551
Muscle fibres and efferent nerves in a crustacean
receptor muscle
By MELVIN J. COHEN
(From the Department of Biology, University of Oregon, Eugene, Oregon, U.S.A.)
With 4 plates (figs. 2 to 5)
Summary
The accessory flexor muscle of the crab myochordotonal organ consists of two kinds
of muscle-fibres. In one type the fibrillar material has a uniform punctate appearance
in cross-section. The Z bands are often broken across the fibre width and are spaced
2 to 3/n apart. These resemble the 'Fibrillenstruktur' muscle of vertebrates. In the
second fibre type the fibrillar material appears as large clumps in cross-section and it
is often difficult to distinguish the outlines of individual fibres. The sarcomere length
is 10 to i2/x. These resemble the 'Felderstruktur' muscle of vertebrates.
Two types of expanded structures are seen in conjunction with the two efferent
neurones innervating this muscle. With fresh material under phase microscopy one
type appears as a 20 by 40 fj. rectangular plaque containing vacuoles as well as dark
clumps and granules. The other kind of enlargement consists of a group of spheres
giving the appearance of a grape cluster and measuring 40 /x in its greatest dimension.
It is suggested that these expanded structures may be associated with efferent nerve
terminations in this muscle.
Introduction
T H E accessory flexor muscle in the walking legs of decapod crustaceans was
described by Barth (1934) as forming part of a sense organ called the myochordotonal organ. During a physiological investigation of this sensory system
(Cohen, 1960, 1963) the accessory flexor muscle was seen to have several
unique morphological characteristics. Two distinct structural types of
muscle-fibres were observed. The efferent neurones innervating this muscle
seem to have large specialized expansions unlike the fine tapered terminals
commonly described for crustacean motor terminations (Mangold, 1905; van
Harreveld, 1939; Hoyle, 1957; Wiersma, 1961).
The accessory flexor muscle plays a specialized role in a sensory system very
much analogous to the intrafusal fibres of the vertebrate muscle-spindle
(Cohen, 1963). The muscle seems to have been modified both functionally
and structurally for this special role. The present study describes the peculiar
morphology of this muscle and its efferent innervation and suggests some
functional implications of this unique structure.
Methods and materials
The local market crab Cancer magister Dana was used throughout this study.
Males ranging in weight between 900 and 1700 g were used. The animals
were obtained and kept in the laboratory as described by Cohen (1963).
[Quart. J. micr. Sci., Vol. 104, pt. 4, pp. 551-9, 1963.]
552
Cohen—Crustacean muscle and nerve
Walking legs 2, 3, and 4 were cut off either at the joint between the body
and the coxipodite or between the coxipodite and ischiopodite. The body
stump was packed with soft wax to reduce bleeding. The anterior cuticle of
the meropodite was removed and the accessory flexor muscle and associated
sensory structures exposed. All dissection was carried out with the limb
immersed in sea water kept between io° and 12° C.
The methylene-blue preparations were made after the method of Alexandrowicz (1951). For phase contrast examination of muscle and efferent nerve
junctions the fresh material was teased out on a slide and then gently flattened
with a cover slip.
Two types of silver stains were used on paraffin sections cut at 10 and 20/x.
The protargol silver technique of Stotler (1951) proved excellent for showing
the various types of muscle-fibres, while the modified Bielschowsky technique
of Weiss (1934) proved most suitable for examination of efferent neurones
and their endings.
A modification of Masson's trichrome technique was also used for examination of muscle-fibres. The technique was essentially that described by Masson
(1929) except that the light green stain was made up as a 0-5% solution in
90% ethyl alcohol rather than in acetic acid. The slides were transferred from
the green stain directly into 95% ethyl alcohol, dehydrated through xylene
and mounted in xylene damar. The tissue was fixed in Bouin's fluid.
For the cholinesterase staining, fresh material was teased out on a slide and
excess water blotted from around the preparation. It was then fixed in a solution of 10% formalin in sea water for 14 to 24 h and stained for cholinesterase
by the technique of Koelle (1951, 1955).
Results
Gross anatomy
The gross anatomy of the accessory flexor muscle and the entire myochordotonal organ has been described in detail by Cohen (1963) and will only
be reviewed briefly here for purposes of orientation. As seen in fig. 1, the
accessory flexor muscle consists of a spindle-shaped proximal head and a
broad flat distal head. The proximal head originates at the ventral proximal
edge of the meropodite (figs. 1; 2, A) and sends a fine tendon the full length
of the meropodite to insert on the tendon of the main flexor muscle. The
distal head originates from the anterior cuticle of the meropodite and inserts
on the accessory flexor tendon just before the latter joins the tendon of the
main flexor muscle.
A thick and a thin efferent nerve-fibre innervate the proximal and distal
accessory flexor heads (figs. 1, 4). As far as can be determined from examination of well over 100 methylene-blue-stained preparations, these two neurones
do not innervate any other musculature of the leg and provide the sole efferent
innervation for both heads of the accessory flexor.
The following results are obtained primarily from the proximal head of the
accessory flexor, but the situation in the distal head seems essentially similar.
Cohen—Crustacean muscle and nerve
553
Two types of muscle-fibres
If one looks at the proximal head of the accessory flexor (fig. 2), the musclefibres along the dorsal edge in the region of the attached elastic strand are seen
to be in loose bundles and less densely packed than in the remainder of the
muscle. A transverse section of the muscle taken through the distal edge of the
FIG. 1. Anterior view of a right walking leg with the anterior cuticle removed to expose the
myochordotonal organ sensory system and the main limb musculature. Dorsal is above and
medial is to the right. View of the ischiopodite (i), meropodite (m), and carpopodite (c) to show
the relationship of the accessory flexor system to limb musculature and joints. The accessory
flexor tendon (taf) runs the full length of the meropodite to attach to the main flexor tendon
((/) near the m-c joint. Note the relationship between the proximal head of the accessory
flexor muscle (afp) elastic strand (es) and sense cell bodies (sc) in the region of the ischiomeropodite (i-m) joint. Two efferent nerve-fibres (en) leave the leg nerve (In) to innervate
both the proximal and distal (afd) accessory flexor heads. The main extensor tendon (te) is
also seen (modified from Cohen, 1963).
elastic strand (fig. 2, A) and stained with Masson's trichrome (fig. 2, B) shows
two distinct types of muscle-fibres. Along the dorsal edge adjacent to the
elastic strand, there are a group of 18 to 30 loosely packed muscle-fibres ranging from 10 to 25 ju in diameter. The fibrillar material gives a punctate
appearance in cross-section and seems to be more or less uniformly distributed throughout the fibre. Individual units of this material range from 1 to
5/x in diameter and presumably constitute single muscle fibrils. In the
remainder of the muscle-fibres, the fibrillar material appears in cross-section
as large clumps separated by irregular canals. The canals often contain a fine
green line in the Masson's stain, which may be sarcolemma. In this part of the
554
Cohen—Crustacean muscle and nerve
muscle it is difficult to determine the outline of individual muscle-fibres
because there is very little extracellular space and the peripheral bounding
membrane seems of the same type as that lining the internal canals.
Longitudinal sections further indicate the presence of two different kinds
of muscle-fibres, as seen in fig. 3, A. Fibres showing the punctate fibrillar
distribution in cross-section are seen at the right of the figure to have short
striation intervals. The fibres with the large fibrillar clumps are to the left
in the figure and have very long striation intervals.
In fig. 3, B, c, fresh teased muscle-fibres are shown in phase contrast. A
single fibre with long striation interval is shown in fig. 3, B to have two kinds
of alternating dark bands composing the striations. One band is dense and
forms a continuous structure across the width of the fibre. The other band
also extends across the full width of the fibre, but is more diffuse. Without
a polarized light study it is not possible to say conclusively which of these two
dark regions is the A band and which is the Z band. However, judging from
other phase contrast muscle studies (Hess, 1961a) it seems that the dense
continuous line is most probably the Z band and it will be considered so here.
As the two kinds of bands are evenly spaced, with the distance between dense
bands equal to that between diffuse bands, one can measure sarcomere length
on the basis of the interval between like bands; it is about 12/u. This fibre
type has the large clump-like fibrillar distribution in cross-section. Fig. 3, c
shows a fibre with short striation intervals of 2 to 3 JX. Here the dark transverse
bands appear fragmented and do not extend unbroken across the width of the
fibre. This type of fibre with the short broken striations has the punctate
appearance of the fibrils in transverse view.
Innervation of accessory flexor muscle
Efferent axons. The accessory flexor muscle is innervated by two efferent
nerve-fibres, a thick one and a thin one, branching from the leg nerve and
enclosed in a connective tissue sheath as seen in figs. 1 and 4, A. The thick
fibre averages about 14^ in diameter and forms a characteristic 'Y' configuration when it sends a branch as the stem of the 'Y' ventrally across the flexor
muscle to join the proximal head of the accessory flexor. The thin nerve-fibre
averages 2 to 3/x in diameter and its branching from the leg nerve is much
less regular than for the thick fibre. It sometimes accompanies the thick fibre
FIG. 2 (plate). A, fresh preparation of the proximal head of the accessoryflexormuscle (afp)
in the same orientation as seen in fig. 1. The elastic strand (es) is seen to pull the loosely
packed 'Fibrillenstruktur' fibres out from the main muscle mass. The white area (en) is a bit
of connective tissue attached to the efferent nerves just as they enter the: muscle. The nerves
are not in the plane of focus. A section through the muscle at plane 'p is seen in the picture
below.
B, transverse section taken through the accessory flexor muscle at the level indicated above
by line 'p'. Stained with Masson's trichrome. 'Fibrillenstruktur' muscle-fibres are seen at
the right. Note the punctate distribution of the fibrillar material. These fibres are well
defined by a distinct outer limiting membrane and are loosely packed. The 'Felderstruktur'
fibres are seen to the left, and show the fibrillar material clumped into larger irregular units
separated by wide channels.
v.
M. j . coiii:\
Cohen—Crustacean muscle and nerve
555
branch along the proximal limb of the 'Y' (fig. 4, A) and other times it runs
along the distal limb. The initial branching of the thin fibre often occurs deep
-within the leg nerve and is not readily observed. As the two efferent fibres
run toward the proximal accessory flexor head, they start simultaneous and
repeated branching about 3 mm after leaving the leg nerve and enter the
muscle as a tangle of many nerve twigs (fig. 4, D). These twigs continue
branching profusely as they enter the muscle and turn to run proximally and
distally along its length.
After giving off the branch to the proximal head, the thick and thin fibres
run distally within the leg nerve almost the entire length of the meropodite to
enter the posterior surface of the accessory flexor distal head where they both
terminate with profuse branching (fig. 1).
Efferent nerve expansions. As seen in fig. 4, D the thick and thin efferent
fibres branch profusely upon entering the proximal accessory flexor head.
Numerous varicosities and expansions are seen along the nerve-fibres and at
first view these were thought to be artifacts commonly found in methylene
blue staining. However, under closer inspection, it is seen that many of the
nerve expansions occur at the ends of branches rather than as characteristic
beading along the length of the nerve-fibre. Under high magnification, as
seen in fig. 4, B, E, short branches are observed to come off a long fibre and
these branches end as well-defined terminal expansions averaging 10 to 15^,
in their longest dimension. These expansions at nerve terminals look quite
different from the usual tapering free motor endings described in crustacean
muscle (Mangold, 1905; van Harreveld, 1939; Hoyle, 1957). They give the
appearance of an efferent nerve terminal apparatus.
Sections of the proximal accessory flexor head were cut and stained with
silver according to the technique of Weiss (1934). As seen in fig. 4, c, F,
definite structured expansions occur along a nerve-fibre where it is in contact
with muscle. These expansions often appear slightly granular and correspond
roughly in size to the expansions seen with methylene-blue staining. They
give the impression of a specialized terminal structure.
However, the most convincing evidence for an expanded efferent terminal
apparatus in this muscle comes from phase contrast observation on fresh
teased material. Such observations show the occurrence of two definite types
of specialized enlargements associated with nerves as seen in fig. 5. Fig. 5, A
shows an efferent nerve-fibre apparently terminating on a muscle-fibre by
FIG. 3 (plate). All figures from proximal head of accessory flexor muscle.
A, longitudinal section showing the broad striation type muscle-fibres on the left and the
narrow striation muscle-fibres on the right. Stotler protargol silver stain.
B, fresh teased preparation showing a portion of a single muscle-fibre with long striation
intervals viewed in phase contrast. This type of fibre shows the 'Felderstruktur' organization
of the fibrillar material in cross-section.
c, fresh teased preparation of a single short striation muscle-fibre seen in phase contrast.
Note the irregular broken nature of the transverse bands and the short interval between them.
This kind of fibre has the 'Fibrillenstruktur' type of organization in the sarcoplasm.
556
Cohen—Crustacean muscle and nerve
forming a rectangular plaque-like structure 20 to 3 o /x long and 1 o to 15 /x wide.
These plaques are filled with dense granules and have a few large, clear,
vacuolar areas.
The second type of expansion associated with efferent nerve is seen in fresh
material under phase contrast in fig. 5, c. A nerve-fibre merges into a grapelike cluster of spheres. Each sphere is 8 to 10 JU, in diameter and the entire
mass of spheres ranges from 30 to 40 \L in diameter.
If teased preparations of this muscle are stained for cholinesterase according
to the technique of Koelle (1951, 1955) the two types of expansions can be
seen. Fig. 5, B shows a teased preparation incubated in an acetyl-thiocholine
substrate and viewed under phase contrast. The definite rodlets and granules
occupying the terminal are similar to those seen with phase microscopy in the
fresh material (compare fig. 5, A and B). Fig. 5, D shows a 'grape cluster' type
of expansion viewed from above, after incubation in a butyryl-thiocholine
substrate and stained for cholinesterase. Fine granules may be seen at the
periphery on the right side of the structure and dark clumps of material appear
near the centre. In general, the 'plaque' enlargements are more clearly seen if
incubated in acetyl-thiocholine and the 'grape cluster' structures seem to show
up better if incubated in butyryl-thiocholine. The lack of cleanly differentiated
intensely darkened areas after staining for cholinesterase leaves it open to
question whether or not these regions do indeed contain cholinesterase. All
that can be said at present is that the structures are not readily evident with
bright field microscopy before treatment with the cholinesterase technique
and that they can be seen following Koelle's procedure. This may be due to
non-specific density changes induced by the fixation and staining processes.
The material is included here simply as another piece of evidence substantiating the existence of these specialized structures.
Discussion
The striking morphological differentiation of two muscle-fibre types in the
proximal accessory flexor head is remarkably similar to the two kinds of
FIG. 4 (plate). A, methylene-blue stained whole mount of the thick and thin efferent nerve
fibres to the proximal head of the accessoryflexormuscle showing the typical 'Y1 configuration
where the thick fibre sends a branch toward the muscle. Proximal is to the left and ventral
(toward the muscle) is to the top.
B to F are taken from proximal head of accessory flexor muscle.
B, a part of D at higher magnification, showing an efferent nerve-fibre giving off branches
which end in definite terminal expansions.
c, longitudinal section showing dense black efferent nerve with definite expansion along its
length.
D, whole mount stained with methylene blue, showing efferent innervation just after the
nerves enter the muscle. Note the appearance of terminal expansions particularly in lower
right corner even at this low magnification.
E, a part of D at high magnification, showing an efferent nerve-fibre ending in an expansion resembling an arrow-head.
F, another longitudinal section showing a loop of nerve-fibre with a definite structured
expansion believed to be an efferent nerve junction.
E to F stained with the Weiss silver technique.
Cohen—Crustacean muscle and nerve
557
striated muscle-fibres described in the vertebrates. Kruger (1949) and his
group have devoted considerable study to the comparative histology of extrafusal muscle-fibres in frogs and birds. He divides striated muscle-fibres into
two groups; those in which the muscle fibrils are distinct and give a punctate
appearance in cross-section ('Fibrillenstruktur'), and those muscle-fibres in
which the fibrils are grouped together in clumps and present an areal or diffuse
pattern in transverse view ('Felderstruktur'). He presents evidence substantiating the hypothesis that muscle-fibres with' Fibrillenstruktur' are those
which produce a 'fast' or twitch-like response while the 'Felderstruktur' fibres
are those of the 'slow' or tonic system. Further histological work has confirmed this concept (Gray, 1957, 1958; Hess, 1960a). Work by Kuffler and
Vaughan Williams (1953) presents evidence that there are two functional types
in frog extrafusal muscle-fibres. The so-called 'fast' muscle-fibre gives a
twitch-like response, and the 'slow' muscle-fibre produces graded contractions. The latter, together with its innervation, has often been referred to as
the small motor system. The recent combined histological and functional
study of Peachey and Huxley (1962) has conclusively demonstrated in the frog
that 'Fibrillenstruktur' fibres are of the 'twitch' type while the 'Felderstruktur' is associated with slow muscle-fibres.
Gray (1957, 1958) and Hess (1960 a, b) also point out that there are two
distinct kinds of motor-nerve terminations on the different vertebrate muscle
types. The 'fast' or 'Fibrillen' muscle-fibres are innervated by classical motor
end-plates which are often referred to as the 'en plaque' type of termination.
There is generally only one such terminal per muscle-fibre. The slow or
'Felderstruktur' fibres, on the other hand, are innervated by nerve-fibres
terminating in small round grape-like masses which have been termed 'en
grappe' motor endings. A single slow muscle-fibre may have up to 13
such endings (Hess, 1960a). Hess also demonstrated the presence of the two
muscle-fibre types and endings in chicken (1961&) and guinea-pig (1961a) and
has suggested a correlation with 'fast' and 'slow' type muscle-fibres.
The same two kinds of muscle-fibres and motor terminations have also been
observed in the intrafusal fibres of the amphibian muscle-spindle (Gray, 1957).
Boyd (1962) described two types of muscle in the mammalian muscle-spindle
but raises some question as to whether they fit into Kruger's classification.
FIG. S (plate). All figures are from the proximal head of the accessory flexor muscle.
A, fresh teased preparation in phase contrast showing an efferent nerve-fibre merging with
an expanded 'plaque'. Note granules, vacuoles, and large dark clumps in the terminal
expansion.
B, preparation similar to A seen in phase contrast after staining for cholinesterase in an
acetyl-thiocholine substrate. Note the definite structured aspect of the terminal expansion.
C, 'grape cluster' efferent nerve expansion seen in profile in fresh teased preparation, phase
contrast. Note nerve-fibre coming from the left and entering a cluster of spheres which is
believed to be part of a nerve terminal region.
D, teased preparation showing 'grape cluster' expansion seen from above after staining for
cholinesterase in butyryl-thiocholine substrate and viewed in bright field illumination. The
nerve enters the terminal region from above and large dark masses are seen in the lower left
portion of the structure while small granules are seen along the periphery at the right.
558
Cohen—Crustacean muscle and nerve
The muscle types found in the crustacean accessory flexor muscle system
correspond in many ways to the two kinds of vertebrate muscle-fibres
described above. In the accessory flexor fibre with short striations, the Z disc
extends broken across the fibre and the sarcoplasm is divided into small
regular longitudinal units giving a rather uniform punctate appearance in
transverse section. This crustacean fibre type therefore has many of the
major structural characteristics of vertebrate'Fibrillenstruktur' muscle-fibres.
The unbroken appearance of the Z disc in the long striation muscle-fibre and
the grouping of fibrillar material into large clumps closely resembles the
structural specialization of the vertebrate' Felderstruktur' muscle-fibre. Comparison of figs. 2 and 3 in this oaper with figs. 1 and 2 in the paper by Hess
(1961a) shows a remarkable similarity between the two kinds of muscle-fibres
found in the crustacean system and the two kinds of muscle-fibres found in the
extra-ocular muscles of the guinea-pig. The strong correlation in the vertebrates between structure and function in striated musculature (Ginsborg,
i960; Peachey and Huxley, 1962) leads one to believe that a similar functional
relationship may exist for the two distinct types of fibres found in the accessory flexor muscle. Experiments are in progress to test this hypothesis and
it appears that the 'Fibrillenstruktur' crustacean fibres may be 'fast' while
those with 'Felderstruktur' may be 'slow', just as in the vertebrates.
It should be pointed out, however, that transitional stages between the two
structural extremes do appear (fig. 2, B) and the clean separation of these
muscle-fibres into two categories may be an over-simplification. This point
has also been made by Boyd (1962) for vertebrate muscle.
The appearance of large, well-differentiated expansions associated with the
efferent nerves innervating the accessory flexor muscle is unlike the fine tapering motor endings commonly described for crustacean muscle (see Wiersma,
1961, for review). Maynard and Maynard (i960) have described two kinds
of efferent nerve terminations on muscle-fibres of the muscle receptor organs
in the lobster. These endings, as seen with cholinesterase staining techniques,
seem to consist of delicate arborizations and appear unlike the large structures
described here. The electron microscopic studies of Peterson and Pepe
(1961 a, b) on the muscle-receptor organs in the crayfish also indicate that the
efferent nerve endings on the receptor muscles are in the neighbourhood of
1 to 2/n and probably are similar to the fine tapering crustacean motor terminations previously seen with the light microscope. The expanded structures associated with efferent nerve terminations described here appear to
form some type of specialized nerve terminal apparatus in the accessory flexor
muscle. The functional role played by these structures as well as their precise
relationship to the axon terminals and muscle membrane await further
investigation.
This investigation was supported by PHS research grant B-1624 from the
National Institute of Neurological Diseases and Blindness, U.S. Public Health
Service.
Cohen—Crustacean muscle and nerve
559
The technical assistance of Mr. Herbert Swick is much appreciated. He
aided this work under the sponsorship of the National Science Foundation
Undergraduate Research Participation Program.
Mrs. Ruth Day of the Hallmark Fisheries, Charleston, Oregon, has generously provided crabs for this work and her aid is gratefully acknowledged.
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