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A M . ZOOLOGIST, 7:527-551 (1967).
Crustacean Neuromuscular Mechanisms
HAROLD L. ATWOOD
Department of Zoology, University of Toronto, Canada
SYNOPSIS. Properties of crustacean muscle fibers and neuromuscular synapses are
discussed, with particular reference to the problems of fast and slow contraction,
synaptic diversity, and peripheral inhibition.
Electrical and mechanical responses of crustacean muscle fibers are variable, and
govern to a large extent the muscle's performance. Fast and slow contractions are
often mediated by distinct "phasic" and "tonic" muscle fibers, as in abdominal
muscles, in which such fibers are segregated into two parallel sets of muscles. In
leg muscles the fibers are often heterogeneous in properties and innervation. In
doubly-motor-innervated muscles of crabs the axons producing fast and slow contractions preferentially innervate rapidly and slowly contracting fibers, respectively.
Crustacean neuromuscular synapses vary greatly in electrical behavior and in
ultrastructural characteristics. Some motor axons possess both facilitating and nonfacilitating synapses. The proportion of the different types of synapse associated
with a motor axon probably determines in large measure the properties of the
postsynaptic potentials evoked by that axon.
Pre-synaptic and post-synaptic inhibition both occur, sometimes in the same muscle.
The latter type is more common. Pre-synaptic inhibition is thought to be mediated
by the action of an inhibitory transmitter-substance on receptors of the motor nerve
terminals.
Crustacean muscles differ from those of
the higher vertebrates in the sparseness of
their efferent innervation, which may consist of as little as two axons for muscles
such as the opener of the crayfish claw. A
common feature is the presence of at least
one inhibitory axon, activity of which can
weaken or abolish the contractions evoked
by the motor axons. Muscles receiving
more than one motor axon often generate a
fast or a slow contraction depending upon
which axon is stimulated; thus the different
axons have been termed "fast" or "slow".
The innervation patterns of decapod leg
muscles are known from the work of
Wiersma, van Harreveld, and collaborators
(see review by Wiersma, 1961). Two of
the known patterns, the brachyuran and
the astacuran, are shown in Figure 1. Although the motor innervation is the same
in these two and in other Reptantia as well,
there are differences in the inhibitory innervation. For instance, in the brachyurans
the opener and stretcher muscles receive
double inhibitory innervation, whereas in
the crayfish there is only a single inhibitory
axon supplying each muscle.
Numerous questions were raised by the
early work on the innervation and physiology of the crustacean leg muscles
(Wiersma, 1961). Attention will be focused
here mainly on three of these:
1) What mechanisms are responsible for
fast and slow contraction?
2) Why do synapses of different axons
show differences in electrical behavior?
3) What mechanisms are responsible for
peripheral inhibition?
Recent work has produced tentative answers to the above questions, but has left
others unsolved, as will be indicated later.
The present account will be confined to
the Decapoda Reptantia, which have been
the most thoroughly investigated of the
crustaceans. No attempt will be made to
present an exhaustive review; rather, a
point of view which has arisen from certain
recent observations will be developed.
Some of the work reported here was supported
by a grant (A-2352) from the National Research
Council of Ganada.
Crustacean muscle fibers
FAST AND SLOW CONTRACTION
(527)
The contractions of any muscle are
528
HAROLD L. ATWOOD
B
B
F
A
1
L
i
*
B
FIG. 1. Efferent innervation of brachyuran (A) and
uslacuran (B) leg muscles. Solid lines represent
motor axons, and dotted lines, inhibitory axons.
The muscles are : O, opener; C, closer; R, bender;
S, stietcher; F, flexor of carpopodite; A, accessory
flexor; E, extensor of carpopodite. (After Wiersma,
1961.)
dependent on the properties of the responding muscle fibers. This is readily apparent in such material as the muscles of
the frog, in which twitch and tonus fibers
have been shown to be responsible for fast
and slow contractions, respectively (Kuffler
and Vaughan Williams, 1953a, b) . Until
recently, relatively little attention was
given to the contractile and morphological
characteristics of crustacean muscle fibers.
In attempts to explain crustacean fast and
slow contractions, most workers concentrated on the properties of the different
axons, including differences in their electrical and chemical effects (e.g., Hoyle and
Wiersma, 1958 u-c).
However, there were early indications
from the work of Jasper and Pezard (1934)
and Alexandrowicz (1951, 1952) that crustacean muscle fibers are not uniform in
their morphological and contractile properties. Only recently have these findings
been more fully developed and elaborated.
It is worth emphasizing here the importance of the diversity of muscle fibers which
occurs in crustacean material.
The diversity extends to fine structure,
contractile behavior, and electrical responsiveness. In different fibers, responses to
depolarization range from all-or-nothing
spikes in some crab leg fibers and in crayfish abdominal muscle fibers (Atwood,
1965«, b; Atwood, et al., 1965; Abbott and
Parnas, 1965), through the large or small
graded responses of the majority of crustacean fibers (Fatt and Katz, 1953a; Atwood, et al., 1965), to delayed rectification
and absence of graded responsiveness (Atwood, 1963fl, b; Atwood, et al., 1965). In
certain leg muscles of crabs, the entire
range of electrical responsiveness may be
encountered. In Figure 2, representative
responses to depolarization of fibers in the
closer of Chionecetes tanneri are shown. In
other muscles, the range may be more
restricted. The fibers of the deep abdomi-
529
CRUSTACEAN XF.UROMUSCUI.AR MFXHANISMS
TABLE 1. Characteristics of muscle fibers in the Ca rein us closer muscle
(Alter Atwood, 19636)
Fiber type
(Fig. 21)
Motor
innervation
A
Mainly fast axon
B
Mainly slow axon
C
Both Cast and slow
axons
Membrane responsheness
Large graded active membrane
responses
Little or no graded responsiveness: delayed rectification
Variable small graded responses
nal muscles of the crayfish and rock lobster
show responses ranging from all-or-nothing
spikes to large graded responses, whereas
the fibers of the superficial abdominal muscles show small graded responses or none
(Kennedy and Takeda, 1965c/, b; Parnas
and Atwood, 1966).
L
FIG. 2. Membrane responses of three fil>ers in the
closer muscle of Chionecetes with intracellular stimulation. Current is monitored in the lower traces.
The three libers show spikes (A), graded response
(15), and delayed rectification (C). Calibration:
voltage, 20 mV; cm lent, 2 ^A (A), 1 ^A (15, C):
time, 100 msec (A). 200 msec (15), 400 msec (C,
top), 1 sec (C, bottom). After Atwood (19656).
In crab leg muscles, the fibers which
generate large graded responses or spikes
show certain differences in their cable
characteristics from those which lack such
responses (Table 1; Atwood, 1963o, b).
These differences are reflected in the shapes
of the post-synaptic potentials set up in
the fibers by indirect stimulation. However,
Membrane
length
constant
Sarcomere
length
ij-10 msec
0.:") mm
2-5 M
40-200 msec
2.5 mm
6-12 M
10-30 msec
1.5 mm
5-10M
Membrane
time
constant
values for cable constants must be interpreted with caution in view of the complex
morphology of crustacean muscle fibers
(Selverston, 1967).
Speeds of contraction and relaxation
may differ many-fold in crustacean muscle
fibers. For example, the deep abdominal
muscles of the crayfish produce contractions
which reach peak amplitude in 10-20 msec
and relax in 30-40 msec, whereas the depolarized superficial abdominal muscles
develop tension slowly over the course of
many seconds and require about 10 sec to
relax (Abbott and Parnas, 1965; Parnas
and Atwood, 1966). In decapod leg muscles, fibers with this degree of difference in
speed of contraction often occur in the
same muscle. This was demonstrated by
Atwood and Dorai Raj (1964) in the accessory flexor muscle of the crab, Cancer
magisler. Jn this muscle, there are fibers
which contract very slowly when depolarized; tension may continue to increase after
depolarization has terminated (Fig. 3).
These fibers do not show spikes or graded
responses as a rule. Elsewhere in the muscle, such electrical responses are common,
and the contraction of the fibers in which
they appear is faster. The same differences
L
FIG. 3. Contraction of a tonic fiber in the accessory
flexor muscle of Cancer, showing slow development
and relaxation of tension. Calibration: voltage,
40 mV; tension, 0.04 g; time, 1 sec (after Atwood
and Dorai Raj, 1964).
530
HAROLD L. ATWOOD
ous muscle fibers. The slow-acting fibers of
the crab accessory flexor muscle, and the
superficial extensor muscles of the crayfish
abdomen, remain contracted for many
minutes when depolarized by a potassiumrich solution (Atwood and Dorai Raj,
1964; Parnas and Atwood, 1966). The fastcontracting fibers of the crab accessory
flexor, and fibers in the leg muscles of the
A
B
crayfish, relax after several seconds or someFIG. 4. Tension developed by spiking (A) and
what longer, depending on the amount of
gradedly-responding (B) fibers in the stretcher
muscle of Cancer magister. In (A), tension does depolarization (Atwood and Dorai Raj,
1964; Zachar and Zacharova, 1966). The
not appear at depolarizations below the spiking
level. Calibration: voltage, 20 mV; time, 100 msec fast-acting fibers of the deep abdominal
(after Atwood, el al., 1965).
extensor muscles of the crayfish relax in a
are found in other crab leg muscles. Con- few seconds (Parnas and Atwood, 1966).
tractions of a spiking fiber and of a There is apparently a wide spectrum of
gradedly-responding fiber from the stretch- contractural responses in crustacean fibers.
er muscle of Cancer magister are shown in There would appear to be some justificaFigure 4. The contractions of these fibers tion for terming those which can normally
are quite different from that shown in maintain prolonged tension "tonic fibers",
and those which can maintain only tranFigure 3.
It is worth noting that many of the sient tension "phasic fibers", particularly
spiking crustacean muscle fibers develop since some of the former (in the tonic
little or no tension at depolarizations be- abdominal muscles) are known to receive a
low the threshold for spiking (Fig. 4), as is "tonic" (steadily maintained) input from
true also in frog twitch fibers (Hodgkin the central nervous system, whereas some
and Horowicz, 1960). By contrast, in some of the latter (in the phasic abdominal
of the non-spiking crustacean fibers, the muscles) are known to lack this input
threshold membrane potential for produc- (Kennedy and Takeda, 1965a, b). Howtion of tension may be very close to the ever, it must be borne in mind that innormal resting potential (Atwood, et al, termediate types of fiber occur between the
1965; Reuben, et al., 1967).
two extremes and the terminology is thereThere are also differences in the normal fore somewhat limited in usefulness.
The differences in speed of contraction
tension-maintaining capabilities of vari-
L
TABLE 2. Sarcomere length, relaxation time, and electrical responsiveness of some crustacean muscle fibers.
Muscle
Deep abdominal extensor of crayfish
Extensor muscle of crab
Contractor epimeralis
of crayfish
Accessory flexor of crab
Superficial abdominal
extensor of crayfish
Sarcomere Relaxation
length of time after
fiber
contraction
Electrical responsiveness
Authors
2-3 M
30 msec
Spike or large graded response
Abbott & Parnas
1965
4-5 M
40 msec
Spike or large graded response
Selverston
1967
9^
2 sec
Small to large graded response
Orkand
1962
10-12/i
2-5 sec
Delayed rectification
Atwood & Dorai Raj
1964
8-14 M
6-10 sec
Delayed rectification or small
graded response
Abbott & Parnas
1965
Parnas & Atwood
1966
CRUSTACEAN NEUROMUSCULAR MECHANISMS
531
correlate reasonably well with differences
in the resting length of sarcomere. This
correlation was suggested earlier by Jasper
and Pezard (1934) and by Huxley and
Niedergerke (1954). Some of the data for
crustacean muscle fibers are tabulated in
Table 2, in which it is apparent that the
slower fibers have the longer sarcomeres. It
will be noted, however, that there is not a
rigid correlation between speed of contraction and electrical excitability. While
it is true that many of the fast-acting fibers
produce spikes or large graded responses,
some of the slower ones do also. However,
spike generation is undoubtedly less common, in the statistical sense, in the slow-
FIG. 5. Longitudinal section through a crayfish
phasic abdominal muscle fiber; note the relatively
short sarcomeres (2 JJ), myoftbrils separated by
sarcoplasmic reticulum (S), and diads
(After Jahromi and Atwood, 1967.)
(arrows).
S32
HAROLD L. ATWOOD
contracting and tonic fibers.
In the crayfish abdominal extensor muscles, very fast-acting, phasic fibers occur in
the deep muscles in parallel with very
slow-acting, tonic fibers in the superficial
muscles (Parnas and Atwood, 1966). The
former have much shorter sarcomeres than
the latter (Table 2; Figs. 5, 6).
This degree of difference in length of
sarcomere is not found in vertebrate twitch
and tonus fibers (e.g., Page, 1965). However, in the vertebrate fibers there are
FIG. 6. Longitudinal section through a crayfish
tonic abdominal muscle fiber; note the relatively
long sarcomere (7.5 p), wide Z-lines (Z), the separation of the myofibrils by elements of the sarco-
plasmic reticulum (arrows), and diads (D). Mitochondrion (M) is indicated. (After Jahromi and
A l wood, 1967.)
CRUSTACEAN XEUROMUSCULAR MECHANISMS
533
FIG. 7. Transverse sections through crayfish phasic
(A) and tonic (B) abdominal muscle fibers, with
diads indicated (arrows). The myofibrils are not
greatly different in size in the two fibers but the
myofilaments are differently arranged (after
Jahromi and Atwood, 1967.)
considerable differences in the amount of
sarcoplasmic reticulum and in the number
of contacts (triadic, pentadic, etc.) between
the T-system tubules and the sarcoplasmic
reticulum, the tonic fibers being poorer in
both respects (Page, 1965; Hess, 1965;
Hoyle, et al, 1966). One can postulate that
the slowness of the vertebrate tonic fibers
arises from the sparseness of the sarcoplasmic reticulum, or perhaps from lack of
some factor associated with it {e.g., relaxing
factor; Gruener and Abbott, 1965).
In the crayfish abdominal muscles, the
situation may be different. Both phasic and
tonic fibers have well-defined myofibrils
separated by layers of sarcoplasmic reticulum; the average minimum transverse
distance from the center of the myofibril
to the sarcoplasmic reticulum is about 0.4 ^
(Figs. 5, 6, 7; Jahromi and Atwood, 1967).
The number of diadic contacts between the
T-tubules and the sarcoplasmic reticulum
is about the same in both types of sarcomere, but since the phasic fibers have
much shorter sarcomeres, the number of
diads per unit length of fiber is greater in
them. The more rapid onset of contraction
in the phasic fibers could result at least in
part from more effective activation of the
sarcoplasmic reticulum by the closely
spaced diads. However, the contractile material of both types of fiber is equally accessible to the sarcoplasmic reticulum, and
if relaxation is brought about by movement
of calcium from the contractile material
into the sarcoplasmic reticulum, it may be
necessary to postulate a difference in the
nature of the sarcoplasmic reticulum, or
of the contractile filaments, to explain the
very considerable difference in the rate of
relaxation.
The arrangement of the contractile filaments is also different in the two types of
fibers, there being a 3:1 ratio of thin to
thick filaments in the phasic fibers and a
5:1 to 6:1 ratio in the tonic fibers. The arrangement in the phasic fibers is similar to
that in insect flight muscle (Huxley and
Hanson, 1957), whereas the arrangement
in the tonic fibers is like that in insect leg
or visceral muscles (Hagopian, 1966; Smith,
et al., 1966), The major ultrastructural dif-
534
HAROLD L. ATWOOD
ferences between the phasic and tonic fibers
in the crayfish abdomen are the length of
sarcomere, the longitudinal spacing of the
diads, and the arrangement of the myofilaments, rather than the relative amount of
sarcoplasmic reticulum. From this it would
seem that the mechanisms underlying the
different speeds of contraction in these
crustacean fibers are different from those
which operate in vertebrate fibers. However, more observations are required to
elucidate the factors which limit the speed
of contraction in both vertebrate and crustacean fibers.
From the above discussion, it is at least
apparent that there is within crustacean
muscles a substrate for production of fast
and slow contractions, in the form of fastand slow-acting muscle fibers. The diversity
of fibers within a single crustacean muscle
(especially certain crab leg muscles) may
exceed that in any known vertebrate muscle. On the other hand, there may be uniformity of substrate, as in the medial deep
abdominal extensor muscle of the crayfish
(Abbott and Parnas, 1965). Even homologous muscles in different appendages of
one animal may have different contractile
substrates. In the American lobster, many
of the fibers of the fast-acting cutter-claw
closer muscle have sarcomeres of about
3 jx, whereas all the fibers of the same muscle in the slow-acting crusher-claw have
sarcomeres of 7-10 fi (Jahromi and Atwood,
unpublished).
The next step in pursuing the discussion
of fast and slow contraction is to consider
the ways in which the central nervous system can call the different muscle fibers
into play.
Neuromuscular physiology of
abdominal muscles
The abdominal muscles of crayfish and
rock lobster (Abbott and Parnas, 1965;
Kennedy and Takeda, 1965a, b; Parnas
and Atwood, 1966) provide a case in which
phasic and tonic contractile elements have
been separated into distinct muscles. One
set of muscles, the deep, contains only
phasic fibers, and the other set, the super-
M
SEL
mm
LI L2
FIG. 8. Diagram o£ the preparation of the abdominal extensor muscles of the crayfish, showing the
medial deep extensor muscle (DEAM), the lateral
deep extensor muscles (L 1, L 2), the medial and
lateral superficial extensor muscles (SEM, SEL), the
abdominal segmentation (S 2-4), stimulating electrodes positioned on the nerves to the two sets of
muscles (El, E2), and a recording microelectrode
(M) positioned in the medial deep extensors. (After
Parnas and Atwood, J. Cell. Physiol. 68, 1966.)
ficial, contains only tonic fibers. The gross
anatomy of the abdominal extensor muscles in the crayfish is shown in Figure 8.
It is not surprising, in view of the nature
of the contractile elements in the two sets
of muscles, that the deep muscles produce
rapid twitches and tetani with indirect
stimulation, whereas the superficial muscles
produce smooth, slowly developing contractions (Kennedy and Takeda 1965a, b;
Parnas and Atwood, 1966). Another feature,
linked to the first, is the specialization of
the motor axons supplying the two sets of
muscles.
The innervation of the deep extensor
muscles of the crayfish is shown in Figure
9. From each of the middle segmental
ganglia, six axons supply the deep muscles,
five being excitatory and one inhibitory.
Some of these axons supply not only the
muscles in their own segment, but those in
the next posterior segment also. Thus each
of the medial deep extensors receives a
triple excitatory innervation and a double
inhibitory innervation. This type of innervation is also found in Lx of the lateral
extensors, but is reduced to double ex-
CRUSTACEAN NEUROMUSCULAR MECHANISMS
L
FIG. 9. Diagram o[ the deep extensors in segments
3 and 4 of the crayfish, showing the distribution
of the innervation entering segment 3. Tracings of
representative electrical responses are also shown.
Axons are numbered 1 to 6; axon 5 is inhibitory.
Calibration: 1-4, 20 mV; 5, 4 mV; time, 20 msec
(after Parnas and Atwood, 1966).
citatory and single or double inhibitory
in L2. In the rock lobster, the pattern of innervation differs in detail from that in the
crayfish, but there are many features in
common (Parnas and Atwood, 1966; Fig.
10).
In both rock lobster and crayfish, the inhibitory axons have the most widespread
distribution. The electrical responses which
they produce in the muscle fibers are in
the form of small depolarizing or hyperpolarizing, post-synaptic potentials (p.s.p's;
Fig. 9, 10). Activity of these axons is effective in reducing the electrical and mechanical responses set up by the motor
axons, as will be discussed more fully in
a later section.
The motor axons generate spike-shaped
responses or large p.s.p's with each stimulus, as shown in Figures 9 and 10. The
mechanical response is a sharp twitch.
Mechanical activity does not occur unless
some form of graded or spike-shaped response is present; the p.s.p's alone are not
sufficient to produce contraction.
Transmission of the motor axons is sub-
535
ject to rapid fatigue with repetitive stimulation. Apparently the axons are specialized
for a large output of transmitter over a
brief period of time, in keeping with their
functional role in rapid swimming.
The superficial muscles also receive five
excitatory axons and one inhibitory axon.
The axons are distributed in a complex
manner to various fibers within the muscles
(Kennedy, et al., 1966), as in the superficial
flexor muscles (Kennedy and Takeda,
19656). Some fibers receive no inhibitory
innervation. Most receive at least three
motor axons, which give p.s.p's of various
sizes in response to single stimuli. The
p.s.p's are seldom as large as those encountered in the deep muscles, but some
have considerable powers of facilitation
(Fig. 11). Perhaps the outstanding difference between these axons and the ones supplying the deep extensors is their ability to
resist fatigue with prolonged stimulation.
They seem specialized for prolonged, lowlevel output of transmitter substance,
which fits them well for their normal role
in control of abdominal posture and tone.
The superficial muscles do not produce
any rapid contractions, although some of
the fibers can generate an occasional
graded response (Fig. 11). As mentioned
previously, development of tension and
relaxation is always very gradual. There
is no comparable slow change in the electrical response (Fig. 11).
In comparing the two sets of abdominal
muscles, it can be seen that both the muscle fibers and the motor axons are specialized for either fast or slow contraction.
This clear-cut separation is not found in
the leg muscles, but it may be profitable
to approach them with the example of
the abdominal muscles as a reference point.
Nenromuscular physiology of
singly-motor-innervated leg muscles
Many of the singly-motor-innervated leg
muscles have a variety of muscle fibers
within them. This was first found in the
accessory flexor muscle of the crab in which
structural diversity was shown by Cohen
(1963), and in which Dorai Raj (1964)
536
H A R O L D L.
L 2 L,
A
^ ^ >
LI
4
M
ATWOOD
M
L,
L2
•3 ^
4' "
™\ 3
JL
II
J
FIG. 10. Diagram of the inncrvalion of the deep
extensors in segments 3 and 4 of the rock lobstei,
showing the distribution of the inncrvalion entering segment 3. Nfedial (M) and lateral (LI, L2)
mm
divisions of the deep extensors are indicated. Axon
5 is inhibitory. Calibration: voltage. 1-4, 20 mV;
5, 4 mV; time, 20 msec (after 1'arnas and Atwood,
I9G6).
distinguished slow-contracting, long-sarcomered, electrically passive fibers in the
proximal part of the muscle, and fastcontracting, short-sarcomered, spiking or
gradedly-responding fibers in the distal
part, with intermediates of various sorts in
between. All fibers receive the same motor
axon, together with an inhibitor.
The density of innervation of the different fibers may be variable since some
have much larger p.s.p's than others of
similar input resistance (Dorai Raj, 1964;
Atwood and Dorai Raj, 1964). However,
the association of different muscle fibers
with qualitatively distinct motor axons, as
in the crayfish abdomen, does not occur
here.
Repetitive stimulation of the single
motor axon produces a slow, sustained contraction at one end of the muscle, and a
series of twitches or an incompletely fused
tetanus at the other end. Both fast and
slow contractions are generated, but by the
same axon.
The same general finding has been made
in other singly-motor-innervated crab muscles, such as the stretcher of Cancer magister and the opener of Chionecetes ianneri (Atwood, 1965ft; Atwood, et al., 1965).
In these, the phasic, tonic, and interme-
537
CRUSTACEAN NEUROMUSCULAR MECHANISMS
L
FIG. 11. Electrical responses recorded from a fiber
of the superficial abdominal extensors of the crayfish. A: Excitatory p.s.p's evoked by three motor
axons. B: Facilitation and summation of one of
the p.s.p's with repetitive stimulation; note single
graded response. Calibration: voltage, A, 10 raV;
JS, 20 mV; lime. A, 20 msec, B, 1 sec. (After I'arnas
and Atwood, 1966.)
diate types of muscle fiber can be distinguished on the basis of observations of
morphological and electrical and contractile performance.
In Chionecetes some of the tonic fibers
start to contract with indirect stimulation
at 3/sec. The excitatory p.s.p's are very
large and slow in these fibers, requiring as
long as 1 sec to decay completely. At relatively low frequencies of stimulation, a substantial depolarization plateau is built up
(Fig. 12, D-F), and a slow contraction of
the fiber is observed.
The phasic fibers in the same muscle
have relatively small and brief p.s.p's (Fig.
12, A), which facilitate with repetitive
stimulation (Fig. 12, B). No contraction is
observed until fairly high frequencies of
stimulation are applied. Spike-shaped electrical responses and twitch contractions
then result (Fig. 12, C).
Once again it is evident that the power
of fast and slow contraction exists in these
crab muscles, and that both types of contraction can be brought about by the same
axon. However, only the slow contraction
appears at low frequencies of stimulation.
Presumably, short bursts of stimulation at
a high frequency bring in the fast contraction without much of the slow, and
this may occur normally when rapid movement is required.
In the opener muscles of the crayfish leg
and claw, there has been no evidence so far
for diversity of muscle fibers. Since these
muscles have been used extensively by
many investigators, features of the type
described above would probably have attracted attention. Possibly, in these muscles
the contractile substrate and the innervation are more uniform than in the crab
muscles, but direct evidence for this has
not been given. Similarly, in the opener
muscle of the hermit crab, Pa gurus
bernhardi, Wiersma and Bobbert (1961)
concluded that all fibers behaved similarly
in response to motor axon stimulation and
were capable of generating spike-like electrical responses and fast contractions as
well as a maintained depolarization and
slow contraction.
Neuromuscular physiology of doubly-motorinnervated leg muscles
Even though fast and slow contraction of
crustaceaii muscles was originally described
in the doubly-motor-innervated decapod
leg muscles (Lucas, 1917), the mechanisms
responsible for the two types of contraction
B
L
FIG. 12. Responses of two fibers (A-C, D-F) in the
opener muscle of Chionecetes, during stimulation of
the motor axon with single shocks (A, D) and with
trains of stimuli at 25/sec (B), 75/sec (C), 1.4/sec
(E), and 6/sec (F). Calibration: \oltage, 20 mV;
time, 40 msec (A), 100 msec. (D), I sec (II, C, E,
F). (After Atwood, 19656).
538
HAROLD L. ATWOOD
A
B
FIG. 13. Whole-muscle tension (lower traces) and
electrical response from a single muscle fiber (upper
traces) in the closer muscle of Randallia, during
stimulation of the fast (A) and slow (B) axons at
30/sec, showing the "paradox" phenomenon. Calibration: voltage, 20 mV; time, 2 sec. (After Atwood
and Hoyle, 1965.)
have remained difficult to ascertain in this
material. Several confusing observations
have contributed to the difficulty. Observations by van Harreveld (1939) indicated
that all fibers in some of the doubly-motorinnervated muscles receive both motor
axons and an inhibitor. This gave rise to
the idea that each muscle fiber could
produce two types of contraction, the fast
and the slow (Wiersma, 1961). In some
muscles, particularly the closer of the crab,
Randallia, and the crayfish claw closer,
which were thought to have a uniformlyinnervated population of muscle fibers, the
electrical responses set up by the slow
axon in the muscle as a whole (Wiersma
and van Harreveld, 1938) or in impaled
single fibers (Hoyle and Wiersma, 1958c)
were smaller than those set up by the fast
axon at the same frequency; yet the slow
contraction was larger (Fig. 13). This observation, termed the "paradox", led to
the hypothesis that contraction is mediated
by direct action of transmitter substances
on the contractile apparatus, the chemical
of the slow axon being more effective, and
the membrane electrical events being unimportant (Hoyle and Wiersma, 1958a;
Wiersma, 1961).
This hypothesis has been proven wrong
by several more recent observations. First,
it has been possible to record tension of
single innervated muscle fibers while stimulating the motor axons. When the depolarization set up by nerve stimulation is
not large enough to exceed the threshold
membrane potential for contraction as determined by direct electrical depolarization,
the muscle fiber develops no tension (Fig.
14 A). When the indirectly induced depolarization is sufficiently large to exceed
the threshold, tension appears, but it can
be abolished by hyperpolarizing current
(Fig. 14 C). Clearly, membrane depolarization past a critical level is part of the
sequence of events leading to production of
tension by the motor axons (Atwood, et al.,
1965). This conclusion was indicated
earlier by experiments of Orkand (1962),
who first employed intracellular depolarization to induce contractions in single
crustacean muscle fibers.
In some of the fibers of the closer of
Cancer magister, tension was recorded
while both fast and slow axons were stimulated. In some cases the electrical responses
were similar, and the tension responses
were also similar. It was not possible to
show that a single muscle fiber could produce two types of contraction when the
electrical events associated with stimulation
of the two axons were similar (Atwood,
et al, 1965).
Re-examination of one of the "paradox"
muscles, the closer of Randallia, showed
that there were fibers within the muscle
L
B
C
FIG. 14. Tension (lower traces) and electrical responses (upper traces) in a single fiber of the
stretcher muscle of Cancer. In (A), stimulation of
the motor axon at 50/sec fails to produce tension.
In (B), the membrane potential is depolarized from
70 mV to 56 mV by excess potassium; injected
depolarizing current is more effective in producing
tension after the shift in membrane potential
(right). In (C), stimulation of the motor axon is
effective in producing tension, but injection of
hyperpolarizing current abolishes the tension. •Calibration: voltage, 20 mV; tension, 0.05 gm; time,
1 sec. (After Atwood, Hoyle, and Smyth, 1965).
539
CRUSTACEAN NEUROMUSCULAR MECHANISMS
which gave larger electrical responses to the
"slow" axon than to the "fast", at a given
frequency of stimulation (Fig. 15). These
fibers were found at the ends of the muscle,
and apparently were not sampled in previous studies (Atwood and Hoyle, 1965).
When the tendon of the muscle was cut
so that only these atypical fibers were left
innervated, stimulation of the slow axon
caused them to contract, but stimulation
of the fast axon was ineffective (Atwood,
unpublished). There seems little doubt that
the tension generated by the slow axon at
"paradox" frequencies is confined to these
atypical fibers, and that the others contract
only at higher frequencies of stimulation.
FIG. 15. Responses of a fiber in the closer of
Randallia to stimulation of the fast (A-C) and
slow (D-F) motor axons at frequencies of 10/sec
(A, D), 25/sec (B, E), and 40/sec (C,F). Wholemusole tension is registered in the lower traces.
Note that in this fiber the slow electrical response
is larger than the fast. Calibration: voltage, 20 mV;
tension, 3 g; rime, 4 sec. (After Atwood and Hoyle,
1965.)
Studies on other crab muscles, such as
the closer of Pachygrapsus (Hoyle and
Wiersma, 1958a), the closer of Carcinus
(Atwood, 1963a, b), and the closer of
Chionecetes (Atwood, 19656), have indicated that diversity of response to indirect stimulation is a common feature. In
Carcinus, and Chionecetes, the more common fibers are doubly-motor-innervated,
showing relatively modest p.s.p's for both
axons (Fig. 16). There are also fibers
which show very large p.s.p's for the fast
B
B
FIG. 16. Slow (b) and fast (c) excitatory p.s.p's in
a fiber of the closer of Carcinus; membrane response to injected current is also shown (a). Timeconstants estimated from a, b, and c are similar,
about 22 msec. (After Atwood, 19636).
axon, and small, or no, p.s.p's for the slow
axon (Figs. 17, 18). Often large graded or
spike-shaped responses appear in these
fibers, even with single stimuli to the fast
axon (Fig. 18 B). Still other fibers resemble
the atpical fibers of Randallia in their relatively large responses to the slow axon,
their small responses to the fast axon, and
the absence of graded responses (Fig. 19).
In some of the latter, the fast electrical response may be entirely absent. Similar
fibers occur in Pachygrapsus (Hoyle and
Wiersma, 1958a).
The difference in form of the fast and
slow contractions is pronounced even in
Randallia (Figs. 13, 15). In Carcinus it is
even more striking (Fig. 19). At low frequencies of stimulation the fast response is
a series of small twitches, while the slow
response is a gradually developing contraction which relaxes many times more
4 0 mV
10 msec
FIG. 17. Responses of a Carcinus closer muscle
fiber to stimulation of the fast axon at 5/sec,
showing the development of large graded responses.
540
HAROLD L. ATWOOD
FFG. 18. Responses of two fibers of the closer of
Chionecetes to single stimuli. In (A), fasl (large)
and slow (small) p.s.p's are registered, and in (B),
a spike-shaped response is generated by the fast
axon. Calibration: voltage, 20 mV; time, 40 msec
(A), 20 msec (B). (After Atwood, 19656).
slowly than the fast contraction. The difference in the speed of relaxation is in itself a strong indication that the two contractions are being produced by different
muscle fibers at this frequency. In Chionecetes and Pachygrapsus, it is possible to
observe visually the fibers which are contracting at the low frequencies of stimulation, and to confirm that only a few fibers—
those with electrical response as shown in
Figures 17 and 18—are participating in the
fast contraction at these frequencies. Similarly, the slow contraction can be seen to
be associated with fibers of the type shown
in Figure 19.
The properties of the contractions suggest that, once again, fibers with different
morphological and contractile characteristics may be involved, with differences
in electrical behavior superimposed. This
situation was confirmed in Chionecetes,
in which short-sarcomered, fast-contracting
fibers and long-sarcomered, slow-contracting fibers were responsible for the fast
and slow contractions, respectively, at low
frequencies of indirect stimulation.
In certain other crustaceans, the range
of muscle fiber types may be less extreme,
and the differences between the fast and
slow contractions therefore less pronounced.
For example, in the crusher-claw closer
of the lobster, in which the short-sarcomered fibers are not present, Wiersma
(1955) found little difference between the
fast and slow contractions, whereas in the
cutter-claw closer, which contains shortsarcomered fibers, the difference is pronounced. In Nephros, the differences between the fast and slow contractions in
walking-leg closers may be explained largely in terms of differences in the form of
the electrical responses set up by the two
axons; no convincing evidence was found
for extreme variation in properties of muscle fibers (Atwood, 1963c). In the crayfish
claw closer, however, the fast and slow
contractions have very different speeds at
low frequencies of stimulation. The fast
axon twitch occupies 50-150 msec, whereas
0.25
q
2 sec
FIG. 19. Responses of a fiber in the closer of
Carcinus to stimulation of the fast and slow axons;
tension of the whole muscle is registered in the
lower traces.
CRUSTACEAN XEUROMUSCULAR MECHANISMS
l_
B
FIG. 20. Responses to stimulation ot the slow axon
in the claw of the crayfish, Aslacus palhpes. In (A),
electrical responses of a single fiber are registered,
along with tension of the whole muscle (lower
trace). In (B-D), tension responses to repetitive
stimulation at 10/sec (15), to a single shock (C),
and to paired shocks (3 msec separation, 2/sec),
arc shown at higher amplification. Note the prolonged lime coiuse of the slow "twitches". Calibration: Voltage. 20 mV; tension, 4 g (A), 0.2 g
(B-D); time, 0.4 sec (A), 1 sec (15), 50 msec (C, D).
(After At wood, 1963fl.)
the slow axon "twitch" which can sometimes be measured by sensitive recording
methods, lasts for at least 500 msec (Fig.
20) . Probably different fibers are involved
in generation of these responses, although
this remains to be shown.
It is important to emphasize that the
differences between the fast and slow contractions, if any, are most apparent at the
low frequencies of stimulation. At higher
frequencies large numbers of fibers are
recruited. Since many of these respond
well to both axons, they may contribute
to both fast and slow contractions. Any
differences in the contractions of these
fibers in response to the two axons would
be mediated through differences in electrical behavior which undoubtedly often
occur (Hoyle and Wiersma, 1958«).
The relative abundance of the doublymotor-innervated fibers is apparent in the
generalized scheme presented in Figure 21,
which is based on findings made in crab
muscles. The behavior of the muscle at
the low frequencies of stimulation is dominated by the extreme types of muscle fiber
(Types A and B). At higher frequencies
of stimulation, the more typical fibers
(Type C) play a greater role.
The general conclusion to be drawn
541
from the above information is that the
contractile behavior of any muscle under
consideration is influenced by the nature
of the responding muscle fibers, the nature
of the nerve endings supplying them, and
the way in which the connections between
the axons and the muscle fibers are set up
(in other words, the "wiring diagram").
Further consideration of the possible
differences between the motor axons involves more detailed study of the behavior
of individual neuromuscular junctions.
PROPERTIES OF EXCITATORY SYNAPSES
Electrical behavior
By means of the technique of extracellular recording with a microelectrode (Fatt
and Katz, 1952), it is possible to study the
electrical behavior of individual crustacean
neuromuscular junctions (Dudel and Kuffler, I961o-c). This technique has now been
FIG. 21. Innervation scheme of a crab closer
muscle (based largely on findings in Carcinus and
Chioneceles; diagrammatic). The postulated distributions of fast (F), slow (S), and inhibitory (I)
axons are indicated. The muscle fibers fall roughly
into three categories: Type A (mainly fast axon
innervation), Type B (mainly slow and inhibitory
innervation), and Type C (mainly triply innervated).
542
HAROLD L. ATWOOD
used to study the properties of the synapses of a number of different crustacean
motor axons, in order to see whether the
pattern of transmission elucidated for the
opener-stretcher motor axon of the crayfish walking leg (Dudel and Kuffler,
1961a, b) is present at other crustacean
motor synapses.
In using this technique, care was taken
to record differentially between two microelectrodes, one placed at the synapse and
the other a few microns above it. This
procedure was necessary to eliminate nonspecific potentials arising from nearby junctions and muscle fibers. The criteria used
in deciding whether a junctional region
had been located included discreteness of
localization, and presence of extracellularly recorded spontaneous miniature
potentials. Sometimes a nerve terminal
potential was also present, but this feature
was often lacking in records obtained at
what appeared to be well-localized junctions.
In the deep extensor muscles of the crayfish abdomen, the potentials set up by
the synaptic currents were quite different
from those reported for the crayfish leg
opener (Dudel and Kuffler, 1961a, b; Takeuchi and Takeuchi, 1964, 1966b). At
low frequencies of stimulation of any of
the motor axons, the synaptic currents were
of fairly large size, usually several times
the quantal unit, as estimated from the
spontaneous miniature potentials (Fig.
22C). Failures of transmission were rare
except in fatigued preparations. At some
junctions there was appreciable fluctuation
in magnitude of the synaptic current with
successive stimuli (Fig. 22 A,B), but often
there was rather little fluctuation (Fig. 22
C,D). Furthermore, the facilitation of the
synaptic current at higher frequencies of
stimulation was modest. In all these respects, there was a contrast with the synaptic currents of the crayfish leg opener
muscle, which were smaller, showed a high
rate of failure at low frequencies of stimulation, and exhibited pronounced facilitation. The observations on the phasic abdominal muscles, therefore, show that the
FIG. 22. Measurements of synaptic currents (upper
traces) and post-synaptic potentials (lower traces)
in crayfish phasic abdominal extensor muscles, at
1/sec stimulation. (A) and (B) are from a fiber in
muscle L2, showing variation in the synaptic current. (C) and (D) are from the medial muscle,
showing synaptic currents several times the size
of the spontaneous miniature potential (arrow),
and with little fluctuation in magnitude. Calibration: voltage, top traces, 400 ^V (A, B), 1 mV (C,
D); bottom traces, 20 mV; time, 4 msec.
events at individual junctions reflect those
recorded with internal electrodes, and in
particular, that the large post-synaptic
potentials result from a relatively large
output of transmitter substance at each
junction (by crustacean standards), and
not merely from the occurrence of large
numbers of endings on the muscle fiber.
Recordings were also obtained from the
superficial abdominal extensor muscles
(Fig. 23). However, the records of synaptic currents were not as satisfactory as in
most of the other muscles, perhaps because
of the inherent difficulty in getting an
electrode tip close enough to the synapses
on these muscle fibers (see Fig. 28). Spontaneous miniature potentials were seldom
seen. The synaptic currents were much
smaller than those in the deep extensors.
For some axons the amplitude of the
synaptic current was remarkably constant
at frequencies of occurrence of 1-10/sec,
although for other axons it increased with
frequency. The internally recorded p.s.p's
set up by these axons reflected the behavior
of the synaptic currents.
It was frequently possible to record two,
or even three, synaptic currents at one
543
CRUSTACEAN NEUROMUSCULAR MECHANISMS
synaptic facilitation in some cases in which
an impulse in one excitatory axon arrived
just before an impulse in a second one
(Fig. 23). A phenomenon which may be
related to this is the potentiation of the
fast contraction by the slow in the crayfish
claw, and vice versa (Wiersma and van
Harreveld, 1939). The mechanism underlying this phenomenon awaits further
study.
Comparison of the synaptic currents
measured in the crayfish leg opener and
in the abdominal muscles indicates that
there can be considerable variation in
performance among endings of different
axons. This variation extends also to
different endings of the same axon in many
cases. Indications of this situation appear
in records of internally recorded p.s.p's,
which in some fibers show facilitation,
and in others little or none, even within
the same muscle and in response to the
same axon. An example has been described in the closer muscle of Randallia
for the fast axon (Atwood and Hoyle
1965). Even better examples can be found
in the opener and stretcher muscles of
TV
B
Pachygrapsus (Fig. 24).
L
FIG. 23. Synaptic currents and post-synaptic potentials in a crayfish tonic extensor muscle fiber. In
(A), one of the motor axons was stimulated at 10/
sec, producing a synaptic current of small but
constant amplitude. In (B), stimulation of a second axon was timed to set up a response just
before that of the first axon. A second synaptic
current of small amplitude (arrow) was recorded.
The synaptic current and p.s.p. of the first axon
were facilitated. Calibration: voltage, top traces,
400 jtV; bottom traces, 20 mV; time, 10 msec.
locus by stimulating various axons (Fig.
23 B). From this observation, one would
suspect that synapses of the different axons
occur close together, and this is borne
out by electron microscopy (Fig. 28).
An interesting phenomenon, as yet unexplained, was the appearance of hetero-
Recordings of synaptic currents in the
latter muscles have shown that, whereas
some synapses behave much like those of
the crayfish opener-stretcher axon, and are
characterized by frequent failures and low
amplitudes at frequencies of stimulation
of 1-2/sec. (Fig. 25 A,B), others are more
like those of the axons supplying the phasic
abdominal muscles, and show few or no
failures at low frequencies of stimulation
(Fig. 25 C). In the latter type, the currents
C
D
FIG. 24. Poorly facilitating (A, B) and strongly
facilitating (C, D) p.s.p's from two fibers in the
opener muscle of Pachygrapsus. Calibration: voltage, 10 mV; time, 1 sec.
544
HAROLD L. ATWOOD
transmission occur at low frequencies,
whereas at others, there are no failures
in fresh preparations. The fluctuation in
amplitude of the synaptic currents is much
more pronounced in this preparation than,
for example, in the crayfish abdominal
muscles. However, on the average the endings show a higher probability of transmitter output (Fig. 27) than the endings
of the opener-stretcher motor axon.
FIG. 25. Synaplic currents from facilitating (A, Ii)
and poorly facilitating (C) junctions, in Pachygrapsus. J11 (A) and (15), currents of one and two
quanta! units, respectively, are shown. In (C). the
currents are several times the quantal unit (the
size of which is comparable to the spontaneous
potential indicated by the arrow). Calibration:
voltage, top traces, 400 ^V; Ixittom traces, 20 mV,
time, 4 msec.
are typically several times the size of the
quantal unit. They may vary little in
amplitude (Fig. 31), and frequently they
show little or no facilitation with an increase in the frequency of stimulation.
The fast closer axon of Pachygrapsus
has also now been thoroughly investigated
in this respect. Here, too, different endings of the axon show differences in behavior (Figs. 26, 27). At some, failures of
7
FIG. 26. Synaptic currents at a fast axon junction
in the Pachygrapsus closer muscle. In (A), there
was failure of transmission at this junction. In (B)
and (C), synaptic currents of variable size were
generated. Note the occurrence of spontaneous
potentials in (A) and (B). Calibration: voltage, top
traces, 1 mV; bottom traces, 20 mV; time, 10 msec.
FIG. 27. Synaptic currents at a junction of the fast
axon in the Pachygrapsus closer, showing the great
variation in amplitude encountered at 1/sec stimulation. Calibration; voltage, top traces, 400 ^V;
bottom traces, 20 mV; time, 4 msec.
In the doubly-motor-innervated muscles
of the lobster and the rock lobster, it is
sometimes possible to record both fast-axon
and slow-axon synaptic currents at one
locus, but just as often, only one can be
registered. Thus the synaptic arrangements
are probably variable (Atwood, Dorai Raj,
and Parnas, unpublished). In these muscles
relatively little difference in behavior of
synapses of the two motor axons was
apparent.
A general conclusion which emerges
from this comparative work is that crustacean motor synapses can vary a great
deal in their performance. The properties
of a particular motor axon may depend
very much upon the relative numbers of
facilitating and poorly facilitating synapses associated with it. The question which
must next be answered is: what causes the
variation in behavior? An initial step in
CRUSTACEAN NEUROMUSCULAR MECHANISMS
FIG. 28. Electron micrograph through a tonic
fiber of the crayfish abdominal extensor muscles
cut in longitudinal section, showing five axons, of
which four contain synaptic vesicles and three
answering this question has been made
with electron microscopy.
545
synaptic densities (arrows). Other structures include the sarcolemma (S), extensive non-filamentous
sarcoplasm (N), a Z line (Z), and the contractile
filaments (K). (After Jahromi and Atwood, 1967.)
muscles of the crayfish abdomen are supplied by five excitatory axons and one inhibitor, and not all fibers receive the latter
Morphological features
axon, so there is at least an 85% chance
In studying the morphology of crusta- on a priori grounds that any given synapse
cean synapses, the problem of identifying is excitatory. Since the motor axons of
those associated with the different axons the tonic extensors are all of the relatively
supplying a particular muscle fiber imme- low-output, fatigue-resistant type, whereas
diately presents itself. For example, how the Pachygrapsus fast closer axon is of the
does one tell a slow axon ending from a high-output, fatigue-sensitive type, an infast in a doubly-motor-innervated muscle structive comparison can be made.
fiber?
The synapses in the tonic abdominal
As a start, therefore, it was necessary muscles often occur on several closely assoto choose material in which the identity ciated axons (Fig. 28; Jahromi and Atwood,
of the synapses was reasonably certain. In 1967). Very often the synapses are made
the closer of Pachygrapsus, there are fibers near the surface of the muscle fiber with
which respond only to the fast axon arm-like extensions of non-contractile sar(Atwood, et al., 1967). The tonic extensor coplasm. Sometimes two or more synapses
546
HAROLD L. ATWOOD
occur close together. The presynaptic vesicles, which average about 600 A in diameter, are often rather closely packed. Dense
accumulations of them appear at the synaptic membranes.
The fast axon synapses on Pachygrapsus
fibers sometimes occur at the surface, but
more usually they are found in sarcolemmal clefts a few microns away from the
surface (Fig. 29; Atwood and Johnston,
unpublished). There is typically a small
amount of non-contractile sarcoplasm associated with the synapse. The endings are
usually single, in confirmation of the physiological data (Atwood, et al., 1967). Often
there are many synaptic areas on the ending, the apparent region of synaptic contact being fairly extensive (Fig. 30). The
vesicles average about 600 A in diameter,
but they are usually much less densely
packed than in the endings on the tonic
abdominal muscles.
The main features of contrast between
the two types of ending are: the larger
diameter of the Pachygrapsus fast axon
endings; the higher density of synaptic
FIG. 29. Electron micrograph through a fiber in
the closer muscle of Pachygrapsus, showing an axon
(AX) located in a sarcolemmal invagination (IN); a
large nucleus (X) appears near the surface of the
fiber. Possible synaptic region is indicated by
arrows. (Electron micrograph by H. Johnston.)
CRUSTACEAN NEUROMUSCULAR MECHANISMS
547
The experiments of Marmont and Wiersma (1938), Dudel and Kuffler (1961c),
Dudel (1965), and Takeuchi and Takeu-
chi (1965, 1966) have provided convincing
evidence for a process of pre-synaptic inhibition in the opener muscle of the crayfish
leg and claw. The basic observations are:
first, that the excitatory p.s.p. and the output of transmitter substance (as indicated
by measurements of synaptic current) are
reduced when the excitatory p.s.p. is closely
preceded by an inhibitory impulse; secondly, that the extracellularly-recorded
nerve terminal potential is also reduced
in amplitude; and thirdly, that this reduction is probably associated with an increased permeability of the nerve terminal
to chloride ion, since the reduction is prevented by replacing chloride with an impermeant anion. The mechanism of presynaptic inhibition is thought to involve
receptors on the excitatory axon which
FIG. 30. A nerve terminal of the Pachygrapsus fast
axon (AX), showing areas of synaptic contact
(arrows), with associated non-contractile sarcoplasm
(N). T tubules (T) leading into the muscle, are
also in evidence. (Electron micrograph by H.
Johnston.)
vesicles in the tonic abdominal axons; the
larger ratio of synaptic membrane to available vesicles in the Pachygrapsus endings;
and the differences in the amount and arrangement of non-contractile sarcoplasm.
The first and third of these features could
relate to the differences in transmitter output at low frequencies of stimulation, while
the second point could have something to
do with the different rates of fatigue.
There is no evidence in these findings for
qualitatively different transmitters in the
two kinds of axons.
PERIPHERAL INHIBITION
Pre-synaptic inhibition
548
HAROLD L. ATWOOD
are sensitive to the transmitter substance
released by the inhibitory axon, and which,
when activated, bring about the increase
in chloride conductance in the axonal
membrane. The resulting decrease in amplitude of the nerve terminal potential is
linked to a reduction in output of the
excitatory transmitter agent.
We have extended the observations on
pre-synaptic inhibition to the opener and
stretcher muscles of crabs. In PachygrapsuSj the dual reduction of the nerve terminal potential and of the synaptic current
is well marked when impulses of the specific inhibitor axon are timed to precede
the excitatory events (Fig. 31). The inhibition affects both the synapses which normally show no failures of transmission and
little fluctuation in amplitude of the synaptic current, and those which show frequent
failures of transmission at low frequencies
of stimulation. To date, comparable information on the common inhibitor axon
of crab muscles is not available.
L
Post-synaptic inhibition
In crustacean muscle fibers, post-synaptic
inhibition was established earlier than presynaptic inhibition as an important physiological phenomenon. The work of Fatt
and Katz (19536) and of Boistel and Fatt
(1958) demonstrated that crustacean muscle
fibers undergo an increase in membrane
conductance under the influence of inhibitory impulses, and that this change is
most likely due to an increased membrane
permeability to chloride ion. The basic
chemical mechanism may thus be similar
to that responsible for pre-synaptic inhibition, differing only in location.
The post-synaptic mechanism is considerably more widespread than the pre-synaptic
mechanism. The crayfish and crab opener
muscles, in which the pre-synaptic mechanism has been demonstrated, also show
post-synaptic inhibition for the same inhibitory axons. Kennedy and Evoy (1966)
could find no evidence for pre-synaptic inhibition in the crayfish abdominal flexor
muscles, and similarly, Atwood, et al.
(1967) were not able to demonstrate it in
B
J'IG. 31. Synaptic currei.ts and nerve terminal
potentials (upper traces), and excitatory postsynaptic potentials (lower traces) in a muscle fiber
o£ the stretcher of Pachygrapsiis. (A) Responses to
3 successive stimuli to the excitatory axon at 1/sec.
The first deflection in the top trace is the nerve
terminal potential, and the second deflection is
produced by the synaptic current. At this synapse
there is little variation in amplitude of the synaptic
current. (B) Effect of preceding stimulation of the
specific inhibitory axon on the excitatory responses.
The first deflection in the top trace is the inhibitory
nerve terminal, and the second and third deflections
are the excitatory responses, as in (A). Note that
the excitatory nerve terminal potential, the synaptic current, and the internally recorded post-synaptic
potential are all reduced, and that the synaptic
current shows considerable fluctuation. Calibration:
voltage, 400 ^V (top), 20 mV (bottom); time, 4 msec.
the deep abdominal extensor muscles of
the crayfish. In the latter instance, the
post-synaptic mechanism is well developed,
for inhibitory stimulation can alter the
shape of excitatory p.s.p's and eliminate
spike-shaped or graded responses (Fig. 32).
549
CRUSTACEAN NEUROMUSCULAR MECHANIS:NIS
CONCLUSION
L
FIC. 32. Inliibition in crayfish deep extensor
muscles. In (A) and (15), simultaneous records
were made from M (top) and L2 (bottom) muscles.
Axon 4 produces a p.s.p. in L2 (A). Axon 5 produces an inhibitory p.s.p. in M, and accelerates the
decay of the excitatory p.s.p. without reducing its
amplitude greatly (B). Initiation of the excitatory
p.s.p. at various times relative to the inhibitory
p.s.p. (C, arrow) fails to produce attenuation of
the excitatory p.s.p. In another preparation (rock
lobster, L,), Lhe electrically excited membrane responses set up by the p.s.p. are attenuated when
preceded by an inhibitory impulse (arrow). Calibration: voltage, 10 mV (A-B), 20 mV (CD); time,
20 msec. (After Atwood, et al., 1967).
Often the excitatory p.s.p's in these muscles
show little change in amplitude, indicating little if any pre-synaptic action (Fig.
32).
In crab leg muscles, not all of the fibers
react physiologically to stimulation of inhibitory axons. In the doubly-motor-innervated muscles, the most phasic fibers, which
respond well to the fast axon, are affected
only slightly, or not at all, by stimulation
of the inhibitory axon (Fig. 33; Atwood,
19656; Atwood, et al, 1967). This finding partly explains the previous ones by
Wiersma and Ellis (1942) that the fast
contractions of the doubly-motor-innervated muscles are less influenced by inhibitory stimulation than the slow contractions. The presumed lack of inhibitory
innervation of certain fibers is incorporated
into the scheme of Figure 21. A parallel
situation is described by Usherwood and
Grundfest (1965) in muscles of insects.
The above discussion has been directed
mainly toward the problem of the organization of crustacean neuromuscular systems
and the gross properties of the components.
A striking feature of these systems is their
variety and apparent functional adaptability, as exemplified by the two claws
of the lobster. The properties of the muscle fibers and of the axons supplying them
are extremely variable, and can be utilized
in combinations which permit generation
of a great variety of responses. The questions may be asked, how the different
muscle fibers and axons acquire their properties during development, and how they
TTTT
rf
B
l_
FIC. 33. Effects of stimulating the inhibitory axon
at 15/sec on membrane voltage responses to applied
current pulses in two fibers of Chionecetes (in the
closer muscle). Current recording is shown in the
lower traces, membrane voltage in the upper traces,
and duration of inhibition is indicated by the
thick bars. In (A) there is a marked increase in
membrane conductance; in (B) there is very little.
Calibration: voltage, 20 mV; current, 1.5 /tA; time,
2 sec. (After Atwood, 1905b).
550
HAROLD L. ATWOOD
come to be associated together in useful
ways. The observations of Dorai Raj
(1964) would seem to rule out a specific
trophic relationship between nerve and
muscle which would determine the characteristics of the muscle fiber (e.g., Buller
et al., 1960a, b), for in the accessory flexor
muscle of the crab, several different muscle
fibers are all supplied by the same axon.
The problem thus raised remains open
to investigation.
Another problem which has not yet
been fully explored concerns the chemical
mechanisms involved in neuromuscular
transmission and in responsiveness of muscle fibers. There is strong evidence, from
the recent work of Takeuchi and Takeuchi
(1964, 1966a, b) that glutamate stimulates
excitatory synapes and may be the excitatory transmitter in crustacean leg muscles.
There is so far no good evidence bearing
on the question of the identities of the fast
and slow transmitters, and whether they
are the same, or different. Since the phasic
abdominal muscles as well as the leg muscles respond to glutamate (Ozeki and
Grundfest, 1967), it is tempting to assume
that the same transmitter may be involved
in all these muscles.
Gamma-aminobutyric acid is a strong
candidate for the inhibitory transmitter.
The experiments of Kravitz, et al. (1963),
Dudel (1965a, b) and Takeuchi and Takeuchi (1965, 1966a) have demonstrated that
this substance is present in greater concentrations in crustacean inhibitory neurons than in the excitatory neurons, and
that it duplicates the action of the inhibitory transmitter substance in all known
respects. The fact that certain crab muscle
fibers are not much affected by this substance (Florey and Hoyle, 1961), is explained by the finding that they probably
receive little or no inhibitory innervation
(Atwood, 19656; Atwood, et al., 1967;
Fig. 21).
The chemical mechanisms underlying
the electrical behavior of crustacean muscle
fibers have been explored by many workers.
A few of the more recent investigations
include those of Hagiwara and Naka
(1964), Hagiwara and Takahashi (1967),
Abbott and Parnas (1965), Ozeki, et al.
(1966a, b), and Ozeki and Grundfest (1967).
This work has shown that, while spike
potentials are dependent on a gradient of
calcium ions across the membrane, the excitatory postsynaptic potentials are probably dependent on transfer of sodium ions
The chemical make-up of crustacean muscle fiber membranes is apparently complex,
and perhaps variations can easily arise,
which would help to explain the variety
of types of electrical responsiveness. The
large size of crustacean muscle fibers makes
them promising experimental material for
further investigation.
A final problem concerns the normal
use made by the animal of the peripheral
mechanisms. The complexity of the neuromuscular apparatus can be rationalized as
resulting from the relative paucity (by
vertebrate standards) of total neurons.
The presence of relatively few efferent
axons implies that the pattern of activity
generated in each of them is of great importance, and investigations by Wilson and
Davis (1965) and Kennedy and Takeda
(1965a, b) support this idea. Certain
strange and apparently illogical features,
such as the distribution of the common
inhibitor in crabs (Fig. 1), invite further
investigation in this area.
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