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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. 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