Muscle Fibers in Regenerating Crayfish Motor Nerves

Joanne Pearce1, Kristin M. Krause2, and C. K. Govind1

1 Life Sciences Division, University of Toronto at Scarborough, Scarborough, Ontario M1C 1A4, Canada; and 2 St. Thomas Aquinas College, Sparkill, New York 10976

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Pearce, Joanne, Kristin M. Krause, and C. K. Govind. Muscle fibers in regenerating crayfish motor nerves. J. Neurophysiol. 78: 3498-3501, 1997. Single discrete muscle fibers were found in regenerating motor nerves in adult crayfish. The regenerating nerves were from native or transplanted ganglia in the third abdominal segments and consisted of several motor axons. The proximal end of these motor axons showed numerous sprouts. Muscle fibers in these regenerating nerves appeared newly developed and were innervated by excitatory nerve terminals. A likely source of these novel muscle fibers may be blood cells in the nerve or satellite cells from neighboring muscle. Contacts made by axon sprouts with other axon sprouts, glia, and muscle fiber, in the form of a dense bar with clustered clear vesicles, characterized the regenerating nerve. These contacts may provide a possible signaling pathway for axon regeneration and myogenesis.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The trophic influence of nerve on muscle is well known. In mammals, removing the nerve early in development retards muscle growth and differentiation, resulting in grossly abnormal muscle development, whereas in adults, denervation brings about atrophy and degeneration of muscle (McLennan 1994). These findings demonstrate a permissive role for the nervous system in the development and maintenance of muscle. In insects, early muscle development may occur in the absence of motor nerves (Broadie and Bate 1993), although later muscle development is seriously compromised by denervation (Consoulas and Levine 1997). Nerves clearly have a profound influence on muscle development, and they may also influence where muscle forms. Here we report an unusual location for muscle, within a regenerating adult crayfish motor nerve, where hemocytes or migratory satellite cells may be the source of these fibers. Regenerating crayfish motor nerves may therefore have an instructive as well as a permissive role in muscle development.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Adult crayfish Procambarus clarkii measuring 10-15 cm in total length were used. Regeneration of the phasic motor axons to the deep abdominal flexor muscles was induced in native or transplanted nerves of the third root in the third abdominal segment of crayfish. Regeneration of the native nerve was induced by transecting the deep branch via a small ventral incision in cold anesthesized crayfish to induce axon sprouting at its cut end. Regeneration of a transplanted phasic nerve entailed isolating the deep branch of the third root attached to its ganglion, as well as the next posterior ganglion, from a donor crayfish and inserting this nerve complex into a host crayfish. This allotransplantation technique is described in detail for the tonic branch of the third root (Krause and Velez 1995); here we used the phasic branch of the third root. The donor ganglia and attached nerves were implanted into the host's third abdominal segment between the exoskeleton and the superficial flexor muscle by pulling them through a cavity created by cutting the fourth swimmeret (Krause and Velez 1995).

Regeneration of the native or transplanted nerves was allowed to proceed for 8-10 wk before they were prepared for electron microscopy by exposing the ganglion and its roots in the third abdominal segment. The preparation was superfused with primary fixative for 30 min before the regenerating nerves were dissected free and further processed by standard procedures (Krause et al. 1996). Regenerating nerves were examined before they contacted the superficial flexor muscle on the dorsal surface in both the native and transplant preparations.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

The third root from the abdominal ganglia in crayfish, soon after its exit from the ventral nerve cord, subdivides into a very thin superficial branch that carries the 6 motor axons to the superficial flexor muscles and a thick deep branch that carries the 10 motor axons to the deep flexor muscles (Kennedy and Takeda 1965a,b). The deep branch further subdivides into several smaller branches, each of which carries a subset of the 10 phasic motor axons (Selverston and Remler 1972). We examined a deep branch that carries six axons each represented by a single profile (Fig. 1A). When branches of the third root are cut before they enter the muscle, the proximal cut end of the motor axons, whose cell bodies are located in the ganglion, regenerate via sprouting (Krause et al. 1996). Such nerve regeneration was examined with the electron microscope in the third abdominal segment of adult crayfish when transected branches to the deep flexor muscle belonged either to the native or allotransplanted ganglia. Altogether, three preparations were examined, one from a native ganglion and two from transplanted ganglia. In each case the regenerating nerve was composed of six motor axons (Fig. 1B). Each of these motor axons was surrounded by a glial lamellated sheath of several layers that enclosed a single large profile, representing a branch of the main axon, and many smaller axon profiles, representing sprouts of the axon. This method for sprouting at the cut proximal end of axons has been previously shown for crayfish limb motor neurons (Kennedy and Bittner 1974; Nordlander and Singer 1972). Both the axon and its sprouts were filled with microtubules and had a peripheral ring of mitochondria, features typical of crustacean axons (Atwood 1976).


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FIG. 1. A: cross-section of a branch of the 3rd root to the deep flexor muscle from a control ganglion showing 6 single axon profiles (arrows) each enclosed by a glial sheath and indicative of 6 motor neurons. B: cross-section of a regenerating branch of the 3rd nerve root to the deep flexor muscles from a transplanted ganglion showing 6 complex axons (arrows) and a single muscle fiber (double arrow). Each of the complex axons is composed of a single large profile (representing a single axon) and several smaller profiles (representing sprouts) enclosed in a glial sheath. C: oblique section of a regenerating branch of the 3rd root to the deep flexor muscles from a transplanted ganglion showing many axon profiles (a), glial nuclei (n) and a few scattered muscle fibers (arrows). Magnification in A, ×600; B, ×1,000; C, ×1,200. Scale bars 20 µm.

In these regenerated nerves we made an unusual finding: the presence of muscle occurring as separate, individual fibers (Fig. 1B), a few being scattered along the lengths of the nerve (Fig. 1C). The fibers were small, 5-10 µm in diameter and up to 25 µm in length and becoming tapered at their ends. They appeared mostly in cross-section running parallel with the nerve, although occasionally in longitudinal view (Fig. 2A) as well, demonstrating that they did not always follow a particular orientation along the nerve. Nor did they show signs of tendinous attachment, but were attached to the surrounding connective tissue.


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FIG. 2. A: oblique section of a regenerating branch of the 3rd root to the deep flexor muscles from a transplanted ganglion with axon profiles (a) and muscle fibers in cross-section (arrow) and longitudinal section (double arrow), the latter with distinct sarcomeres between adjacent Z-lines (arrowheads) and a nucleus (n). B: cross-section of novel muscle fiber composed of myofibrils with scattered mitochondria (m) and occasional diad (arrows). C: cross-section of myofibril showing linearly arranged thick filaments each surrounded by 8-10 thin filaments. D: motor nerve terminal (t) recognized by clear, spherical synaptic vesicle (v) and a synaptic contact with presynaptic dense bar (arrowhead) and parallel, densely stained opposing membranes (between arrows) forming a synaptic gap that has a filamentous substructure. A contact with a presynaptic dense bar (double arrowhead) and surrounding synaptic vesicles but without specialization of the opposing membranes is also seen. Magnification in A, ×5,000; B, ×23,600; C, ×85,500; D, ×63,000. Scale bars: A, 5 µm; B, 1 µm; C and D, 0.25 µm.

In cross-section these muscle fibers were composed of myofibrils each bounded by tubules of sarcoplasmic reticulum (Fig. 2B), which did not appear as elaborate as in normal muscle. At points along the myofibril, close juxtapostion of sarcoplasmic reticulum and transverse tubules gave rise to well-defined diads. Thick filaments within the myofibrils were arranged in a fairly linear manner, and each was encircled by thin filaments that numbered from 8 to 10, in places where they could be clearly counted (Fig. 2C). The longitudinal arrangement of thick and thin filaments gave rise to distinct sarcomeres bounded by relatively thick, wavy Z-lines, enclosing A and I bands and measuring ~4 µm in length (Fig. 2A). Profiles of mitochondria were found scattered within the myofibrils (Fig. 2B), although large accumulations at the periphery of the fiber were not observed. Muscle nuclei were relatively large compared with the size of the fiber. Most of these features indicate that the muscle was newly formed.

The novel muscle fibers were innervated by small nerve terminals containing clear, spherical synaptic vesicles (Fig. 2D) indicative of excitatory axons (Atwood 1976). These nerve terminals made synaptic contacts with the muscle membrane adjacent to granular sarcoplasm, contacts that were defined by darkly stained presynaptic and postsynaptic membranes and presynaptic dense bars indicative of active zones (Pearce et al. 1986). Contacts were also seen that lacked the parallel alignment and intense staining of the paired opposing membranes but possessed the presynaptic dense bar surrounded by clear synaptic vesicles.

These novel muscle fibers appeared in the sheath surrounding the nerve, in areas possessing higher order branches of the sprouting nerve. Many of the higher order axon sprouts showed small, spherical vesicles clustered about a dense body (Fig. 3, A and B), reminiscent of a synaptic active zone. These contacts were often seen between axon sprouts (Fig. 3A) or between axon sprouts and glial tissue (Fig. 3B), and, as mentioned previously, between axon sprouts and muscle (Fig. 2D). We also occasionally found, adjacent to these muscle fibers, a proliferation of very small axon profiles lacking microtubules and filled with clear spherical synaptic vesicles and an occasional dense core vesicle (Fig. 3C). Dense bar contacts were prominent in this collection of very small axon profiles.


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FIG. 3. A and B: dense bar contacts in the form of clear spherical vesicles clustered about a dense bar (arrow) from an axon (a) sprout onto an adjacent axon sprout (A), or from an axon (a) sprout onto glial (g) tissue (B). C: area in a transplanted regenerating nerve between a large axon profile (Ia) and a muscle fiber (arrows) populated by many small axon profiles (a) filled with clear spherical vesicles and making dense bar contact (arrowheads) with other axon profiles. Magnification in A, ×34,800; B, ×90,000; C, ×33,600. Scale bars: A and C, 0.5 µm; B, 0.25 µm.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Muscle is not normally found in crustacean or insect nerves, although it does occur in molluscan nerves (Coggeshall 1967; Umitsu et al. 1987). What role if any these muscle fibers play in regenerating crayfish motor nerves is unknown, but receiving excitatory innervation as they do suggests that they are capable of contracting. Such contractions may serve to tighten the regenerating nerve and reduce the tendency for axonal sprouts to branch widely.

How do muscle fibers come to be located in regenerating crayfish nerves? One possibility is that they migrate from neighboring flexor muscles in the form of satellite cells that subsequently differentiate into muscle fibers. Such cells have been identified in crayfish limb extensor muscles (Novotová and Uhrík 1992) and in the supericial flexor muscles (unpublished observations), where they appear as spindle-shaped, mononuclear cells usually located between basal lamina and plasma membrane. They are also found in a similar location in vertebrate muscle where they give rise to new muscle during regeneration (Konisberg 1979), and may likely have the same function in crustacean muscle (Uhrík et al. 1989). There is the question of how satellite cells from the denervated muscle get transported to the nerve. In mammals, they are known to leave their ensheathing external lamina and migrate to areas of injury where they participate in regeneration of new fibers (Lipton and Schultz 1979). It is therefore possible that satellite cells from the denervated crayfish abdominal flexor muscles would migrate to the regenerating nerve and form new muscle fibers in this unusual location.

Another possible avenue for muscle to be located within regenerating nerves is if blood cells transformed into fibers. Blood cells in the form of amoebocytes have been shown to penetrate damaged areas of the limb extensor muscle in crayfish where they engulf degeneration products and subsequently transform, via degranulation, into muscle cells by producing small areas of contractile filaments (Uhrík et al. 1989). Blood cells are also known to transform into myoblasts in regenerating mammalian muscle after injury (Bateson et al. 1967). Moreover, during remodeling of the claw closer muscle in adult snapping shrimps, hemocytes appear to perform several roles; they phagocytose fast muscle fibers, mobilize protein stores in the form of crystalline bodies, and act as stem cells for genesis of slow muscle fibers (Govind and Pearce 1994). The pluripotent nature of hemocytes and their availability make them prime candidates as precursors of muscle fibers in regenerating crayfish nerves.

Whether muscle precursors are satellite cells or hemocytes, they must nevertheless be instructed, most likely by the nerve itself, to change their identity. Although the nature of such signaling is unknown, a potential site for signal transfer is offered by the dense bar contacts observed in the regenerating nerve; these contacts have the appropriate orientation from axon to target tissue be it either axons, glia, or muscle. Dense bar contacts between axon sprouts or between axon sprout and glia have been extensively described in regenerating crayfish limb motor axons (Nordlander and Singer 1976), where, interestingly enough, they were found in large numbers in the proximal rather than distal branches of the lesioned motor axon, and before the cut proximal end had reconnected to the distal segment. Similar dense bar contacts were also seen in developing motor axons to a crayfish limb muscle (unpublished observations), and hence they may be endemic to growing axons. These dense bar contacts may provide a means of communication between the motor axon and its surroundings and thus possibly act as a signaling device for the motor neuron. The ability of neurons to enhance the proliferation of myogenic cells in insect nerve-muscle cultures occurred only in regions of physical overlap between neurons and precursor muscle cells (Luedeman and Levine 1996). Such close range interaction may be provided by the dense bar contacts that we observed in the bundle of very small axon sprouts with each other, with glia, and especially with muscle.

Nerves are not essential for the embryonic development of insect larval muscle, but are necessary for the normal development of adult muscle. During metamorphosis from larva to adult in the moth, myoblasts are generated and migrate to appropriate regions in the leg but fail to proliferate and aggregate in the absence of its innervation showing the permissive role of motor nerves in muscle formation (Consoulas and Levine 1997). Because muscle does not normally develop in arthropod nerves, our observation of muscle fibers in regenerating crayfish nerves would suggest a possible instructive role for motor neurons in addition to their permissive role in myogenesis.

    ACKNOWLEDGEMENTS

  We thank R. Coulthard and two anonymous reviewers for criticism of the manuscript.

  This work was supported by the Natural Sciences and Engineering Council of Canada.

    FOOTNOTES

  Address for reprint requests: C. K. Govind, Life Sciences Division, University of Toronto at Scarborough, 1265 Military Trail, Scarborough, Ontario M1C 1A4, Canada.

  Received 14 July 1997; accepted in final form 12 August 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society




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