Regeneration of Phasic Motor Axons on a Crayfish Tonic Muscle: Neuron Specifies Synapses

Kristin M. Krause1, Joanne Pearce2, and C. K. Govind2

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

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Krause, Kristin M., Joanne Pearce, and C. K. Govina. Regeneration of phasic motor axons on a crayfish tonic muscle: neuron specifies synapses. J. Neurophysiol. 80: 994-997, 1998. Motor neurons are matched to their target muscles, often forming separate phasic and tonic systems as in the abdomen of crayfish where they are used for rapid escape and slow postural movements, respectively. To assess the role of motor neuron and muscle fiber in forming synapses we attempted a mismatch experiment by allotransplanting a phasic nerve attached to its ganglion to a denervated tonic muscle. Regenerating motor axons sprouted 10-30 branches (typical of phasic motor neurons, as tonic ones sprout far fewer branches) to reinnervate muscle fibers and form synapses that produced large excitatory postsynaptic potentials (typical of phasic motor neurons, as tonic synapses give small potentials). Therefore motor neurons, not muscle fibers, appear to specify one of the major properties of regenerating neuromuscular synapses.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Precise patterns of neural connections arise during development via interactive mechanisms for both pathway and target selection and for the refinement and remodeling of synaptic terminals (Goodman and Shatz 1993). Thus motor neurons find their correct target muscles with the use of molecular cues and form synaptic connections via inductive interactions between the nerve and muscle (Hall and Sanes 1993), generating highly characteristic neuromuscular systems. Two separate and very divergent neuromuscular systems, the phasic and tonic, occur in the crayfish abdomen where they bring about extension and flexion of the individual segments (Kennedy and Takeda 1965a,b). The phasic system, used only occasionally for escape, brings about rapid, powerful tail flipping, whereas the tonic system is used continuously for slow postural movements. The phasic motor neurons fire in brief bursts, generating large excitatory postsynaptic potentials (EPSPs), which give rise to twitch contraction of the muscle fibers. In contrast tonic motor neurons fire continuously, generating small EPSPs that result in gradual buildup of tension in the muscle fibers. Such divergence in neuromuscular systems provides an opportunity to probe the role of the neuron and its target muscle by wiring them in a mismatched manner, e.g., phasic neurons to tonic muscle. We were able to make precisely this mismatch in adult crayfish Procambarus clarkii and find that the regenerated neuromuscular connections generate large EPSPs with single impulses. Because initially large EPSPs are typical of phasic axons, our results suggest that this property of synapses is governed by the motor neuron.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Crayfish motor neurons have remarkable powers of regeneration, not only to their own muscle (Ely and Velez 1982) but when transplanted to a homologous muscle either in an adjacent segment (Worden et al. 1988) or in another animal (Krause and Velez 1995). Allotransplantation of motor axons provided an opportunity for testing whether the neuron or target muscle specifies the nature of regenerating synapses by transplanting a phasic nerve onto the tonic superficial flexor muscle. To permit allotransplanted phasic axons to innervate a tonic muscle, we began by first denervating the host superficial flexor muscle in the third abdominal segment of one side (Fig. 1). This is done by cutting the superificial flexor nerve of the third root proximal to the medial edge of the muscle field (Clement et al. 1983). The severed distal stumps of the tonic axons degenerate within 3 wk. The proximal stump is too short to reconnect to the superficial flexor muscle, and we did not observe regeneration of these severed tonic axons to the muscle. A day or two after the host tonic nerve is cut, the donor phasic nerve is transplanted into this segment. The transplant consists of the third and fourth abdominal ganglion with the connecting ventral nerve cord and part of the third root from the third ganglion that carries the motor axons to the phasic deep flexor muscles. The entire transplant was introduced into the abdominal cavity through an opening created by removing a swimmeret (Krause and Velez 1995). To guide the phasic nerve into position on the ventral face of the superificial flexor muscle, a human hair was pushed through a pinprick opening in the ventral cuticle and out through the swimmeret opening. Here it was attached to the phasic nerve with surgical thread and, by gently pulling the hair from the cuticle side, the nerve could be maneuvered into position near the denervated host muscle.


View larger version (48K):
[in this window]
[in a new window]
 
FIG. 1. Superficial flexor muscle in an abdominal segment of crayfish is normally innervated by tonic motor neurons whose axons travel in a thin branch of the 3rd root of the ganglion. Cutting this nerve denervates the muscle and allows the newly transplanted phasic branch of the 3rd root to regenerate and form new innervation. The phasic nerve is transplanted along with its 3rd ganglion as well as the 4th ganglion and the intervening nerve cord to increase the probability of reinnervation. The transplanted nerve regenerates onto the muscle dorsal surface where it is stimulated to test for reinnervation while recording intracellularly from muscle fibers.

Routine electrophysiological techniques were used for recording EPSPs from muscle fibers impaled with glass microelectrodes while the regenerated nerve was stimulated with a suction electrode (Krause and Velez 1995). Following these recordings the tissue was prepared for electron microscopy by techniques standard to our laboratory (Krause et al. 1996).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Regeneration of the transplanted nerve was examined after 8-10 wk and found to extend partway across the dorsal muscle surface, in the path of the original nerve but much larger (Fig. 1). We used electron microscopy to determine whether the regenerating nerve was phasic or tonic. Cross sections of the regenerated nerve provided striking evidence for sprouting in a phasic nerve. The nerve has between five and six complex axons (Fig. 2A), each defined by a thin lamellated glial sheath within which there is usually a single large axon profile and numerous smaller profiles. This is in stark contrast with the branches of the intact phasic nerve (Fig. 2B), which normally shows distinct single profiles, each surrounded by a glial sheath indicative of single motor neurons. The numerous smaller profiles of the complex axons of the regenerated nerve indicate sprouting of the axon similar to that found in crayfish limb motor neurons (Kennedy and Bittner 1974; Nordlander and Singer 1972). The phasic nature of the transplanted nerve is seen by the relatively large number of sprouts in the complex axons, from as few as 10 sprouts in the smaller axons to as many as 50 in the larger axons (Fig. 2A). Transplanted tonic nerves to the superficial flexor muscle also show evidence of sprouting in the form of complex axons (Fig. 2C) compared with the native tonic nerve (Fig. 2D), but are characterized by far fewer sprouts (between 4 and 8) compared with the transplanted phasic nerve. These same phasic motor neurons grown in culture typically sprout four to five times as many branches as their tonic counterparts (Arcaro and Lnenicka 1995), a difference most likely caused by differences in target area between phasic and tonic motor neurons. Another feature that suggests the phasic nature of the regenerating nerve is its relatively thin glial sheath (Fig. 2A), similar to that of the native phasic nerve (Fig. 2B) but very different from that of the regenerating or native tonic (Fig. 2, C and D) nerve where it is considerably thicker (Krause et al. 1996). Therefore the allotransplanted phasic nerve retains its character during regeneration on the superficial tonic muscle.


View larger version (148K):
[in this window]
[in a new window]
 
FIG. 2. Cross sections of a transplanted (A) and native (B) phasic nerve, the former showing 6 complex axons (black-down-triangle ), each with a single large axon profile and several smaller profiles indicative of sprouting of main axon and the latter showing 5 single axon profiles (1 large and 4 medium) denoting 5 motor neurons. Cross sections of the transplanted (C) and native (D) tonic nerve, the former with 5 complex axon profiles (black-down-triangle ) and the latter with 6 single axon profiles (1 large, three medium, and two small). Bar 25 µm. Magnification A: ×600; B: ×380; C and D: ×900.

The transplanted ganglia also showed structural integrity, as electron microscopy revealed nucleated cell bodies, characteristic of motor neurons from intact ganglia, blood vessels and lacunae indicative of vascularization, and healthy looking neuropil with dendritic profiles with synaptic contacts and clear and dense vesicles, similar to our previous findings with tonic transplants (Krause et al. 1996). Within the muscle there were well-formed motor nerve terminals that were populated with clear, round synaptic vesicles characteristic of excitatory axons and that contained synaptic contacts with presynaptic dense bars or active zones. The muscle fibers retained their tonic identity judging from the high thin-to-thick myofilament ratio (8-12 thin filaments surrounding a single thick filament) and relatively long (>6 µm) sarcomere length. Thus electron microscopic examination suggests that transplanted motor neurons remain alive, regenerate their axons via sprouting to the host muscle, and form neuromuscular synapses.

The nature of the synaptic connections formed by the regenerating phasic nerve was examined with electrophysiology. Electrical stimulation of the regenerated nerve gave rise to EPSPs in the impaled muscle fibers, demonstrating functional innervation by the transplanted nerve. Eight out of 10 transplants showed EPSPs with stimulation of the regenerated nerve (Fig. 3, A and B). Clearly, muscle reinnervation by the transplanted nerve was highly successful. Moreover, reinnervation was fairly comprehensive, as the majority of fibers tested showed an EPSP. Thus out of 63 fibers tested in 8 preparations only 4 lacked an EPSP, i.e., 6%, and these were found in only 2 preparations.


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 3. A and B: excitatory postsynaptic potentials (EPSPs) recorded from tonic superficial flexor muscle fibers in response to stimulation of the regenerated phasic nerve. Increasing stimulus intensity evokes three successive sizes of EPSPs (1, 2, and 3), denoting innervation by three axons; the 3rd one in A is 25 mV, whereas the 3rd one in B gave an attenuated active response (arrow) that caused contraction of the muscle fiber, dislodging the recording electrode (double arrow). C: comparison of amplitude distribution of EPSPs in the tonic superficial flexor muscle fibers from transplanted tonic (open bars: 103 EPSPs from 7 preparations) and transplanted phasic (filled bars: 102 EPSPs from 6 preparations) nerves. Allotransplantation of the phasic nerve is that described in the text, whereas a second set of animals was used for allotransplantation of the tonic nerve. Bars = 5 mV, 1 ms.

The tested fibers were innervated usually by two to three axons, judging from increments in EPSP amplitude elicited by increasing stimulus intensity to the regenerated nerve (Fig. 3, A and B). This was the case for six of the seven preparations; a single preparation showed innervation by a lone excitatory axon, and we were not able to detect innervation by an inhibitory axon in any of the preparations. Innervation of these tonic muscle fibers by two to three transplanted excitatory axons is comparable with normal innervation of these muscles (Kennedy and Takeda 1965b). EPSPs generated by these transplanted axons showed a wide range in amplitude, from 0.5 to 25 mV, although >60% were >5 mV, and >35% were >10 mV (Fig. 3C). In contrast, innervation of the superficial flexor muscle by allotransplanted tonic axons gave considerably smaller EPSPs, ranging from <0.1 to 9 mV (Fig. 3C) (Velez and Wyman 1978). The average amplitude of the regenerated phasic EPSP was 8.01 ± 7.08 (SD) mV (n = 102) compared with the regenerated tonic EPSP, which was 2.3 ± 1.41 mV (n = 103). Thus EPSPs were significantly larger (P < 0.0005, Student's t-test) from the transplanted phasic axons than those from the transplanted tonic axons. Because initially large EPSPs are typical of the native phasic axons to the deep flexor muscle (Kennedy and Takeda 1965a), our findings strongly indicate that the transplanted phasic axons regenerated phasic synapses on a tonic muscle.

Convincing proof of the phasic nature of these synaptic connections was that these EPSPs were large enough, 20-25 mV (Fig. 3A), to trigger an active response (Fig. 3B), which is the equivalent of an action potential in crustacean muscle fibers (Atwood 1976). These active responses also led to a brief (twitch) contraction in a few muscle fibers. Active responses are not normally triggered by the small tonic EPSP, although they may occasionally be generated by firing of the largest of the tonic axons (Evoy et al. 1967), nor is twitch contraction of the muscle fibers seen in the superficial flexor muscles; both active responses and twitch contraction characterize the normal phasic deep flexor muscle (Kennedy and Takeda 1965a). The electrophysiological evidence suggests that the transplanted phasic nerve regenerates phasic-type synapses on a tonic muscle.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Phasic motor nerve teminals generate large EPSPs with a single stimulus, but these depress with repetitive stimuli, whereas tonic terminals have an initially small EPSPs, which facilitate with repetitive stimuli (Atwood 1976). These two properties characterize differences between phasic and tonic synapses (Lnenicka 1991). Our experiment of mismatching a phasic nerve to a tonic muscle in crayfish shows that the transplanted phasic motor neuron regenerates nerve terminals that give rise to large EPSPs with a single stimulus. Thus for the initial release of transmitter, our results suggest that synapse specification is governed largely by the neuron, not the muscle.

However, our findings do not rule out the possibility that the target muscle may influence synapse specificity during primary development when motor neurons are first forming, and there is considerable circumstantial evidence for this view for both central and peripheral synapses. For peripheral synapses, single motor neurons to crustacean limb muscles give rise to motor nerve terminals that vary in the degree of facilitation among individual muscle fibers (Atwood 1976; Atwood and Wojtowicz 1986), or a single motor neuron gives rise to synapses that facilitate on one muscle and depress on another muscle (Katz et al. 1993). For central synapses in the cricket, single cercal sensory neurons generate facilitating synapses on one interneuron and depressing synapses on another (Davis and Murphey 1993). In the leech, single sensory neurons express different levels of facilitation on different target motor neurons (Muller and Nichol 1974). In the cat, single Ia afferents make facilitatory synapses on some target cells and depressing synapses on other target cells (Koerber and Mendell 1991). Therefore single neurons generate synapses that vary in their presynaptic properties of facilitation and depression among different regions of the target or on different targets, implying that the target influences the expression of these synaptic properties. Such retrograde influence may involve differential expression of the genomic blueprint of the motor neuron via activity-dependent factors (Goodman and Shatz 1993).

Activity-dependent factors may influence the development of synaptic properties of single motor neurons. For example, prolonged stimulation of the phasic motor neuron to the claw closer muscle in juvenile crayfish converts its synaptic connections toward those of the tonic motor neuron, although the conversion is incomplete (Lnenicka 1991; Lnencika et al. 1986, 1991). This partial conversion suggests that refinement of synaptic properties within the genomic blueprint of the phasic motor neuron is possible but that switching to the tonic genome is improbable. Phasic and tonic neuromuscular connections remain distinct even when they occur on single muscle fibers in the crayfish limb extensor muscle (Bradacs et al. 1997; King et al. 1996), where the target is unlikely to differentially influence their character; rather their distinctiveness must reside in the motor neurons.

In sum, deductions concerning the role of the neuron and its target in specifying synapses are based largely on circumstantial evidence. Our attempts at transplanting a phasic nerve onto a tonic muscle provides one of the first direct tests of the interaction between these two elements and shows that one of the major properties of the synapse, the initial release of transmitter, is defined by the motor neuron during regeneration in adult crayfish.

    ACKNOWLEDGEMENTS

  We thank H. L. Atwood, M. P. Charlton, and two anonymous reviewers for critiquing of the manuscript and S. J. Velez for introducing the experimental preparation.

  Financial support was provided by Natural Sciences and Engineering Research Council of Canada to C. K. Govind.

    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 12 January 1998; accepted in final form 28 April 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Krause, K. M.
Articles by Govind, C. K.
Articles citing this Article
PubMed
PubMed Citation
Articles by Krause, K. M.
Articles by Govind, C. K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online