Download Role of Activity in Determining Properties of the Neuromuscular

AMER. ZOOL., 27:977-989 (1987)
Role of Activity in Determining Properties of the
Neuromuscular System in Crustaceans1
H. L. ATWOOD AND G. A. LNENICKA
Department of Physiology, University of Toronto,
Toronto, Ontario M5S 1A8, Canada
SYNOPSIS. Crustacean muscle fibers, like those of higher vertebrates, are diversified in
physiology, morphology, and biochemical attributes. However, unlike motor units of
mammals, those of crustaceans usually do not contain fibers of uniform type. Motor neuron
activity acts as a unifying force for the motor units of mammalian muscles, but its role in
determining properties of crustacean motor units is less well defined. In certain crustacean
muscles, differential activity of sensory-motor systems is important for establishing muscle
fiber properties during early development. In freshwater crayfish, neuromuscular junctions of a phasic motor neuron are altered physiologically and morphologically by chronic
stimulation; the adapted junctions release less transmitter per impulse and are more
fatigue-resistant than naive junctions. The muscle fibers may also adapt to chronic stimulation, but less dramatically and at a slower rate. The adaptive responses of the neuromuscular junction can be achieved through manipulation of sensory input and with little
increase in motor impulse activity. This suggests that altered protein synthesis is triggered
centrally by synaptic input to the motor neuron. In general, present evidence suggests
that long-term adaptation of neuromuscular junctions and muscle fibers of crustaceans
can occur in response to altered activity in the nervous system, in spite of the fact that
certain muscle fiber properties appear to be genetically predetermined. Some aspects of
matching between neuromuscular junction and muscle fiber appear to be determined in
response to growth of the muscle fiber; other features are activity-dependent; and some
may result from expression of inherent neuronal properties.
INTRODUCTION
Study of muscle fiber and neuromuscular properties in both vertebrates and
arthropods has entered a new phase in the
past few years with the application of
molecular techniques and growing emphasis on the means by which specific genes
are turned on and off during development
or in response to environmental and physiological challenges. The study of nervemuscle interaction can be seen as part of
the broad field of developmental biology,
with molecular assays employed as a major
technique.
Altered activity of motor axons and their
attached muscle fibers has come to the fore
as a perturbing factor that triggers adaptive responses in the mammalian neuromuscular system. It seems that the properties of motor neurons and muscle fibers
respond to activity levels both during and
1
From the Symposium on Muscle Fiber Typing as a
Bioassay of Nerve-Muscle Interaction: Comparison of
Arthropod and Vertebrate Systems presented at the Annual
Meeting of the American Society of Zoologists, 2730 December 1985, at Baltimore, Maryland.
after development. Indeed, the very survival of specific motor neurons seems linked
to their developmental experience (Oppenheim and Chu-Wang, 1983).
How general are these findings? Can the
principles of development and alteration
worked out for mammalian motor units be
shown to apply to other major groups of
animals, or must they be viewed as special
adaptations restricted to certain classes of
vertebrates?
Considerable work has been done
recently on developmental neurobiology
of arthropods in general (Bastiani el ai,
1985), and on development of crustacean
neuromuscular systems in particular (Govind, 1984). So far, the volume of experimental material relating to the role of
activity in specifying properties of neuromuscular system in arthropods is not large
compared to that available for mammalian
neuromuscular systems. Nevertheless, it is
possible to compare results as they now
stand, and to look for general principles,
if such exist.
We will begin by comparing data for
mammalian and crustacean motor systems,
977
978
H. L. ATWOOD AND G. A. LNENICKA
and then introduce new observations on
the role of activity in specifying neuromuscular properties in crustaceans. A complete picture of the deterministic role of
activity is not yet available, but evidence to
date indicates that its effect is far from
insignificant.
ACTIVITY-RELATED ALTERATION OF
MAMMALIAN MOTOR UNITS:
A BRIEF REVIEW
Muscle fibers
Several recent reviews deal with properties of mammalian muscle fibers (Buller
and Pope, 1977; Pette, 1985; Pette and
Vrbova, 1985). It is clear that physiological
differences are correlated with definable
morphological features and with molecular speciation. Differences in the myosin
molecule have been of particular interest
in recent work (Pette and Schnez, 1977;
Pierobon-Bormioli et al., 1981), but other
molecules such as troponin (Bronson and
Schachat, 1982) have also been found to
vary with fiber type.
Physiological features that can be readily
measured include contraction and relaxation times of a single twitch, maximal isometric force, and rate of decline in tension
(fatigue) during maintained stimulation.
These differences allow broad distinction
between slow-twitch, fatigue-resistant or
"slow oxidative" (Type I or SO) and fasttwitch (Type II) muscle fibers. Fast-twitch
fibers can be further categorized as
endowed with oxidative and glycolytic
enzymes, and able to resist fatigue (fast oxidative-glycolytic or FOG fibers), or mainly
with glycolytic enzymes, and fatigable (fast
glycolytic or FG fibers).
Structural features known to be correlated with physiological fiber type (reviewed
by Eisenberg, 1985) include: mitochondrial content (low in FG fibers, higher in
FOG and SO fibers); sarcoplasmic reticulum (relatively plentiful in FOG and FG
fibers, sparser in SO fibers); Z-line width
(broad in SO fibers, thin in FG fibers); and
fiber diameter (FG fibers are usually large,
FOG and SO fibers small to intermediate
in size). Oxidative fibers are better supplied by capillaries than glycolytic fibers.
Generally, these features suggest that oxi-
dative fibers are specialized for low level,
maintained activity, while glycolytic (FG)
fibers are well adapted for brief, rapid,
intense activities, since most of their crosssectional area is devoted to myofilaments.
At the molecular level, it is known that
the heavy and light chains of the myosin
molecule vary between fast- and slow-twitch
fibers. In addition, variation in myosin light
chains is found in subclasses of fast-twitch
fibers. These differences account for variations in myosin ATPase activity and for
differences in pH stability (reviewed by
Pette and Vrbova, 1985). Differences in
troponin of fast and slow-twitch fibers are
also known. The calcium-binding protein
parvalbumin is present in much higher
amounts in fast-twitch than in slow-twitch
fibers (Celio and Heizmann, 1982).
Taken together, the relatively small
amounts of sarcoplasmic reticulum and of
parvalbumin correlate well with the slower
relaxation rate of slow-twitch fibers, and
the lower ATPase activity of myosin correlates with their slower rate of contraction.
Most mammalian muscles are heterogeneous in their muscle fiber composition.
The precise allotment of fiber types appears
well adapted to the normal activity of the
muscle (Pette, 1985).
Motor units
Through studies of the muscle fibers
controlled by individual motor neurons, it
has been found that all fibers comprising
a single motor unit are of uniform type.
Motor units have been classified as slow
fatigue-resistant (S), fast fatigable (FF), and
fast fatigue-resistant (FR) by Burke and his
collaborators (review, Burke, 1980; Pette
and Vrbova, 1985). The slow (S) motor
units comprise SO muscle fibers and generate small forces, but are active much of
the time (for postural maintenance). FF
motor units are large, seldom recruited,
and comprise FG muscle fibers, while FR
motor units are recruited often in locomotion and comprise mainly FOG muscle
fibers.
It has been argued (Burke, 1980) that
the relative occurrence of different types
of motor unit in a particular muscle rep-
CRUSTACEAN NEUROMUSCULAR DETERMINATION
resents an adaptive adjustment to the normal use of the muscle. Slow motor units
are more efficient at maintaining low-level
force, but consume more energy for maintenance. Fast motor units are less efficient
in maintaining force, but can develop more
force per unit area of fiber cross-section
and permit much more rapid movement.
Motor neurons
In general, motor neurons of slow motor
units are more readily recruited than those
of fast motor units, and can sustain prolonged firing of impulses more readily
(reviewed by Freund, 1983). Part of this
difference is related to the size of the motor
neuron (the "size principle" of recruitment). In addition, differences in membrane electrical properties have been
reported, leading to a longer after-hyperpolarization in slow motor neurons, and to
a correlated difference in firing pattern.
More recently, it has been shown that
oxidative metabolism of motor neurons
serving S motor units is better developed
than that of motor neurons serving FF
motor units, while FR motor neurons are
intermediate (Sickles and Oblak, 1984).
This seems to represent an adaptation to
the more sustained firing of the motor neurons involved in posture and routine locomotion.
In all of the above properties, there is
considerable overlap among members of
the population of -motor neurons. However, the sub-populations emerge as different in large samples of measurements.
Developmental role of activity
The end-point of mammalian neuromuscular development is the emergence of
motor units within which the muscle fibers
are of uniform type and matched to the
functional roles of the innervating motor
neurons. This condition is established with
time during development, since at birth
muscle contraction is generally slower than
in the adult. After birth, both fast and slow
motor units undergo changes in contraction speed and fatigability, but the alterations are much more dramatic in the case
of fast motor units (Lowrie et ai, 1982;
Vrbova et ai, 1985).
979
There is convincing evidence for the
hypothesis that the influence of the motor
neuron unifies the properties of the innervated muscle fibers, and that a major component of this effect is the pattern of impulses in the motor neuron (Vrbova et al.,
1985). During development, the activity
patterns of the motor neurons change, and
muscle fiber properties change along with
them. For example, in the soleus muscles
of rodents, the pattern of impulses in motor
units of newborn animals is intermittent
and phasic in nature, while in the adult lowfrequency sustained discharges are seen.
Correspondingly, muscle fibers become
more fatigue resistant in the adult. Interference with motor neurons supplying
developing muscles has profound effects
on the properties of the muscle fibers, particularly fast muscle fibers. The normal
complement of FG muscle fibers may be
lost after such interventions.
The argument for a role of impulse
activity in determining properties of
the motor unit during development is
strengthened considerably by the well
established transformations of muscle fiber
type that can be effected in adult muscles
by experimentally imposed activity patterns (Eisenberg, 1985).
The fate of the motor neurons themselves is determined to at least some extent
by the activity they experience during early
development. At the time that initial neuromuscular connections are being established, about half of the motor neurons are
eliminated (Oppenheim and Chu-Wang,
1983). Blocking impulse activity between
the central nervous system and the muscle
retards the elimination of the extra motor
neurons; thus, feedback from muscle to
nerve of a factor related to muscle activity
is implicated (reviewed by Fawcett and
O'Leary, 1985). Motor neurons selected
for survival may be those which are more
active (perhaps due to early establishment
of sensory connections within the central
nervous system). Activity also plays a major
role in elimination of polyneuronal innervation within a muscle. These findings
taken as a whole imply use-dependent
determination of the properties of both
motor neurons and muscle fibers.
980
H. L. ATWOOD AND G. A. LNENICKA
respective motor neurons (Pette and
Vrbova, 1985). The changes observed are
A considerable body of research over the very similar to those produced by direct
past 20 years supports the general view that experimental manipulation of impulse
fiber types in mature mammalian muscles activity.
are adapted to the type of activity they perMorphology and transmitter-releasing
form (Burke and Edgerton, 1975). Altera- capabilities of the motor end-plate are also
tion of the impulse pattern in the motor correlated adaptively with muscle fiber
axon transforms the properties of its inner- type. End-plates of slow fibers are smaller,
vated muscle fibers in an adaptive fashion release less transmitter per impulse, and
(reviewed by Jolesz and Sreter, 1981). Pro- fatigue less rapidly than those of fast glyvision of regularly spaced impulses at a low colytic fibers (Duchen, 1971; Korneliussen
frequency over a period of several weeks and Waerhaug, 1973).
causes fast fibers to transform to the slow
Mammalian muscle fibers appear to be
type (Eisenberg, 1985); reversion occurs strongly influenced in their properties by
when the imposed low-frequency stimula- the ongoing activity of the central nervous
tion is discontinued. Conversely, interfer- system, expressed at the neuromuscular
ence with the normal low-frequency pat- level through the impulse pattern of the
tern of impulses normally carried by S-type motor neuron. Muscle fiber type can be
motor neurons permits the innervated altered to meet new demands imposed by
muscle fibers to drift in the fast direction. the motor system. During development and
Within the fast fiber population mitochon- in adult life, impulse activity is a major
drial content can be made to change determinant of motor unit properties.
through altered muscle exercise, even
without artificial manipulation of motor
impulse pattern (Andersen and HenriksCRUSTACEAN MUSCLES
son, 1977).
Muscle fiber types
Experimental transformation of fiber
type proceeds in a sequential fashion, with
Early recognition of structural differmuscle membrane systems (T system and ences among crustacean muscle fibers, as
sarcoplasmic reticulum) showing the ear- evidenced by differences in length of the
liest changes (starting within hours). sarcomere (Jasper and Pezard, 1934), was
Metabolic systems, the calcium binding sar- eventually followed by recognition of physcoplasmic protein parvalbumin, and con- iological differences and their correlation
tractile proteins are transformed or with structure (reviews by Atwood, 1973a,
adjusted at a slower rate, with complete 1976).
change requiring several weeks (EisenCrustacean muscle fibers have a wide
berg, 1985). This sequence of events sug- range of physiological, structural, and biogests that ionic fluxes (particularly regu- chemical properties. At one end of the
lation of intracellular calcium) could be spectrum, we can discern the slow-acting
signals that regulate genetic processes or "tonic" type of fiber, which has long
involved in the phenotypic alteration (Pette sarcomeres, correspondingly long thick filand Vrbova, 1985). Thus, early changes in aments, a high ratio of thin to thick filacalcium-regulating membrane systems may ments, low myosin ATPase activity, eleclead to local increases or decreases of intra- trically inexcitable membrane, relatively
cellular calcium, which in turn could influ- sparse sarcoplasmic reticulum, and charence phosphorylation and activity of reg- acteristically slow contraction. In contrast,
ulatory proteins and ultimately influence the extreme fast-acting or "phasic" fiber
activity of selected parts of the genome.
has short sarcomeres, short thick filaThe well-studied transformation of mus- ments, high myosin ATPase activity, eleccle fiber type by cross-reinnervation of fast trically excitable surface membrane, relaand slow muscles seems to depend largely, tively generous sarcoplasmic reticulum, and
or perhaps entirely, on the influence of the fast contraction (Atwood, 1973a; Chappie,
different activity patterns carried by their 1982). Usually the slow-acting fibers are
Transformation by activity
CRUSTACEAN NEUROMUSCULAR DETERMINATION
well endowed with mitochondria; fast-acting fibers vary considerably in this respect
(Silverman and Charlton, 1980). Between
the extremes, a wide variety of combinations of structural, metabolic, and electrical properties has been observed.
Although the much-used correlation of
sarcomere length with speed of contraction may serve as a useful preliminary guide
to a muscle fiber's properties, fiber-by-fiber
analysis of certain crustacean muscles has
indicated that contractile response can vary
considerably among fibers of similar sarcomere length. This is well illustrated in
the closer muscles of the American lobster
(Jahromi and Atwood, 1971; Costello and
Govind, 1983). Stated simply, this indicates
that there are several determinants of
overall contractile performance, and any
one factor by itself cannot be relied upon
as an accurate predictor of physiology.
Variation of membrane excitability or
amount of sarcoplasmic reticulum can produce large differences in performance
among fibers of similar sarcomere structure. In addition, metabolic properties, as
judged by relative mitochondrial content
show a wide range, seemingly adapted to
muscle function and conferring differing
abilities to maintain contraction for long
periods.
Detailed biochemistry of contractile proteins is beginning to appear (Meiss et al.,
1981; Maier et al., 1984; Mykles, 1985),
and it is clear that considerable variation
occurs, as in mammalian muscles. Some
features of physiological performance may
soon be explained at the molecular level.
A general statement about these findings
is that crustacean muscle fibers, like those
of mammals and insects, show great diversity of structural, biochemical, and physiological properties, which match them to
a variety of functional roles.
981
motor axons control the muscle, and the
muscle fibers within a motor unit may be
quite diverse (Atwood, 1973*, 1976). Thus,
the unifying role of the motor axon and
its activity in controlling the overall properties of a motor unit seem less easy to
accept for crustacean (and other arthropod) muscles.
A survey of available data on crustacean
neuromuscular systems shows that some
muscles are fairly uniform in fiber composition. Examples include fast extensor
and flexor muscles in the abdomens of
crayfish and lobsters. Other muscles, particularly limb muscles which have a variety
of functions to perform, are generally
found to contain a range of fiber types,
even though only one or two excitatory
motor axons supply the entire set of muscle
fibers.
As for the motor axons, their physiological differences are well established.
Extreme types can be found in the "fast
axons" of limb closer muscles (Atwood,
1973a, b), and the "motor giant" axon of
the crayfish fast flexor muscles. Such phasic
axons are of large size, and normally silent.
Their neuromuscular junctions generate a
large excitatory postsynaptic potential
(EPSP) during initial activation, but exhibit
rapid depression. In contrast, the motor
axons supplying postural muscles such as
the slow flexors of the crayfish abdomen
(Kennedy and Takeda, 1965) are smaller
in diameter and normally tonically active.
Their neuromuscular junctions generate
small to moderate-sized EPSPs and are
highly resistant to depression. "Slow axons"
of limb muscles are intermediate, but
resemble tonic motor axons more closely.
It is known that the threshold of recruitment of the more tonic motor axons is much
lower than for phasic motor axons,
although the "size principle" is in some
cases less important than differences in
membrane properties of central processes
Motor units
(Wiens, 1976).
A general matching of fast-acting muscle
The most significant difference between
neuromuscular systems of higher verte- fibers with phasic motor axons, and of slowbrates and mandibulate arthropods is in acting muscle fibers with tonic motor axons,
the organization of motor units. In the for- certainly occurs among crustacean muscles
mer, numerous motor axons supply a mus- (Atwood, 1973a). However, in the closer
cle, and the muscle fibers within a motor muscles of the American lobster, which
unit are of uniform type. In the latter, few have been extensively investigated, the
982
H. L. ATWOOD AND G. A. LNENICKA
matching is not perfect: examples of slow
fibers innervated primarily by a "fast" axon
have been found in the crusher claw (Costello et al, 1981; Costello and Govind,
1983). This suggests that some properties
of the muscle fiber could be specified independently, rather than by the innervation.
(It should be noted, however, that the
"fast" axon of the lobster crusher claw
often fires sustained bursts and is not completely phasic in its properties; thus the
argument for mismatch of*properties is not
entirely convincing in this case.)
There are many cases in which the range
of muscle fiber properties is great, even
though only one or two motor axons supply the muscle. Does this mean that the
motor axon does not determine the properties of the muscle fibers? Since all fibers
supplied by a single motor axon will experience the same pattern of impulses, why
do their properties diverge?
Individual neuromuscular junctions of a
single motor axon often show quite diverse
transmitter-releasing properties and pattern sensitivity (Bittner, 1968; reviewed in
Atwood, 1976). These presynaptic refinements, together with differences in postsynaptic properties such as contraction
threshold and membrane excitability
(Atwood et al., 1965), account for variable
recruitment of muscle fibers by a single
motor axon. Thus, muscle fibers may experience different patterns of activation even
when the entire muscle is innervated by
one motor axon.
The pattern of matching of synaptic
properties and muscle fiber type has been
explored in a number of muscles. In several crab opener and stretcher muscles, the
single excitatory axon provides the longersarcomered fibers with neuromuscular
junctions that evoke large EPSPs at low
frequency (Sherman, 1977). These slowacting fibers are recruited first during normal locomotion. But in the crayfish opener
muscle, large low-frequency EPSPs occur
in a number of fibers with relatively short
sarcomeres, as well as some with longer
sarcomeres (Thompson and Atwood, 1984;
and unpublished observations). In still
other muscles, EPSPs of different amplitude occur in fibers with no apparent post-
synaptic differences (Frank, 1973). Thus,
a generalizable pattern of pre-and postsynaptic matching remains elusive. Of course,
we do not yet have detailed knowledge of
possible molecular differences in presynaptic or postsynaptic elements.
In principle, it is conceivable that programmed presynaptic properties at the
neuromuscular junction could act to induce
postsynaptic features, perhaps during early
stages of development (Atwood, 1973a).
Alternatively, the fate of the muscle fibers
could be pre-specified and the properties
of the neuromuscular junctions dictated by
a postsynaptic influence (Frank, 1973). Or
perhaps a compromise situation exists, with
both pre- and postsynaptic developmental
programmes playing a role. In such a case,
is there any role for activity in determination of the properties of the muscle fibers
and neuromuscular junctions?
Experimental approaches to this problem involve study of pattern formation and
activity-dependent perturbation of pattern
during early development, and alterations of activity or cellular properties after
development has been completed to observe effects on the components of the neuromuscular system.
NEURAL INFLUENCE IN
CRUSTACEAN MUSCLE
Neuromuscular development
Recent experiments on decapod crustaceans with asymmetric claws have provided
evidence for a neural influence in determination of muscle fiber properties. In the
American lobster Homarus (reviewed by
Govind, 1984) and in fiddler crabs of the
genus Uca (Yamaguchi, 1977) determination of claw asymmetry occurs during a
limited period in early development. After
that time, one claw becomes larger (the
"major" claw) and is apparently more specialized. If it is removed by autotomy, it
eventually reappears as before. In contrast,
snapping shrimps (Alpheus) retain the ability to develop a specialized major claw
throughout adulthood; removal of the
original major claw causes the contralateral minor claw to transform to a major
claw (Mellon, 1981). In some way, a time
window of plasticity is allowed for deter-
CRUSTACEAN NEUROMUSCULAR DETERMINATION
mination of claw type; the duration of this
window varies from one species to another.
That neural mechanisms are involved in
determination of claw type is shown by the
effects of denervation. In Homarus denervation of one claw early in development
invariably results in contralateral appearance of the major, or crusher, claw (Govind, 1982). In Alpheus, denervation of the
major claw results in contralateral transformation of the minor claw (Mellon, 1981).
Muscle proteins specific to the major claw
appear during transformation provided the
nerve supply is intact (Quigley et al., 1985).
In Homarus, experimental manipulations
that reduce activity of one claw or treatments that reduce sensory input such as
tenotomy favour appearance of the major
claw on the other side (Govind, 1982,
1984). Rearing of small animals under conditions in which use of both claws is minimal leads to a large percentage of animals
which do not have a major claw. These
experiments clearly demonstrate a role for
activity during development in establishing claw lateralization. The activity-dependent developmental stimulus leads to differentiation of the paired homologous
excitatory motor axons, so that their firing
patterns and neuromuscular synaptic properties diverge. In the lobster, the "fast"
axon of the crusher claw produces longer
trains of impulses during reflex activation
and generally is more active than its more
phasic counterpart in the minor or cutter
claw; also, its neuromuscular synapses are
more resistant to fatigue. The motor neuron differences are correlated with an
overall preponderance of slow muscle fibers
in the crusher claw, and of fast fibers (with
a small population of slow fibers) in the
cutter claw. Although properties of individual fibers within these muscles may follow their own developmental trajectory to
some extent (Costello and Govind, 1983),
the importance of neural activity in setting
the sequence in motion seems clear.
Retrograde muscle-to-nerve influence in
determination of neuromuscular properties is indicated by experiments in which
muscle fiber growth in crayfish slow
abdominal flexor muscles is slowed experimentally (Lnenicka and Mellon, 19836).
983
Transmitter output at neuromuscular synapses is adjusted appropriately to the size
of the muscle fiber. The same phenomenon is seen during normal growth in crayfish (Lnenicka and Mellon, 1983a) and lobster (DeRosa and Govind, 1978; Govind et
al., 1982) neuromuscular junctions.
Some synaptic properties may however
be adjusted to match activity. In regenerating limbs, the newly formed synapses
release less transmitter, and are more fatigable, than at later stages of limb formation
(Govind et al., 1973). The motor axons in
the developing limb bud appear to be silent,
and activity commences near the time of
emergence of the limb during molting.
Thus, synaptic properties change in parallel with motor activity. Cause and effect
has not been shown experimentally.
Evidence for inherent developmental
patterns within the motor neurons themselves, and for differential interaction with
target muscle fibers, comes from the work
of Velez and collaborators (Ely and Velez,
1982; Hunt and Velez, 1982; Clement et
al., 1983). In crayfish superficial flexor
muscles, connections and synaptic properties of some of the innervating axons are
altered during regeneration by surgical
removal of muscle fibers or contralateral
transposition of the nerve. Different members of the slow flexor motor neuron pool
diverge in their connectivity patterns and
synaptic properties when presented with
similar population of target muscle fibers.
The situation is rendered complex by possible competition among axons for target
sites.
Once synaptic connections have been
established, the properties of both muscle
fibers and motor neurons "drift" with age,
sometimes producing apparent mismatch
between nerve and muscle. Thus, in the
crayfish claw, the fibers of the closer muscle gradually become more uniformly slow
in type, as judged by myosin ATPase activity (Govind and Pearce, 1985). The synapses of the "fast" motor axon become
increasingly "phasic" with age, and less
plastic (Lnenicka and Atwood, 1985a). The
functional discrepancy between synaptic
properties of the fast axon and contractile
properties of the muscle fibers it innervates
984
H. L. ATWOOD AND G. A. LNENICKA
Neuromuscular transmission of the "fast"
axon generates a large EPSP that normally
declines with repetitive stimulation at a relatively low frequency (Atwood, 1982). This
neuron can be selectively stimulated in situ
with implanted electrodes, and it is therefore possible to apply long-lasting conditioning stimulation for days or weeks at a
Neuromuscular alterations after
time, and to follow the resulting effects on
development
neuromuscular synapses and muscle fibers.
Direct activation of the "fast" axon
Crustacean muscle fibers, unlike those of
mammals, can withstand degeneration with transforms the properties of the neurolittle apparent change in morphology muscular junctions: a smaller EPSP is pro(Boone and Bittner, 1974; Velez et al., duced initially during test stimulation, and
1981). In part, this stability may be due to it is more resistant to fatigue (Lnenicka and
long-term survival of severed distal ends of Atwood, 1985a, b). This physiological
the motor neurons, which probably rely on change (referred to as "long-term adapglial cell support. However, since motor tation") is accompanied by altered morimpulse activity is absent, muscle fiber phology of the synaptic terminals. The typproperties (at least morphological ones) ically slender, filiform nerve endings are
seem not to require activity for their main- replaced by more varicose ones with a
tenance. In contrast, tenotomy results in larger mitochondrial volume. Synapses and
drastic shrinkage of crayfish muscle fibers mitochondria are concentrated in the var(Boone and Bittner, 1974), suggesting an icosities (Atwood et al., 1985). (See Fig. 1.)
important trophic role for resting tension.
This adaptive response of the neuroIn spite of this apparent insensitivity to muscular junction requires participation of
neural influence, crustacean muscle fibers the cell body or central processes of the
respond to denervation (Lehouelleur et al., motor neuron. Stimulation of the distal end
1983) and possibly to hormonal influences of the neuron after it has been transected
(Atwood et al., 1965) by varying their mem- does not produce long-term adaptation
brane excitability. Thus, it is possible that (Lnenicka and Atwood, 19856; and unpubneural trophic influence on muscle fiber lished observations).
properties may occur, but that it may take
In contrast, increased sensory input to
some time to be expressed in morpholog- the thoracic ganglion shifts neuromuscular
ical terms.
transmission in the adapted direction, while
A possible neural influence on metabolic decreased sensory input has the opposite
properties has been discerned in lobster effect. One way to show this is to immocloser muscles, in which high levels of oxi- bilize one claw. Neuromuscular transmisdative enzymes are found in muscle fibers sion on the immobilized side becomes less
innervated by the "slow" axon, and low fatigue-resistant, while the opposite change
levels in muscle fibers innervated solely by occurs on the other side (Pahapill et al.,
the "fast" axon (Lang et al., 1980). Whether 1985). These effects do not seem to be
this effect is directly linked to the higher directly linked to impulse activity in the
level neural activity impinging upon the fast axon, because the absolute number of
more oxidative fibers has not yet been impulses in the axon remains very low.
determined. The need for direct experiDirect influence of sensory input on neumental testing of the possible role of neural romuscular adaptation without a large
activity is apparent.
change in motor impulse activity was demonstrated through direct stimulation of
Long-term adaptation
claw afferents capable of generating the
The closer muscle of the crayfish (Pro- claw closing reflex (Lnenicka and Atwood,
cambarus clarkii) claw receives "fast" 1985c). Such stimulation evokes one or two
(phasic) and "slow" (tonic) excitatory axons. fast-axon impulses for two or three trials
increases with age, and it could be argued
that the inactive fast axon gradually
becomes senescent, while the more active
slow axon takes over all functional duties.
Whether the fast axon's senescence is activity-mediated or genetically programmed
remains to be determined experimentally.
985
CRUSTACEAN NEUROMUSCULAR DETERMINATION
8-
0 6'
•Q
a
1 4.
a.
(A
a.
"" 25
10
15
20
25
Duration of 5 Hz stimulation (min)
30
B
Long-term adaptation
FIG. 1. Long-term adaptation of synaptic transmission in a phasic motor axon ("fast" axon of the claw of
the crayfish Procambarus clarkii). A. Excitatory postsynaptic potentials (EPSPs) followed during 30 min of 5
Hz stimulation in representative muscle fibers of a conditioned claw ("Experimental"; solid triangles) and the
contralateral unconditioned claw ("Control"; solid circles). Each point on the graph represents the mean value
for ten muscle fibers. Conditioning was achieved by activating the "Experimental" fast axon at 5 Hz for 2
hr a day over a 14-day period. EPSPs in the "Experimental" claw are initially smaller, but decline less with
continued stimulation, than EPSPs in the "Control" claw. Scale: 4 mV, 20 msec. (From Lnenicka and Atwood,
1965a.) B. Morphological transformation of nerve terminals of the fast axon after 2 wk of conditioning, as
observed in serial electron micrographs. Varicosities are more pronounced and contain a greater volume of
mitochondrial material after conditioning. (From Atwood et at., 1985.)
of a series, along with a larger number of
slow-axon impulses. The former soon disappear altogether. Repetition of conditioning stimulation 2 hr a day for 3 days
or more leads to typical long-term adaptation of the neuromuscular junction.
There is a strong indication here that the
central part of the neuron is able to adjust
the properties of its peripheral synapses for
altered activity, perhaps by exporting
material down the motor axon to the neuromuscular junctions. (See Fig. 2.)
986
H. L. ATWOOD AND G. A. LNENICKA
Sensory input
o
Motor output
CNS
Muscle fiber
Closer
FCE
J
10 ms
SCE
0
2 mV
30 min
FIG. 2. Sensory stimulation produces long-term neuromuscular adaptation without many impulses in the fast
axon. A. Diagrammatic representation of input from claw tactile receptors onto the closer motor neurons in
the central nervous system. A short-latency closing reflex is produced by these receptors. B. Experimental
stimulation of the tactile receptors of the inner surface of the claw is achieved with stimulating electrodes
embedded in metallic paint on the claw surface. C. Myographic recording monitors activity of fast and slow
axons during a 0.5 sec burst of stimuli at 5 Hz, applied once every second for 30 min. During the first few
bursts, a few impulses are triggered in the fast axon; thereafter, only slow axon impulses appear, as evidenced
by the myogram. (FCE, myogram of fast closer excitor; SCE, myogram of slow closer excitor). Long-term
neuromuscular adaptation appears after several days of sensory stimulation with minimal additional activity
in the fast axon. (After Lnenicka and Atwood, 19856.)
Preliminary examination of claw closer
muscle fibers after chronic stimulation of
the fast axon suggests that small but perceptible changes do occur. The number of
mitochondria found at the periphery of the
muscle fiber is larger in conditioned than
in control muscles, suggesting a shift to
more oxidative metabolism. Thus, muscle
CRUSTACEAN NEUROMUSCULAR DETERMINATION
fibers may adapt to conditioning stimulation, albeit at a slower rate than the neuromuscular junction.
CONCLUSION
The role of activity in regulating properties of muscle fibers and neuromuscular
junctions remains less clearly defined in
crustacean than in mammalian neuromuscular systems. In the latter, the muscle
genome seems to be very responsive to
changes in neural activity, and the properties of the muscle fibers are continuously
adjusted to meet the demands imposed
upon the motor system. In crustaceans,
there is growing evidence for an important
role of activity during development of certain neuromuscular systems, and in longterm adaptation of neuromuscular performance. There is also some evidence for
mutual interaction between muscle fiber
and motor neuron. Adaptive responses of
muscle fibers to activity probably occur on
a slower time scale than in mammalian
muscles, and the full extent of such adaptations remains to be elucidated.
Specification of properties of both motor
neurons and muscle fibers by preset genetic
programmes may be more complete in
crustacean neuromuscular systems, since
no simple pattern has emerged to account
for the details of matching between motor
neuron and muscle fiber. Within the preset
genetic limits, a certain amount of adaptive
adjustment can occur. In this light, the
mammalian motor system represents an
extreme specialization in favour of rapid
adjustment to new environmental conditions. Even here, species-specific genetic
limits are in place (Burke, 1980). These
have been arrived at through natural selection and probably act most strongly at the
level of sensory-motor connections within
the central nervous system. This locus has
now been shown to be important also in
crustaceans.
ACKNOWLEDGMENTS
The authors received support from the
Natural Sciences and Engineering Research
Council of Canada and the Medical
Research Council of Canada for work on
987
crustacean neuromuscular junctions and
muscle fibers. Ms. Marianne HegstromWojtowicz provided technical assistance
throughout, and helped to prepare the
manuscript, along with Ms. Alma Cull.
REFERENCES
Andersen, P. and J. Henriksson. 1977. Training
induced changes in the subgroups of human type
II skeletal muscle fibers. Acta Physiol. Scand. 99:
123-125.
Atwood, H. L. 1973a. An attempt to account for the
diversity of crustacean muscles. Amer. Zool. 13:
357-358.
Atwood, H. L. 19736. Crustacean motor units. In R.
B. Stein, K. G. Pearson, R. S. Smith, and J. B.
Redford (eds.), Control of posture and locomotion,
pp. 87-104. Plenum Press, New York.
Atwood, H. L. 1976. Organization and synaptic
physiology of crustacean neuromuscular systems.
Prog. Neurobiol. 7:291-391.
Atwood, H. L. 1982. Synapses and neurotransmitters. In H. L. Atwood and D. C. Sandeman (eds.),
The biology of Crustacea, Vol. 3, pp. 105-150. Aca-
demic Press, New York.
Atwood, H. L., G. Hoyle, and T. Smyth. 1965.
Mechanical and electrical responses of single
innervated crab-muscle fibers. J. Physiol. (London) 180:449-482.
Atwood, H. L., G. A. Lnenicka, and L. Marin. 1985.
Morphological responses to conditioning stimulation in a phasic motor axon of crayfish (Procambarus clarkii).]. Physiol. (London) 365:26P.
Bastiani, M. J., C. Q. Doe, S. L. Helfand, and C. S.
Goodman. 1985. Neuronal specificity and growth
cone guidance in grasshopper and Drosophila
embryos. Trends Neurosci. 8:257-266.
Bittner, G. D. 1968. Differentiation of nerve terminals in the crayfish opener muscle and its functional significance. J. Gen. Physiol. 51:731-758.
Boone, L. P. and G. D. Bittner. 1974. Morphological
and physiological measures of trophic dependence in a crustacean muscle. J. Comp. Physiol.
89:123-144.
Bronson, D. D. and F. H. Schachat. 1982. Heterogeneity of contractile proteins. Differences in
tropomyosin in fast, mixed, and slow skeletal
muscles of the rabbit. J. Biol. Chem. 257:39373944.
Buller, A. J. and R. Pope. 1977. Plasticity in mammalian skeletal muscle. Philos. Trans. R. Soc.
London B 278:295-305.
Burke, R. E. 1980. Motor unit types: Functional specialization in motor control. Trends Neurosci. 3:
255-258.
Burke, R. E. and V. R. Edgerton. 1975. Motor unit
properties and selective involvement in movement. Ex. Sports Sci. Rev. 3:31-81.
Celio, M. R. and C. W. Heizmann. 1982. Calciumbinding protein parvalbumin is associated with
fast contracting fibres. Nature (London) 201:535539.
988
H. L. ATWOOD AND G. A. LNENICKA
Chappie, W. H. 1982. Muscle. In H. L. Atwood and
Jahromi, S. S. and H. L. Atwood. 1971. Structural
and contractile properties of lobster leg muscle
Vol. 3, pp. 151-184. Academic Press, New York.
fibres. J. Exp. Zool. 176:475-486.
Clement, J. F., A. K. Taylor, and S. J. Velez. 1983. Jasper, H. H. and A. Pezard. 1934. Relation entre
Effect of a limited target area on regeneration of
la rapidite d'un muscle strie et sa structure hisspecific neuromuscular connections in the craytologique. C. R. Acad. Sci. (Paris) 198:499-501.
fish. J. Neurophysiol. 49:216-226.
Jolesz, F. and F. A. Sreter. 1981. Development,
Costello, W. J. and C. K. Govind. 1983. Contractile
innervation, and activity-pattern induced changes
responses of single fibers in lobster claw closer
in skeletal muscle. Ann. Rev. Physiol. 43:531muscles: Correlation with structure, histochem552.
istry, and innervation. J. Exp. Zool. 227:381-393. Kennedy, D. and K. Takeda. 1965. Reflex control
of abdominal flexor muscles in the crayfish. II.
Costello, W. J., R. Hill, and F. Lang. 1981. InnerThe tonic system. J. Exp. Biol. 43:229-246.
vation patterns of fast and slow motor neurones
during development of a lobster neuromuscular Korneliussen, H. and O. Waerhaug. 1973. Three
system. J. Exp. Biol. 91:271-284.
morphological types of motor nerve terminals in
the rat diaphragm, and their possible innervation
DeRosa, R. A. and C. K. Govind. 1981. Transmitter
of different muscle fiber types. Z. Anat. Entwickl.
output increases in an identifiable lobster motoGesch. 140:73-84.
neurone with growth of its muscle fibers. Nature
(London) 273:676-678.
Lang, F., M. M. Ogonowski, W. J. Costello, R. Hill,
B. Roehrig, K. Kent, and J. Sellers. 1980. NeuDuchen, L. W. 1971. An electron microscopic study
rotrophic influence on lobster skeletal muscle.
of the changes induced by botulinum toxin in the
Science 207:325-327.
motor end-plates of slow and fast skeletal muscle
fibres of the mouse. J. Neurol. Sci.14:47—60.
Lehouelleur, J., J. Cuadras, and J. Bruner. 1983.
Tonic muscle fibres of crayfish after gangliecEisenberg, B. R. 1985. Adaptability of ultrastructure
tomy: Increase in excitability and occurrence of
in the mammalian muscle. J. Exp. Biol. 115:5568.
sodium-dependent spikes. Neurosci. Lett. 37:227.
Ely, P. and S.J. Velez. 1982. Regeneration of specific Lnenicka, G. A. and H. L. Atwood. 1985a. Agedependent long-term adaptation of crayfish phasic
neuromuscular connections in the crayfish. I.
motor axon synapses to altered activity. J. NeuPattern of connections and synaptic strength. J.
roscience 5:459-467.
Neurophysiol. 47:656-665.
Fawcett, J. W. and D. M. O'Leary. 1985. The role Lnenicka, G. A. and H. L. Atwood. 1985ft. Longof electrical activity in the formation of topoterm facilitation and long-term adaptation at syngraphic maps in the nervous system. Trends Neuapses of a crayfish phasic motoneuron. J. Neurosci. 8:201-206.
robiol. 16:97-110.
Frank, E. 1973. Matching of facilitation at the neu- Lnenicka, G. A. and H. L. Atwood. 1985c. Longromuscular junction of the lobster: A possible
term adaptation of crayfish neuromuscular syncase for influence of muscle on nerve. J. Physiol.
apses does not require an increase in the activity
(London) 233:635-658.
of altered synapses. Soc. Neurosci. Abstr. 11:1288.
Freund, H. J. 1983. Motor unit and muscle activity Lnenicka, G. A. and DeF. Mellon, Jr. 1983a. Changes
in voluntary motor control. Physiol. Rev. 63:387in electrical properties and quantal current dur436.
ing growth of identified muscle fibres in the crayfish. J. Physiol. (London) 345:261-284.
Govind, C. K. 1982. Does exercise influence the differentiation of lobster muscle? In C. F. Herreid Lnenicka, G. A. and DeF. Mellon, Jr. 1983ft. TransII and C. R. Fourtner (eds.), Locomotion and enermitter release during normal and altered growth
getics in arthropods, pp. 215-253. Plenum Publ.
of identified muscle fibres in the crayfish. J. Physiol. (London) 345:285-296.
Corp., New York.
Govind, C. K. 1984. Development of asymmetry in Lowrie, M. B., S. Krishnan, and G. Vrbova. 1982.
Recovery of slow and fast muscles following nerve
the neuromuscular system of lobster claws. Biol.
injury during early post-natal development in the
Bull. 167:94-119.
rat. J. Physiol. (London) 331:51-66.
Govind, C. K. andj. Pearce. 1985. Enhanced reappearance of fast fibers in regenerating crayfish Maier, L., W. Rathmayer, and D. Pette. 1984. pH
lability of myosin ATPase activity permits disclaw closer muscles. Develop. Biol. 107:206-212.
crimination of different muscle fibre types in
Govind, C. K., H. L. Atwood, and F. Lang. 1973.
crustaceans. Histochemistry 81:75-77.
Synaptic differentiation in a regenerating crablimb muscle. Proc. Nat. Acad. Sci. U.S.A. 70: Meiss, D. E., T. A. Lyerla, S. Cain, and W. J. Costello.
822-826.
1981. Electrophoretic protein patterns in fast and
slow muscles of the lobster, Homarus amencanus.
Govind, C. K., D. E. Meiss, and J. Pearce. 1982. DifJ. Exp. Zool. 217:361-364.
ferentiation of identifiable lobster neuromuscular synapses during development. J. Neurocytol. Mellon, DeF., Jr. 1981. Nerves and the transfor11:235-247.
mation of claw type in snapping shrimp. Trends
in Neurosci. 4:245-248.
Hunt, W. P. and S. J. Velez. 1982. Regeneration of
specific neuromuscular connections in the cray- Mendelson, M. 1969. Electrical and mechanical charfish. II. Effect of changes in target area. J. Neuacteristics of a very fast lobster muscle. J. Cell.
rophysiol. 47:666—676.
Biol. 42:548-563.
D. C. Sandeman (eds.), The Biology of Crustacea,
CRUSTACEAN NEUROMUSCULAR DETERMINATION
Mykles, D. L. 1985. Heterogeneity of myofibrillar
proteins in lobster fast and slow muscles: Variants
of troponin, paramyosin, and myosin light chains
comprise four distinct assemblages. J. Exp. Zool.
234:23-32.
Oppenheim, R. and I.-W. Chu-Wang. 1983. Aspects
of naturally-occurring motoneuron death in the
chick spinal cord during embryonic development. In G. Burnstock and G. Vrbova (eds.),
Somatic and autonomic nerve-muscle interactions, pp.
57-107. Elsevier Press, Amsterdam.
Pahapill, P. A., G. A. Lnenicka, and H. L. Atwood.
1985. Asymmetry of motor impulses and neuromuscular synapses produced in crayfish claws
by unilateral immobilization. J. Comp. Physiol.
A 157:461-467.
Pette, D. 1985. Metabolic heterogeneity of muscle
fibres. J. Exp. Biol. 115:179-189.
Pette, D. and U. Schnez. 1977. Myosin light chain
patterns of individual fast and slow twitch fibres
of rabbit muscles. Histochemistry 54:97-107.
Pette, D. and G. Vrbova. 1985. Neural control of
phenotypic expression in mammalian muscle
fibers. Muscle and Nerve 8:676-689.
Pierobon-Bormioli.S., S. Sartore, L. Dalla Libera, M.
Vitadello, and S. Schiaflfino. 1981. "Fast" isomyosins and fiber types in mammalian skeletal
muscle.J. Histochem. Cytochem. 29:1179-1188.
Quigley, M. M., G. Cox, and DeF. Mellon, Jr. 1985.
Role of nerve in determining contractile protein
isoform expression in crustacean muscle. Soc.
Neurosci. Abstr. 11:921.
989
Sherman, R. G. 1977. Comparative analysis of an
excitatory motor unit in crustaceans. J. Comp.
Physiol. 114:91-101.
Sickles, D. W. and T. G. Oblak. 1984. Metabolic
variation among a-motoneurons innervating different muscle-fiber types. I. Oxidative enzyme
activity. J. Neurophysiol. 51:529-537.
Silverman, H. and M. P. Charlton. 1980. A fast-oxidative crustacean muscle: Histochemical comparison with other crustacean muscle.J. Exp. Zool.
211:267-273.'
Thompson, C. S. and H. L. Atwood. 1984. Synaptic
strength and horseradish peroxidase uptake in
crayfish nerve terminals. J. Neurocytol. 13:267280.
Velez, S. J., G. D. Bittner, H. L. Atwood, and C. K.
Govind. 1981. Trophic reactions of crayfish
muscle fibers and neuromuscular synapses after
denervation, tenotomy, and immobilization. Exp.
Neurol. 71:307-325.
Vrbova, G., R. Navarrete, and M. Lowrie. 1985.
Matching of muscle properties and motoneurone
firing patterns during early stages of development.]. Exp. Biol. 115:113-123.
Wiens, T. J. 1976. Electrical and structural properties of the crayfish claw motoneurons in an isolated claw-ganglion preparation. J. Comp. Physiol. 112:213-233.
Yamaguchi, T. 1977. Studies on the handedness of
the fiddler crab, Uca lactea. Biol. Bull. 152:424436.