extending the length of the animal. The dorsal body wall was then pinned to one side and a section of body wall and adjacent segmental ganglia removed and pinned onto a small wax block and placed in leech Ringer. Under a binocular microscope, the connective tissue was carefully cleared to expose the muscle fibres. The recording electrodes were pulled from Pyrex glass on a vertical machine and filled with a molar solution of potassium acetate. These electrodes were 987
988
R. J. WALKER,G. N. WOODRUFFANDG. A. KERKUT
selected for a resistance of between 40 and 60 M ~ ; such electrodes were found most suitable for this study. The iontophoretic electrodes were pulled on a horizontal machine and their tips carefully broken to about 1/~ diameter. These were then placed in a solution of molar acetylcholine chloride at a pH of 3-4 and left for a day. The aeetylcholine was found to rise up the tip by capillary action. The shank of the electrode was filled under reduced pressure. A current of 100 nA was used to expel the acetylcholine as a cation. The electrical activity was recorded by means of a Medistor negative capacity electrometer amplifier and displayed on a Tetronix502A oscilloscope. The activity was then either filmed using a Shackman camera or recorded on paper using an Ediswan pen oscillograph. The Ringer used had the following formula: NaCI, 115 raM; KC1, 4 raM; CaCI~, 2 raM; NaHCOa, 5 mM. The volume of the bath was 20 ml. The concentrations of acetylcholine and 5-hydroxytryptamine are expressed as #g/m]. Acetylcholine was used as the chloride and 5-hydroxytryptamine was used as the creatinine sulphate. RESULTS
The muscle fibres have resting potentials in the range of 30-50 mV, the inside being negative. Sometimes penetration is followed by a train of muscle potentials (Fig. 1). These range in amplitude from 50 mV to less than 20 InV. There is always a large after-potential. These muscle potentials have a duration of about 20 msec. Following penetration the muscle fibre usually quickly stops firing, suggesting that the potentials are associated with penetration. However, on occasion, muscle action potentials occur randomly for some time after penetration. When recording in some preparations, small excitatory depolarizations can be seen (Fig. 2). These range in amplitude from less than 1 mV to 10 inV. In addition to these potentials, very small potentials resembling miniature end-plate potentials can be recorded (Fig. 5). Iontophoretic application of acetylcholine results in the depolarization of the leech muscle, resulting in a muscle action potential (Figs. 3 and 4). Figure 4 comprises a series of iontophoretic applications of acetylcholine onto a muscle fibre. In Fig. 4a the top of the spikes is lost due to the high gain; however, they were about 40 mV in amplitude. Repeated applications of acetylcholine resulted in smaller muscle potentials (Fig. 4d-f). The onset of the response was consistently around 3 sec following the start of the application of the acetylcholine. Application to the bath of 0.1 #g/ml acetylcholine sometimes resulted in a single muscle potential similar in shape to those seen in Fig. 4. Higher concentrations of acetylcholine resulted in complete depolarization and the electrode usually came out of the muscle fibre. Application to the bath of 10/~g/ml 5-hydroxytryptamine results in a decrease in amplitude both of the miniature end-plate potentials (Fig. 5), and of the larger excitatory postsynaptic potentials (Fig. 6). On washing the preparation the latter potentials partially recover (Fig. 6).
[ IO,~V ~ C
lOOms¢¢
D
FIG. l. Action potentials recorded from the muscle of Hirudo medicinalis. (a) Resting potential is - 4 4 mV. (b) Resting potential is - 3 4 inV. (c) Resting potential is - 44 inV. (d) Resting potential is - 48 inV.
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FIG. 2. Small excitatory postsynaptic potentials recorded from the muscle of Hirudo medicinalis. The resting potential was - 4 6 mV.
[ IOmV ~ l s ¢ c B
^
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V ACETYLCHOLINE
C
FIG. 3. The effect of the iontophoretic application of acetylcholine on the activity of a muscle fibre. (a) Before the application of acetylcholine. (b) Application of acetylcholine increased the frequency of the small depolarizations and resulted in the production of three muscle action potentials. (c) Following the application ot acetylcholine. T h e muscle fibre resting potential was - 3 8 inV.
A
B
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A C
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FIG. 4. The effect of the iontophoretic application of acetylcholine onto a silent muscle fibre. All recordings are from the same muscle fibre. Trace (a) is at a gain five times greater than (d)-(f). During the experiment the resting potential fell from - 4 8 mV to - 4 0 mV.
A
I imV"
'lsec
B
IO pg/'ml 5- H T C
FIG. 5. The effect of 10/zg/ml 5-hydroxytryptamine on miniature end-plate potentials recorded from leech muscle. The resting potential of the muscle fibre was - 5 0 mV. (a) Before the application of 5-hydroxytryptamine. (b) and (c) After the application of 5-hydroxytryptamine.
JflF~'~CT OF ACETYLCHOLINE AND 5-HYDROXYTRYPTAMINE ON H I R U D O MUSCLE
989
A
A
IOiJg/Zml 5-HT B
[ 5mV ~2sec C . . . .
:, : : . . . .
~. . . . ;...L . . . . L_=,=;h.= a= . . . . . . . . . ,; ,[;=:..=..~-~, ..... ~,
FIG. 6. The effect of 10/~g/ml 5-hydroxytryptamine on excitatory muscle potentials. The 5-hydroxytryptamine greatly reduced the amplitude of the potentials, trace (b); these partially recover in trace (c). The resting potential of the muscle fibre increased following the application of 5-hydroxytryptamine from - 36 to - 4 0 mV. DISCUSSION The characteristics of the muscle action potentials recorded on penetration of the muscle fibres agree with those obtained by Washizu (1967). However, the average resting potential value in this present study, about 40 mV, was about 10 mV more negative than the value obtained by Washizu. T h e average height and duration of the muscle action potentials in both s t u d i e s are similar, around 35 mV and 20 msec respectively. The present electrophysiological investigation confirms the earlier whole-tissue experiments. Acetylcholine excites and depolarizes the resting potential of single muscle fibres, resulting in one or more muscle action potenfi',ds which resemble the muscle action potentials recorded on penetration of the muscle fibres. It has not been possible to locate specifically the site of maximum sensitivity to acetylcholine and to identify such sites with end-plate activity. 5-Hydroxytryptamine depresses the amplitude of excitatory potentials recorded in the muscle fibres, but there is no evidence as to whether this is a general effect on the muscle, altering its membrane resistance and so conductance or whether its site of action is restricted to certain specific areas of the muscle fibre membrane. 5-Hydroxytryptamine is a possible candidate as an inhibitory transmitter at the leech neuromuscular junction. A similar suggestion has been put forward by Myhrberg (1967) for Lumbricus. If 5-hydroxytryptamine is an inhibitory transmitter at the leech neuromuscular junction, then it is possibly liberated from the axon terminals of the Retzius cells which contain 5-hydroxytryptamine (Kerkut et al., 1967) and which run towards the muscle (Hagiwara & Morita, 1962). SUMMARY 1. Intracellular recordings have been made from the body wall muscle fibres of the leech, Hirudo medicinalis. The resting potentials range in size from 30 to
990
R. J. W~U.Kma,G. N. WOODRUFFAND G. A. KmUCUT
50 mV, the inside being negative. T h e action potentials range in amplitude from 20 to 50 mV, with large after-potentials and a duration of around 20 msec. 2. Acetylcholine applied either directly to the bath or iontophoretically produces depolarizations similar in their characteristics to the muscle action potentials. 3. 5-Hydroxytryptamine applied to the bath at a concentration of 10/~g/ml depresses the amplitude of excitatory postsynaptic muscle potentials. 4. It is suggested that acetylcholine is the excitatory neuromuscular transmitter and 5-hydroxytryptamine is the inhibitory neuromuscular transmitter in the leech. REFERENCES
BACQZ. M. ~ COPPEEG. (1937) R6action des vers et des moUusques/~ l'6s6rine. Existence de nerfs cholinergiques chez les vers. Archs int. Physiol. 4.5, 310-324. FUmm'ER H. (1918) Untersuchungen tiber den Synergismus von Giften Chemische Erregbarkeitssteigerung glatter Muskeln. Arch. exp. Path. Pharmak. 82, 57-80. HAOIWARAS. & MORITA H. (1962) Eleetrotonic transmission between two nerve cells in leech ganglion, ft. Neurophysiol. 25, 721-731. I~RKUT G. A., SEDDENC. B. & W;u.g~a R. J. (1967) Cellular localization of monoamines by fluorescence microscopy in Hirudo medicinalis and Lumbricus terrestris. Comp. Biodwm. Physiol. 21, 687-690. M ~ z B, (1932) Pharmakologische Untersuchungen am Blutegelpriiparat, zugleich eine Methode zum biologischen Nachweis von Acetylcholin bei Anwesenheit anderer pharmakologisch wirksamer k6rpereigener Stoffe. Arch. exp. Path. Pharraak. 168, 292-304. MYmm]mo H. E. (1967) Monoaminergic mechanisms in the nervous system of Lurabricus terrestris. Z. Zellforsch. 81, 311-343. POLONIA. (1955) I1 muscolo dorsale di sanguisuga quale test biologico per l'evidenziamento dell'attivit/~ serotoninica nei liquidi organici. Cervello 31, 472-476. RETZlUS G. (1891) Zur Kenntnis des centralen Nervensystems der Witrmer. Biol. Unters (N.F.) 2, 1-28. SCmUN R. J. (1961) Effects of 5-hydroxytryptamine on the dorsal muscle of the leech (Hirudo medicinalis). Br. ft. Pharmae. Chemother. 16, 257-261. WASmZU Y. (1967) Electrical properties of leech dorsal muscle. Comp. Biochem. Physiol. 20, 641-646.
T H E E F F E C T OF A C E T Y L C H O L I N E AND 5-HYDROXYT R Y P T A M I N E ON E L E C T R O P H Y S I O L O G I C A L RECORDINGS FROM MUSCLE FIBRES OF T H E LEECH, HIR UDO M E D I C I N A L I S R. J. WALKER, G. N. W O O D R U F F and G. A. K E R K U T Department of Physiology and Biochemistry, The University of Southampton, Southampton, England (Received 19 August 1967) A b s t r a c t - - 1 . Intracellular recordings from Hirudo muscle fibres indicate
resting potential values of 30-50mV, inside negative; action potentials of 20-50 mV amplitude with a marked after-potential and duration of 20 msec. 2. Iontophoretic application of acetylcholine excites the muscle, producing action potentials; 5-hydroxytryptamine reduces the amplitude of excitatory muscle potentials. INTRODUCTION StNCE the experiments of Fuehner (1918) and Minz (1932) excitation and muscle contraction in the leech has been associated with acetylcholine. The later experiments of Bacq & Coppee (1937) suggested the presence of cholinergic nerves innervating the leech body-wall musculature. The excitatory action of acetylcholine can be antagonized by pretreating the preparation with 5-hydroxytryptamine (Poloni, 1955; Schain, 1961) which itself will relax the muscle. 5-Hydroxytryptamine has been shown to be present in cells in the leech segmental ganglion, including the Retzius cells (Kerkut et al., 1967). These cells send axons down the segmental nerves to innervate the body-wall musculature (Retzius, 1891; Hagiwara & Morita, 1962). The present study was undertaken to investigate the action of acetylcholine and 5-hydroxytryptamine on electrophysiological recordings from the leech body-wall musculature. METHODS
All experiments were performed on the medicinal leech, Hirudo medicinalis. Leeches were pinned on a wax block, dorsal side upwards and a slit made in the lateral body wall extending the length of the animal. The dorsal body wall was then pinned to one side and a section of body wall and adjacent segmental ganglia removed and pinned onto a small wax block and placed in leech Ringer. Under a binocular microscope, the connective tissue was carefully cleared to expose the muscle fibres. The recording electrodes were pulled from Pyrex glass on a vertical machine and filled with a molar solution of potassium acetate. These electrodes were 987
988
R. J. WALKER,G. N. WOODRUFFANDG. A. KERKUT
selected for a resistance of between 40 and 60 M ~ ; such electrodes were found most suitable for this study. The iontophoretic electrodes were pulled on a horizontal machine and their tips carefully broken to about 1/~ diameter. These were then placed in a solution of molar acetylcholine chloride at a pH of 3-4 and left for a day. The aeetylcholine was found to rise up the tip by capillary action. The shank of the electrode was filled under reduced pressure. A current of 100 nA was used to expel the acetylcholine as a cation. The electrical activity was recorded by means of a Medistor negative capacity electrometer amplifier and displayed on a Tetronix502A oscilloscope. The activity was then either filmed using a Shackman camera or recorded on paper using an Ediswan pen oscillograph. The Ringer used had the following formula: NaCI, 115 raM; KC1, 4 raM; CaCI~, 2 raM; NaHCOa, 5 mM. The volume of the bath was 20 ml. The concentrations of acetylcholine and 5-hydroxytryptamine are expressed as #g/m]. Acetylcholine was used as the chloride and 5-hydroxytryptamine was used as the creatinine sulphate. RESULTS
The muscle fibres have resting potentials in the range of 30-50 mV, the inside being negative. Sometimes penetration is followed by a train of muscle potentials (Fig. 1). These range in amplitude from 50 mV to less than 20 InV. There is always a large after-potential. These muscle potentials have a duration of about 20 msec. Following penetration the muscle fibre usually quickly stops firing, suggesting that the potentials are associated with penetration. However, on occasion, muscle action potentials occur randomly for some time after penetration. When recording in some preparations, small excitatory depolarizations can be seen (Fig. 2). These range in amplitude from less than 1 mV to 10 inV. In addition to these potentials, very small potentials resembling miniature end-plate potentials can be recorded (Fig. 5). Iontophoretic application of acetylcholine results in the depolarization of the leech muscle, resulting in a muscle action potential (Figs. 3 and 4). Figure 4 comprises a series of iontophoretic applications of acetylcholine onto a muscle fibre. In Fig. 4a the top of the spikes is lost due to the high gain; however, they were about 40 mV in amplitude. Repeated applications of acetylcholine resulted in smaller muscle potentials (Fig. 4d-f). The onset of the response was consistently around 3 sec following the start of the application of the acetylcholine. Application to the bath of 0.1 #g/ml acetylcholine sometimes resulted in a single muscle potential similar in shape to those seen in Fig. 4. Higher concentrations of acetylcholine resulted in complete depolarization and the electrode usually came out of the muscle fibre. Application to the bath of 10/~g/ml 5-hydroxytryptamine results in a decrease in amplitude both of the miniature end-plate potentials (Fig. 5), and of the larger excitatory postsynaptic potentials (Fig. 6). On washing the preparation the latter potentials partially recover (Fig. 6).
[ IO,~V ~ C
lOOms¢¢
D
FIG. l. Action potentials recorded from the muscle of Hirudo medicinalis. (a) Resting potential is - 4 4 mV. (b) Resting potential is - 3 4 inV. (c) Resting potential is - 44 inV. (d) Resting potential is - 48 inV.
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FIG. 2. Small excitatory postsynaptic potentials recorded from the muscle of Hirudo medicinalis. The resting potential was - 4 6 mV.
[ IOmV ~ l s ¢ c B
^
w
V ACETYLCHOLINE
C
FIG. 3. The effect of the iontophoretic application of acetylcholine on the activity of a muscle fibre. (a) Before the application of acetylcholine. (b) Application of acetylcholine increased the frequency of the small depolarizations and resulted in the production of three muscle action potentials. (c) Following the application ot acetylcholine. T h e muscle fibre resting potential was - 3 8 inV.
A
B
i i~~!~ ii
A C
D
^
!
E
[ 5rnV ,1 5zc
.....................
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JJ
.j~
A
~.
V
FIG. 4. The effect of the iontophoretic application of acetylcholine onto a silent muscle fibre. All recordings are from the same muscle fibre. Trace (a) is at a gain five times greater than (d)-(f). During the experiment the resting potential fell from - 4 8 mV to - 4 0 mV.
A
I imV"
'lsec
B
IO pg/'ml 5- H T C
FIG. 5. The effect of 10/zg/ml 5-hydroxytryptamine on miniature end-plate potentials recorded from leech muscle. The resting potential of the muscle fibre was - 5 0 mV. (a) Before the application of 5-hydroxytryptamine. (b) and (c) After the application of 5-hydroxytryptamine.
JflF~'~CT OF ACETYLCHOLINE AND 5-HYDROXYTRYPTAMINE ON H I R U D O MUSCLE
989
A
A
IOiJg/Zml 5-HT B
[ 5mV ~2sec C . . . .
:, : : . . . .
~. . . . ;...L . . . . L_=,=;h.= a= . . . . . . . . . ,; ,[;=:..=..~-~, ..... ~,
FIG. 6. The effect of 10/~g/ml 5-hydroxytryptamine on excitatory muscle potentials. The 5-hydroxytryptamine greatly reduced the amplitude of the potentials, trace (b); these partially recover in trace (c). The resting potential of the muscle fibre increased following the application of 5-hydroxytryptamine from - 36 to - 4 0 mV. DISCUSSION The characteristics of the muscle action potentials recorded on penetration of the muscle fibres agree with those obtained by Washizu (1967). However, the average resting potential value in this present study, about 40 mV, was about 10 mV more negative than the value obtained by Washizu. T h e average height and duration of the muscle action potentials in both s t u d i e s are similar, around 35 mV and 20 msec respectively. The present electrophysiological investigation confirms the earlier whole-tissue experiments. Acetylcholine excites and depolarizes the resting potential of single muscle fibres, resulting in one or more muscle action potenfi',ds which resemble the muscle action potentials recorded on penetration of the muscle fibres. It has not been possible to locate specifically the site of maximum sensitivity to acetylcholine and to identify such sites with end-plate activity. 5-Hydroxytryptamine depresses the amplitude of excitatory potentials recorded in the muscle fibres, but there is no evidence as to whether this is a general effect on the muscle, altering its membrane resistance and so conductance or whether its site of action is restricted to certain specific areas of the muscle fibre membrane. 5-Hydroxytryptamine is a possible candidate as an inhibitory transmitter at the leech neuromuscular junction. A similar suggestion has been put forward by Myhrberg (1967) for Lumbricus. If 5-hydroxytryptamine is an inhibitory transmitter at the leech neuromuscular junction, then it is possibly liberated from the axon terminals of the Retzius cells which contain 5-hydroxytryptamine (Kerkut et al., 1967) and which run towards the muscle (Hagiwara & Morita, 1962). SUMMARY 1. Intracellular recordings have been made from the body wall muscle fibres of the leech, Hirudo medicinalis. The resting potentials range in size from 30 to
990
R. J. W~U.Kma,G. N. WOODRUFFAND G. A. KmUCUT
50 mV, the inside being negative. T h e action potentials range in amplitude from 20 to 50 mV, with large after-potentials and a duration of around 20 msec. 2. Acetylcholine applied either directly to the bath or iontophoretically produces depolarizations similar in their characteristics to the muscle action potentials. 3. 5-Hydroxytryptamine applied to the bath at a concentration of 10/~g/ml depresses the amplitude of excitatory postsynaptic muscle potentials. 4. It is suggested that acetylcholine is the excitatory neuromuscular transmitter and 5-hydroxytryptamine is the inhibitory neuromuscular transmitter in the leech. REFERENCES
BACQZ. M. ~ COPPEEG. (1937) R6action des vers et des moUusques/~ l'6s6rine. Existence de nerfs cholinergiques chez les vers. Archs int. Physiol. 4.5, 310-324. FUmm'ER H. (1918) Untersuchungen tiber den Synergismus von Giften Chemische Erregbarkeitssteigerung glatter Muskeln. Arch. exp. Path. Pharmak. 82, 57-80. HAOIWARAS. & MORITA H. (1962) Eleetrotonic transmission between two nerve cells in leech ganglion, ft. Neurophysiol. 25, 721-731. I~RKUT G. A., SEDDENC. B. & W;u.g~a R. J. (1967) Cellular localization of monoamines by fluorescence microscopy in Hirudo medicinalis and Lumbricus terrestris. Comp. Biodwm. Physiol. 21, 687-690. M ~ z B, (1932) Pharmakologische Untersuchungen am Blutegelpriiparat, zugleich eine Methode zum biologischen Nachweis von Acetylcholin bei Anwesenheit anderer pharmakologisch wirksamer k6rpereigener Stoffe. Arch. exp. Path. Pharraak. 168, 292-304. MYmm]mo H. E. (1967) Monoaminergic mechanisms in the nervous system of Lurabricus terrestris. Z. Zellforsch. 81, 311-343. POLONIA. (1955) I1 muscolo dorsale di sanguisuga quale test biologico per l'evidenziamento dell'attivit/~ serotoninica nei liquidi organici. Cervello 31, 472-476. RETZlUS G. (1891) Zur Kenntnis des centralen Nervensystems der Witrmer. Biol. Unters (N.F.) 2, 1-28. SCmUN R. J. (1961) Effects of 5-hydroxytryptamine on the dorsal muscle of the leech (Hirudo medicinalis). Br. ft. Pharmae. Chemother. 16, 257-261. WASmZU Y. (1967) Electrical properties of leech dorsal muscle. Comp. Biochem. Physiol. 20, 641-646.