Electrical and mechanical properties and mode of innervation in scorpionfish sound-producing muscle fibres
1 Department of Physiology, School of Medicine, Teikyo University,
Itabashi-ku, Tokyo 173-8605, Japan
2 Department of Anatomy, School of Medicine, Teikyo University, Itabashi-ku,
Tokyo 173-8605, Japan
Author for correspondence (e-mail:
sugi{at}med.teikyo-u.ac.jp)
Accepted 23 July 2004
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Summary |
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Key words: sound-producing muscle, teleost fish, action potential conduction, multiterminal innervation, innervation ratio, endplate potential, scorpionfish, Sebastiscus marmoratus
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Introduction |
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The aim of the present work was to investigate the mode of neural control of mechanical activity in the SBM fibres during sound production. It will be shown that, in the SBM fibres, motor nerve branches run in parallel with the muscle fibres to form many cholinergic neuromuscular junctions, so that action potentials propagate rapidly along the nerve branches but not along the fibre membrane. The SBM consists of about 600 muscle fibres, with low succinic dehydrogenase activity and high ATPase activity, and is innervated by a motor nerve containing about 100 nerve fibres. These results are discussed in connection with the mode of neural control of the SBM during sound production.
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Materials and methods |
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Conventional glass capillary microelectrodes, filled with 3 mol l1 KCl and connected to a high impedance amplifier, were used either to record membrane potentials or to pass rectangular current pulses across the fibre membrane. The motor nerve innervating the SBM was stimulated with single or repetitive 1 ms current pulses given through a pair of platinum wire electrodes. Tension was recorded using a tension transducer (Akers 801, resonance frequency, 5 kHz; Holten, Norway). Current, membrane potential and tension were recorded using an oscilloscope (Tektronix 5113, Beavertown, USA). In each type of experiment, 810 different preparations were used with similar results. All experiments were performed at 1822°C.
Anatomical and histochemical studies
Golgi silver impregnation of nerve branches
The whole SBM and the whole motor nerve entering it were incubated for 24 h
in a mixture of 3% K2Cr2O7, 20%
OsO4 at a ratio of 3:1 (v/v), and then in 0.75% silver nitrate for
24 h (Kobayashi et al., 1989).
After the above procedure, the tissues were dehydrated with ethanol, embedded
in celloidin, and longitudinal sections (thickness, 90110 µm) were
cut for microscopic observation.
Cholinesterase activity at the neuromuscular junction
The SBM fibers were fixed in 10% formalin, incubated in a reaction solution
(Karnovsky and Roots, 1964) at
37°C for 60 min, and observed microscopically.
Succinic dehydrogenase activity
The SBM fiber bundle was quickly frozen at 78°C, and the
cryosections (thickness, 10 µm) were incubated in a reaction solution
at 37°C for 60 min (Barka and Anderson,
1963
). Then the cryosections were fixed in 10% formalin, and thin
sections were cut for microscopic observation.
ATPase activity
The SBM fiber bundle was fixed in 10% formalin, and then quickly frozen at
78°C. Cryosections (thickness, 10 µm) were preincubated in
a reaction solution (pH 10.4 or 4.6) at 4°C for 20 min
(Kahn et al., 1974
). Thin
transverse sections were observed microscopically.
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Results |
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The results are summarized in Table
1. In both the middle and end regions of the SBM fibres, the
length constant (), time constant (
) and specific membrane
resistance (Rm) were much smaller, and specific membrane
capacitance (Cm) was much larger, than the corresponding
values for frog skeletal muscle fibres (
=2.4 mm,
Cm=8 µF cm2,
=34.5 ms,
Rm=4100
cm; Fatt
and Katz, 1951
). The large Cm values in the
SBM fibres reflect the extremely well developed SR
(Suzuki et al., 2003
).
Cm is about twofold larger in the middle region than in
the end region of the fibre, which may be explained by the different triadic
junction distribution in each sarcomere of the SBM fibres; each sarcomere
contains two triadic junctions in the middle region, and one triadic junction
in the end region (Suzuki et al.,
2003
).
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Action potentials in response to direct and indirect stimulation
Depolarization of the SBM fibre membrane by 4050 mV, by passing
outward current pulses, elicited an action potential. The action potential did
not show overshoot, as has been the case in other fish skeletal muscle fibres
(Takahashi, 1959;
Barets, 1961
;
Hagiwara and Takahashi, 1967
;
Hidaka and Toida, 1969
), and
its amplitude increased with increasing current intensity
(Fig. 2), indicating that the
action potential in the SBM fibre is not of an all-or-none type but graded,
depending on the stimulus intensity. The action potential elicited by the
intracellularly applied current pulse was localized around the current
electrode and did not propagate along the fibre.
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Mechanical response to direct and indirect stimulation
Repetitive motor nerve stimulation produced a series of brief twitches,
which did not fuse even at a stimulus of 100 Hz or more. The twitch tension
declined rapidly with time during repetitive stimulation
(Fig. 5A), while the action
potential amplitude did not change markedly. We roughly estimated the maximum
tension per unit cross-sectional area of the SBM (T) by measuring the
whole fibre cross-sectional area (S) as:
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Anatomical and histochemical features
The motor nerve innervating the SBM contained about 100 axons, while the
SBM consisted of about 600 thick muscle fibers (diameter, 100200 µm)
(Fig. 6A,B), giving a low
innervation ratio of 1:6. Golgi silver impregnation of motor nerve showed that
nerve branches run along the fibres to form endplates (neuromuscular
junctions) at many points (Fig.
7A). The endplates exhibited distinct cholinesterase activity,
indicating cholinergic neuromuscular transmission in the SBM
(Fig. 7B). The high density of
endplates is entirely consistent with the present result that both action and
endplate potentials are uniformly recorded along the fibre length in response
to motor nerve stimulation
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Succinic dehydrogenase activity in the SBM fibres (Fig. 8A) was much lower than that of superficial muscle fibres in the animal body (Fig. 8B). On the other hand, the ATPase activity of the SBM fibres was high at pH 10.4 (Fig. 9A) and low at pH 4.6 (Fig. 9B).
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Discussion |
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Mechanical and metabolic properties of the SBM fibres
In fish, slow swimming movement of the animal body is produced by the
mechanical activity of superficial muscle fibres, whereas quick swimming
movement is associated with that of inner muscle fibres
(Bone, 1966;
Rayner and Keenan, 1967
;
Hudson, 1973
). In mammalian
skeletal muscle fibres, succinic dehydrogenase activity is high in tonic (red)
muscle fibres and low in phasic (white) muscle fibres
(Hoyle, 1983
;
Beckett and Bourne, 1973
). The
low succinic dehydrogenase activity of the SBM fibres, compared to that of the
superficial muscle fibres in the animal's body
(Fig. 8A,B), is therefore taken
to indicate that the SBM fibres have mechanical and metabolic characteristics
analogous to those in mammalian phasic muscle fibres.
Meanwhile, the ATPase activity of mammalian phasic muscle fibres is known
to be stable in high pH, and is inhibited at low pH
(Kahn et al., 1974). The
ATPase activity of the SBM fibres is high at high pH and low at low pH
(Fig. 9A,B), which seems to
indicate that the properties of the SBM fibres resemble those of mammalian
phasic muscle fibres.
Contraction-relaxation cycle in the SBM fibres
The contractionrelaxation cycle in muscle is regulated by the
release of Ca2+ from, and its uptake by, the SR
(Ebashi and Endo, 1968). In
fish sound-producing muscles, the maximum frequency of sound produced is
determined by the maximum frequency of twitch fusion, which is primarily
dependent on the rate of relaxation of twitch tension, which is dependent on
the rate of Ca2+ uptake by the SR
(Skoglund, 1959
). In
accordance with this view, the fractional SR volume in the sound-producing
muscle fibres, including the SBM fibres, is much larger than that in skeletal
muscle fibres (Peachey and Porter,
1959
; Fawcett and Revel,
1961
; Revel, 1962
;
Franzini-Armstrong, 1972
;
Appelt et al., 1991
;
Suzuki et al., 2003
).
Twitches produced by repetitive motor nerve stimulation of the SBM tend to
decrease rapidly with time (Fig.
5A), as has also been reported by Hidaka and Toida
(1969). This may result, at
least in part, from the myoplasmic Ca2+ concentration gradually
decreasing during repetitive motor nerve stimulation, due to a large rate of
Ca2+ reuptake by the SR. Considering the small innervation ratio of
the SBM (
1:6; Fig. 6A,B),
the SBM fibres are likely to receive not only multiterminal but also
polyneuronal innervation; i.e. there would be `functional' motor units within
individual SBM fibres. The alternate activation of these `functional'
subcellular motor units by different motor axons would be capable of producing
long-lasting sound production, despite the tendency of twitch tension to
decrease with time.
The maximum twitch tension per unit cross-sectional area in the SBM fibres
was much smaller than the corresponding value in frog skeletal muscle fibres,
while the maximum steady tension of the SBM fibres in response to transverse
a.c. stimulation was comparable to the latter
(Fig. 5B). This may indicate
that only a small fraction of cross-bridges within the SBM fibres can be
activated to interact with the thin filament during brief twitches evoked by
motor nerve impulses, while a much larger fraction of cross-bridges can be
effectively activated by transverse a.c. stimulation. This view is supported
by the report of Rome and Klimov
(2000), who measured rates of
ATP utilization by the SR and cross-bridges in toadfish sound-producing muscle
fibres.
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Footnotes |
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References |
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Appelt, D., Shen, V. and Franziini-Armstrong, C. (1991). Quantitation of Ca ATPase, feet and mitochondria in superfast muscle fibres from the toadfish, Opsanus tau. J. Muscle. Cell. Motil. 12,543 -552.
Barets, A. (1961). Contribution à l'ètude des systèms moteurs lent et rapide du muscle latéral téléostéens. Arch. Anat. Morphol. Exp. 50,91 -187.
Barka, T. and Anderson, P. G. (1963). Histochemistry. New York: Howber.
Beckett, E. B. and Bourne, G. H. (1973). Histochemistry of skeletal muscle and changes in some muscle disease. In Structure and Function of Muscle, vol.4 (ed. G. H. de Bourne), pp.290 -358. New York: Academic Press.
Blaxter, J. H. and Tytler, P. (1978). Physiology and function of the swimbladder. Adv. Comp. Physiol. Biochem. 7,311 -367.[Medline]
Bone, Q. (1966). On the function of the two types of myotomal muscle fiber in elasmobranch fish. J. Mar. Biol. Assn. UK 46,321 -349.
Ebashi, S. and Endo, M. (1968). Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol. 18,123 -183.[CrossRef][Medline]
Fänge, R. (1966). Physiology of the
swimbladder. Physiol. Rev.
46,299
-322.
Fatt, P. and Katz, B. (1951). An analysis of the end-plate potential recorded with an intra-cellular electrode. J. Physiol. 115,320 -370.
Fawcett, D. W. and Revel, J. P. (1961). The
sarcoplasmic reticulum of a fast-acting fish muscle. J. Biophys.
Biochem. Cytol. 10 suppl.,89
-109.
Franzini-Armstrong, C. (1972). Studies of the triad. 3. Structure of the junction in fast twitch fibers. Tissue Cell 4,469 -478.[Medline]
Gainer, H., Kusano, K. and Mathewson, R. F. (1965). Electrophysiological and mechanical properties of squirrelfish sound-producing muscle. Comp. Biochem. Physiol. 14,661 -671.[Medline]
Hagiwara, S. and Takahashi, K. (1967). Resting and spike potentials of skeletal muscle fibres of salt-water elasmobranch and teleost fish. J. Physiol. 190,499 -518.[Medline]
Hidaka, T. and Toida, N. (1969). Biophysical and mechanical properties of red and white muscle fibres in fish. J. Physiol. 201,49 -59.[Medline]
Hiramoto, Y. (1951). Propagation of contraction wave in single muscle fibres. Anat. Zool. Japon. 24,150 -156.
Hodgkin, A. L. and Rushton, W. A. H. (1946). The electrical constants of a crustacean nerve fibre. Proc. R. Soc. Lond. B 133,444 -479.
Hoyle, G. (1983). Muscles and Their Neural Control. New York: John Wiley and Sons.
Hudson, R. C. L. (1973). On the function of the white muscle in teleosts at intermediate swimming speeds. J. Exp. Biol. 58,509 -522.
Kahn, M. A., Papadimitoriou, J. M. and Kakulas, B. A. (1974). The effect of temperature on the pH stability of myosin ATPase as demonstrated histochemically. Histochem. 38,181 -194.
Karnovsky, M. J. and Roots, L. (1964). A `direct cooling' thiocholine method for cholinesterases. J. Histochem. Cytochem. 12,219 -238.[Medline]
Kobayashi, S., Furness, J. B., Smith, T. K. and Pomplo, S. (1989). Histological identification of the intestinal cells of Cajal in the guinea-pig small intestine. Arch. Histol. Cytol. 52,267 -286.[Medline]
Peachey, L. D. and Porter, K. R. (1959). Intracellular impulse conduction in muscle cells. Science 129,721 -722.[Medline]
Rayner, M. D. and Keenan, M. J. (1967). Role of red and white muscles in the swimming of the skipjack tuna. Nature 214,392 -393.[Medline]
Revel, J. P. (1962). The sarcoplasmic reticulum
of the bat cricothyroid muscle. J. Cell Biol.
12,571
-588.
Rome, L. C. and Klimov, A. A. (2000). Superfast
contractions without superfast energetics: ATP usage by SR-Ca2+
pumps and crossbridges in toadfish swimbladder muscle. J.
Physiol. 526,279
-286.
Skoglund, C. R. (1959). Neuromuscular mechanism of sound production in Opsanus tau. Biol. Bull. 117,438 -542.
Skoglund C. R. (1961). Functional analysis of
swimbladder muscles engaged in sound production of the toad fish.
J. Biophys. Biochem. Cytol.
10 Suppl.,187
-200.
Suzuki, S., Nagayoshi, H., Ishino, K., Hino, N. and Sugi, H. (2003). Ultrastructural organization of the transverse tubules and the sarcoplasmic reticulum in a fish sound-producing muscle. J. Elect. Microsc. 52,337 -347.[CrossRef]
Suzuki, S., Hino, N. and Sugi, H. (2004).
Intracellular calcium translocation during contractionrelaxation cycle
in scorpionfish swimbladder muscle. J. Exp. Biol.
207,1093
-1099.
Takahashi, A. (1959). Muscular transmission of fish skeletal muscles investigated with intracellular microelectrode. J. Cell. Comp. Physiol. 54,211 -220.[Medline]