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Axon reflexes in axolotl limbs: Evidence that branched motor axons reinnervate muscles selectively
EXPERIMENTAL
NEUROLOGY
64, 174- 189 (1979)
Axon Reflexes in Axolotl Limbs: Evidence that Branched Motor Axons Reinnervate Muscles Selectively ROBERT Research
Laboratory
of Electronics,
S.
STEPHENSON’
Massachusetts Institute Massachusetts 02139
Received
October
of Technology,
Cambridge,
12. 1978
Numerous axon reflexes were found in grafted forelimbs, regenerated forelimbs, and forelimbs with regenerated nerves in the axolotlAmbys?oma mexicanurn. They were not seen in normal limbs. Each reflex was due to a single motor axon which branched, generally at a point central to the limb. In most cases several consecutive electrical stimuli were necessary to cause a visible contraction or a muscle action potential in the motor units concerned. I observed a total of 13 axon reflexes between the supernumerary limb and the ipsilateral host limb. All but one could be stimulated in either direction. In other animals I demonstrated 13 axon reflexes by cutting a small nerve in the distal limb and stimulating its proximal stump. In at least five, and possibly as many as 14 of these 26 axon reflexes, the two muscles involved were synonymous. In 20 of the 26, the muscles involved were situated in the same limb region and were synergistic or synonymous. Only in five axon reflexes were the muscles in widely different parts of the limb and clearly unrelated in function. Random innervation or mechanical guidance alone cannot account for these muscle-specific axon reflexes. Axon branches innervated muscles selectively, although the mechanism remains unclear. The selectivity was not perfect, though, because axon reflexes between dissimilar muscles did occur occasionally. Although it has been proposed that an axon may remain in an inappropriate muscle but be functionally “repressed,” no evidence of such repressed synapses was found. Abbreviations: AR-axon reflex, SNL-supernumerary limb, HL-host limb, MAPmuscle action potential. ’ Supported in part by NIH grants 5TOlGM0155 and EY07008 and by a grant from the Bell Telephone Co. The author is indebted to Dr. J. Y. Lettvin for his advice and support, to Dr. V. R. Stirling for participating in one of the experiments, and to Drs. P. A. Weiss, W. L. Pak, J. R. Vanable, and J. E. Mittenthal for their valuable comments. I also thank R. Graham, J. Cook, M. Ben Chakri, W. Boyle, L. Winchester, and Drs. R. R. Humphrey, P. Modell, and C. Dalton. The present address of the author is Department of Biological Sciences, Purdue University, West Lafayette, IN 47906. 174 0014-4886/79/040174-16$02.00/O Copyright All rights
Q 1979 by Academic Press, Inc. of reproduction in any form reserved
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INTRODUCTION The nervous system of amphibia and fishes, unlike that of higher vertebrates, has an extraordinary capacity to regenerate itself with return of function (7, 20). In the motor system of lower vertebrates, where this regenerative ability has been much studied, it is not clear to what extent functional recovery after interruption of peripheral nerves depends on the restoration of specific motoneuron-muscle connections. Much of the evidence that it does not comes from the comprehensive experiments of Weiss concerning the “homologous response” of a supernumerary limb (SNL) transplanted to the limb region of a salamander (25, 29-32). The SNL, when innervated, appears to duplicate muscle for muscle the movements of the host limb (HL) beside it (24, 26, 29), although the precision of this duplication has been questioned (2). Weiss concluded that the homologous response depended not on any morphological selectivity in the reinnervation of the transplant (30), but rather on a readjustment or “modulation” occurring more centrally (28). On the other hand, experiments involving reinnervation of the external eye muscles (4, IS, 21) and fin muscles (13) of fish, as well as the reinnervation of frog (3) and salamander (1, 8, 33) limb muscles, suggest that a muscle is preferentially innervated by its original nerve, and foreign nerve fibers are driven out or repressed. Similar experiments on fish yielded conflicting results, however (6, 18). Evidence for specificity at a grosser level is that the distribution pattern of the forelimb nerves is normal only when the limb is innervated by the normal segments of the spinal cord, i.e., the third, fourth, and fifth (17). The apparent inconsistency of these different studies has not been explained. Another experimental approach to the question of selective regrowth of peripheral nerves is to consider the fate of branched axons. It was found that the addition of a SNL to the flank of an amphibian does not cause a significant increase in the number of nerve fibers leaving the spinal cord (27, 30). On the other hand, those nerve fibers branch as they grow peripherally so that each limb receives approximately the normal number of fibers, in proportion to its size (12,27,30). Thus the total number of limb nerve fibers is greater when counted at the bases of the HL and SNL than at their exit from the spinal cord. If the outgrowth of the branched motor fibers is unspecific, as Weiss suggested (28, 30), then the majority of these should innervate two or more unrelated muscles in the periphery. When a branched nerve fiber innervates two different muscles A and B, stimulating muscle A electrically should evoke a contraction in B and vice versa, even after removal of the spinal cord. Such a contraction, which was termed an axon reflex (AR) (lo), provides a test of Weiss’s claim of unspecific
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innervation. In examining his grafted animals, Weiss (30) found only three ARs, apparently between dissimilar muscles, but it was unclear why he didn’t see more. The present study will show that ARs are common in axolotls with regenerated motor nerves, and that ARs between unrelated muscles are exceptional. METHODS Supernumerary forelimbs were grafted onto the shoulder region of axolotls (Ambystoma mexicanum) 3 to 5 months old, according to the procedure of Weiss (29). The fourth or fifth spinal nerve was cut at the time of the grafting, and this provided the major nerve supply to the graft, as shown by stimulation. Ten such grafts were successful, and the SNL moved homologously with the HL in all cases. In other animals I simply amputated a forelimb and allowed it to regenerate. After 3 to 22 months’ recovery, the animal was anesthetized briefly with tricaine methanesulfonate, the medulla was transected, and the animal was decerebrated. 1 exposed the brachial spinal cord by laminectomy, transected all brachial dorsal roots, and skinned the HL and SNL. The preparation was maintained at about 15°C and 100% relative humidity. ARs were evoked electrically with a small bipolar electrode (23)) using trains of five pulses 25 ms apart, repeated once a second. This type of electrode was also used for recording the muscle action potentials (MAPS). To locate ARs I stimulated one forelimb and looked with a dissecting microscope for contraction in a more central muscle or in the other, ipsilateral forelimb. Then I stimulated the muscle which had contracted reflexly and watched for a contraction in the region originally stimulated. By repeatedly stimulating in one direction and then in the other with diminishing stimulus intensity it was possible to isolate each AR present and identify the most sensitive region for stimulating it in each direction. RESULTS Properties of Axon Reflexes. The reflexes so obtained were ARs because (i) they were entirely peripheral, unaffected by ventral root transection and removal of the spinal cord; (ii) they were bidirectional, i.e., they could be stimulated in both directions; (iii) they were persistent, lasting in general as long as the circulation was functional6 days in several cases; and (iv) they disappeared when I cut the limb nerves at a level central to the two muscles involved. The site of the axonal bifurcation-as determined by sectioning more and more distally until the reflex disappeared-was usually central to the SNL and HL and near the region where the brachial nerves had been cut or damaged during grafting or amputation. This is
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consistent with other studies on regenerated nerve (9, 19, 30). Each AR, moreover, involved only one branched axon because (i) as the stimulus intensity increased or decreased continuously the reflex appeared or disappeared in all-or-none fashion; (ii) only one MAP was present and arose from a single end-plate region- usually corresponding to the most sensitive spot for stimulating the reflex-as shown by displacing the recording electrode along the muscle surface (23); finally, (iii) the reflex contraction occurred only in a small group of contiguous muscle fibers. All ARs seemed to involve only two muscles, although one might expect to find axons with three or more peripheral branches as well. This may have been because I grafted only one SNL on each animal and thus did not overload the limb nerve supply excessively. In 80% of the ARs studied, a single stimulus pulse caused neither MAP nor visible contraction. The response facilitated, and when several stimuli followed each other at a 25ms interval, the second and subsequent stimuli evoked MAPS of progressively shorter latency (23). On two occasions I was able to record directly from the branched axon with suction electrodes, and it conducted impulses without failure to a rate of about 60/s with no significant change in latency. Thus, the failure and facilitation of the MAPS seem to be properties of axolotl end-plates, as reported for those of Triturus (1 I). ARs were numerous in grafted limbs, regenerated limbs, and limbs whose nerves had been cut and allowed to regenerate; but none was present in 10 normal limbs examined. Stimulation as described above typically revealed several ARs between different muscles in a regenerated or grafted limb. This procedure could not demonstrate any axon reflexes operating between two parts of a single muscle or between two adjacent muscles, however, because of the difficulty of distinguishing direct from reflex stimulation. To decide how often the latter type of AR occurred it was necessary to look for ARs operating between the HL and SNL. These were present in small numbers in half the grafted animals examined. Such a case is shown schematically in Fig. 1. A study of such interlimb ARs showed that the proportion between synonymous or synergistic muscles was greater than could be due to chance alone. Interlimb Axon Reflexes. I observed a total of 13 ARs between HL and SNL in four different animals. These are shown schematically in Fig. 2. For each reflex the motor units involved are indicated in black on a map of the skeleton: HL at left, SNL at right, dorsal view above, ventral view below. For convenience I will refer hereafter to the group of muscle fibers innervated by one branch of an axon as a motor unit, although strictly speaking they constitute only part of it. The size of the motor units, which in some cases involved as few as six to eight muscle fibers, has been exaggerated for clarity. In the case of AR D4-1, synonymous muscles were
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FIG. 1. A schematic drawing of an axon branching to both the host limb (lower) and the supernumerary limb (upper), giving rise to an interlimb axon reflex.
involved in the HL and SNL: the anconaeus scapular-is mediali+ which is a dorsal division of the extensor of the elbow. Figure 3A shows the response of this axon reflex recorded in the host limb when the SNL was stimulated. The reverse is shown in B. In C a rootlet of the fifth ventral root was stimulated while the recording electrode was placed over the unit in the HL. This excited the same characteristic MAP as in A as well as an earlier, unrelated one. The fifth spinal nerve had been cut at the time the SNL was grafted, and when this nerve was crushed in the vertebral foramen at the conclusion of the experiment the AR disappeared. Trace D was recorded under the same conditions as A but 5 days later, demonstrating the persistence of the ARs. The axon reflex D4-2 in the same animal ran from the anconaeus * I adopt the terminology of Francis (5) from Salamandra salumandra. For its applicability to the axolotl, as well as systematic description of the forelimb muscles in the latter species see
(22).
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coracoideus (a ventral division of the elbow extensor) in the HL to a unit on the ventral side of the anconaeus in the SNL. In both this and the preceding AR the motor units were too weak to produce any gross movement of the limb. It was clear, nevertheless, exactly where the contracting unit lay because (i) it produced a local distortion of the anconaeus, (ii) at 25x I could see a slippage of the active muscle fibers relative to their inactive neighbors, and (iii) the MAP was localized at the spot. A movie showing these two ARs is available from the author. AR D7-1 caused a very slight flexion of digits III and IV (counting from the radial or medial side of the forearm) of the HL when the SNL was stimulated, and when the HL was stimulated, digits I and II of the SNL flexed. The unit in the HL inserted on the overlying palmar tendon, which in turn inserts on the phalanges of each digit, The unit in the SNL inserted on the metacarpals of digits I and II. Because the origins of these units were concealed beneath the palmar tendon, it was not possible to tell which of several small finger or wrist flexors were involved, and therefore I have marked their location on Fig. 2 with an open outline. In animal D8 there were six different ARs between the HL and SNL, all but one involving hand or forearm flexors in both limbs. D8-1 involved a deep flexor of digit II of the HL, and in the SNL the flexor brevis superficialis of digit II. A deep flexor of digit I of the SNL also contracted intermittently when this AR was stimulated. D8-2 involved a unit on the ulnar side of the flexor primordialis communis (the common flexor of the digits and wrist, inserted on the palmar tendon) in the HL, and a wrist flexor on the ulnar side of the SNL forearm. AR D8-3 was between the flexor accessorius lateralis of the HL and the flexor antebrachii et carpi ulnaris of the SNL. The former unit arose from the end of the ulna and inserted on the palmar tendon. The latter arose from the distal end of the humerus and inserted on the ulnar carpus, just distal to the origin of the former. In the case of this and some of the following ARs it was necessary to dissect away the flexor primordialis to expose the active units. D8-4 was a counterpart of D8-3, but situated more toward the radial side of both limbs. In the HL the flexor accessorius medialis contracted, and in the SNL the flexor antebrachii et carpi radialis contracted. Once again, the latter unit inserted just beneath the former on the radial carpus. Reflex DS-5 involved the flexor brevis superficialis of digit IV of the HL, and a flexor of the SNL wrist. Reflex D8-6 was between a central division of the extensor of the HL elbow and an unseen deep muscle on the flexor side of the SNL forearm. The latter produced a slight twisting of the SNL forearm. The remaining four interlimb ARs occurred in animal K7, which had both a transplanted limb and an extra, regenerated limb. I examined K7 only 3 months after grafting, whereas the other animals had a much longer
HOST
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TRANSPLANT
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FIG. 3. Muscle action potentials due to axon reflex D4-1. Each trace is a superposition of the responses to several consecutive stimuli (indicated by the rectangle beneath each trace) 25 ms apart. A-the recording electrode was positioned over the anconaeus scapularis medialis (a division of the elbow extensor) in the host limb, and the anconaeus of the supernumerary limb was stimulated. B-the reverse; the recording electrode was over the anconaeus scapularis medialis of the supernumerary limb, and the stimulus was delivered to the anconaeus of the host limb. The elevation at the start ofthis trace is an unusually large stimulus artifact. C-the recording electrode was as in A, but a rootlet of the ipsilateral fifth ventral root was stimulated with a suction electrode. This rootiet contained the axons of two units in the anconaeus scapularis medialis, but note that the later muscle action potential has the same characteristic shape as, and a slightly smaller latency than that in A. D-same as A, but recorded 5 days later. Differences in latency between A and B or A and D are due to differences in electrode position. The bars at the lower right represent 50 PV and 2 ms and apply to all traces.
recovery before examination. Except for the small size of his limbs the results were the same. In the HL, K7-1 involved a caudal part of the pectoralis (a shoulder muscle which depresses and retracts the humerus), and in the transplant a rudimentary muscle on the coracoid portion of the shoulder girdle, I was unable to identify the latter muscle as it appeared to be attached to the coracoid only and was without effect on the limb. It was probably a shoulder muscle whose other attachment had been severed at the time of grafting. It had roughly the same position relative to the transplanted limb that the unit in the pectoralis had to the HL, but it should
FIG. 2. (opposite) Maps showing location of motor units relative to the forelimb skeleton for each interlimb axon reflex. Each rectangle represents one axon reflex, with the host limb at left and the supernumerary limb at right. Dorsal and ventral views of the limb are shown above and below the horizontal midline, respectively. Extensors of the forearm and hand appear on the upper outline, flexors on the lower. For clarity each darkened area is larger than the motor unit it represents and corresponds in most cases to the entire muscle involved. Where the origin or insertion of a motor unit was unknown an open outline marks the unit’s approximate location. When a motor unit is inserted on the palmar tendon the latter is indicated by stippling. Projection onto a standard outline required distortion in some cases. The regenerated limb of K7 was so abnormal as to be represented individually. Only its dorsal side was visible. Dimensions of the host limb are typically 15 to 20 mm elbow to fingertip; the supernumerary limb was two to three times smaller.
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be noted that although the pectoralis overlies the coracoid it is not attached to it. Reflex K7-3 involved the flexor primordialis communis of the HL, and in the transplant a muscle which ran the length of the forearm and flexed the digits. I could not see the latter unit because the digits of the regenerated limb overlay the forearm of the transplant. Its position and action suggest strongly that it, too, was the flexor primordialis communis. K7-5 involved the anconaeus, the extensor of the elbow, in both HL and transplant. In the transplant, however, the tendon of insertion of the extensor had slipped off the elbow, reversing the action of this muscle and causing the elbow to assume a sharp angle. This happened to the SNLs of several animals. This AR could not be stimulated in the transplant because of the overlying regenerated limb. The last AR, K7-2, was between the HL and the regenerated limb, which was only partially developed as shown in Fig. 2. The unit in the HL was an extensor on the radial side of the forearm. The unit in the regenerate arose from the middle of the humerus and inserted on the distal end of the radius, toward the extensor side. In spite of its rudimentary appearance it was most probably part of the extensor antebrachii et carpi radialis, which in a mature arm arises from the end of the humerus and inserts on the distal end of the radius, as well as on the ulnar carpus. In conclusion, of these 13 interlimb ARs, four--4-1, D4-2, Da-l, and K7-S-involved synonymous muscles in both limbs, and in two additional cases, K7-2 and K7-3, this is likely but not certain. I treat as synonymous all the divisions of the anconaeus, and similarly all the small flexors of a particular digit. With these two conventions, 21 different muscles may be distinguished in the axolotl forearm, and seven different muscles in the shoulder. But there is even more pattern to the distribution of the ARs than this alone would indicate. If one divides all limb muscles into two general categories, flexor-retractors and extensor-protractors, 11 of the ARs respected this partition, and only one, D8-6, involved muscles of both types. The case of K7-2 was unclear. If one makes a dichotomy at right angles to the preceding division, between ventral and dorsal in the shoulder and upper arm, and between radial and ulnar in the forearm, then eight ARs respected this new partition, and only one, D7-1, clearly did not. Cases D8-5, D8-6, K7-3, and K7-5 were unclear. Finally, if one divides limb muscles along a proximal-distal axis into shoulder, upper arm, forearmwrist, and finger categories; nine ARs were confined to muscles of a single category. Two, D8-5 and D8-6, confused two adjacent categories. The cases of D7-1 and K7-2 were unclear. Taken together, the preceding divisions categorized the limb musculature into 16 compartments. Seven of the ARs (D4-1, D4-2, Da-l, D8-2, D8-3, D8-4, and K7-1) involved two motor units within a single such compartment. ARs D7-1, DS-5, and Da-6 did not respect the compartmental boundaries, and the three remaining
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cases were uncertain. Only one AR, D8-6, linked muscles differing widely in location and function. Intralimb Axon Reflexes. The preceding results indicate that, in the case of motor axons branching to both the HL and the SNL, the two branches have a marked tendency to innervate similar, if not synonymous, muscles. Presumably the same is true of branches regenerating to the same limb. This presumption was tested in the following way. I exposed the forearm flexor or extensor nerves by removing the flexor primordialis communis or extensor digitorum communis, respectively, after having skinned the limb and removed the spinal cord. A nerve trunk that supplied only one or two muscles was dissected free for a few millimeters and cut. While stimulating the proximal stump with a suction electrode I examined the limb for ARs. Then I reversed the suction pipet and stimulated the distal stump to see which muscles were innervated by that nerve. This is shown schematically in Fig. 4, where the cross marks the point at which the nerve was cut. This technique could detect ARs between two parts of a single muscle, providing only that the two branches of the axon in question reached the muscle via different nerve trunks. Almost every nerve trunk cut yielded at least one apparent AR, except on the exterior surface of the muscles where most nerves are purely sensory. To rule out the possibility that an apparent AR might really be due to local stimulus spread or to fibers growing centripetally in the nerve, I tied a fine silk thread at or slightly below the shoulder and tightened it until it cut to the bone. If this abolished the contraction when the proximal stump was stimulated, and if stimulating the distal stump showed that the muscle had not failed due to ischemia (this never proved to be the case), the proximal contraction was accepted as a genuine AR. Thus, in Fig. 4 a ligature at the level of the solid line would eliminate the AR. This procedure revealed 13 ARs in five different limbs, shown in Fig. 5. In case E5-A a small extensor nerve, the dorsalis manus intermedius, was cut in the middle of a regenerated forearm. Stimulating the distal stump caused an MAP in the adductor digiti I, shown in Figs. 6A and C. Stimulating the proximal stump caused an AR in the same muscle, Fig. 6B, which disappeared, Fig. 6D, when the arm was ligated at the shoulder. In case E5-B I cut the ulnar nerve just distal to the elbow in the other, regenerated forelimb of the same animal. Stimulating the distal end excited the flexors of digits III, II, and possibly I. Stimulating the proximal end caused ARs in a flexor of digit II, flexor of digit III, in the flexor accessorius lateralis (a flexor of digits II and III), and in the humero-antebrachialis (flexor of the elbow). These were four distinct ARs as they were separable by threshold. In case K3-B the interosseus nerve was cut near its origin just proximal to the elbow in a SNL. Distal stimulation excited the flexor antebrachii et carpi radialis and extensor antebrachii et carpi radialis in the
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FIG. 4. A schematic drawing of an intralimb axon reflex. The nerve trunk is transected at the “X.” Stimulating the proximal stump will cause an axon reflex in the more distal muscle. A tight ligature about the upper arm at the level of the solid line will eliminate this reflex, although ligation at a more proximal level (dotted lines) is without effect. The bipolar electrode is drawn to correct scale over the active muscle.
forearm, whereas proximal stimulation caused ARs in the anconaeus (elbow extensor) and in a trunk muscle. In case K3-D I cut the ulnar nerve of a regenerated limb distal to the elbow. When the distal stump was stimulated the flexor accessorius lateralis contracted. Proximal stimulation gave ARs in the closely related flexor accessorius medialis and in the more proximal flexor antebrachii et carpi radialis. In case KS-A the ulnar nerve of a SNL was cut just distal to the elbow. Stimulating the distal stump caused the flexor antebrachii et carpi ulnaris and flexor antebrachii et cat-pi radialis to contract. Proximal stimulation caused ARs in the former muscle, as well as in the pronator profundus and the anconaeus. There were at least two distinct ARs in the flexor antebrachii et carpi ulnaris which were separable by threshold. Of the 13 ARs elicited by stimulating the proximal stump of a cut nerve,
K5-A
DIRECT
K3-D
REFLEX E5-B
AXON
E5-A
AXON
REFLEX DIRECT K3-B
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REFLEX
FIG. 5. Axon reflexes within single limbs, demonstrated by stimulating forearm nerves with a suction electrode. Within each rectangle the same limb is shown twice. On left-those muscles excited by direct (i.e., orthodromic) stimulation applied to the distal nerve stump. On rightmuscles excited via an axon reflex when the proximal stump was stimulated. The location of the stimulating pipet is shown schematically. Dorsal and ventral views as in Fig. 1.
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FIG. 6. Muscle action potentials recorded from the adductor digiti I in case E5-A. Astimulating the distal stump of the cut nerve trunk shows that it innervates many units in the adductor digiti I. B-stimulating the proximal stump of the same nerve trunk produces an axon reflex in the adductor digiti I. The arm was then ligated tightly at the shoulder level and traces C and D repeat A and B, respectively, showing that the axon reflex was abolished by this ligation (D) whereas the response of the muscle to orthodromic stimulation (C) was unaffected. As in Fig. 3, each trace is a superposition of responses to five consecutive stimuli (indicated by the rectangle beneath each trace) 20 ms apart. The calibration bars represent 20 PV and 2 ms.
seven occurred in the same muscle or muscles innervated by the distal stump (where I consider the flexor accessorius medialis and lateralis as divisions of a single muscle), whereas only four occurred in muscles remote from those innervated by the distal stump. These results, like those of the previous section, give clear evidence of selective innervation. The surgery in these cases was done when the animals were 5 to 21 months old, and they were examined for ARs about 6 months later. These results were in marked contrast to the case of an animal whose limb nerves were cut at 35 months, and examined 5 months later. In the two limbs of this animal I cut a total of five nerves and found eight ARs, none of which involved two parts of a single muscle and seven of which were between muscles widely separated in the limb. It is tempting to speculate that the crucial difference between this case and the others was the age of the animal at the time of surgery, because it is well known that the ability of amphibia to recover function after nerve section declines with age. DISCUSSION As mentioned above, Weiss (30) found a total of three ARs and concluded that they operated between different muscles. Because he did not succeed in stimulating these ARs in both directions and identified the muscles involved on the basis of their actions alone, this conclusion may have been in error. In any case, in the light of the results presented above, it is likely that he observed only a small fraction of the ARs actually present, as he himself surmised (30). It may be relevant to recall here that the majority of the ARs studied here gave no visible response to a single
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stimulus. Weiss concluded that many motor axons were connected to dissimilar muscles, and this posed a serious paradox for his modulation theory (28,30). On the other hand, the present results show clearly that this happens only exceptionally, and so, in salamanders, nerve branching does not present the obstacle to coordination that it does in mammals (9, 20). Mechanical factors alone are clearly insufficient to account for the selectivity demonstrated here. In the case of axons going to both HL and SNL, the branches grew in different directions for a centimeter or more before innervating similar muscles. The SNLs, moreover, were oriented in different directions in different animals with no obvious effect on the selectivity. The results presented here can be explained only by some form of selective affinity between nerve branches and muscles, but the nature of this selectivity is not clear. On the one hand it is possible that the first branch innervates any muscle unselectively, but subsequent branches of the same axon seek out muscles of similar specificity. Alternatively, it is possible that axons which establish themselves on muscles of differing specificity tend to degenerate and be replaced. Although such theories can explain the muscle-specific ARs described above, they cannot explain the homologous response of the SNL. This is because branches between the HL and SNL were rare and were completely absent in two animals whose limbs moved homologously nevertheless. Branching within the spinal cord was excluded also as there were no ARs present in these cases even with the ventral roots and spinal cord intact. The alternative explanation of the selective ARs described above is that each motoneuron is specified ab initio to innervate a particular muscle. This may be the case, for example, in the cockroach (16). Such a theory could account for the homologous response as well. Weiss’s principal argument against such selectivity was that he observed ARs only between dissimilar muscles, but this is invalidated by the present results. Other evidence in support of this hypothesis was cited in the introduction. The modulation theory (28), on the other hand, rests now on two crucial experiments: the homologous movement of SNLs innervated by distal flexor nerves taken from the HL (30) and the homologous response of single muscles innervated by various foreign nerves (26). These experiments deserve to be repeated. Mark et al. (14) proposed that motor axons innervate muscles competitively on the basis of preestablished affinities, and that a synapse between a muscle fiber and the “wrong” axon is “repressed” when the “right” axon arrives. “Repressed” synapses are enduring and morphologically normal, but ineffective. There have been reports that such synaptic repression occurs in axolotl limb muscles (1, 33), although its occurrence has been disputed in other animals (4,6, 18). Such a theory implies that, after nerve regeneration, a great many ineffective axon branches remain in
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“wrong” muscles. It predicts that (i) many ARs (those between dissimilar muscles) should operate in one direction only, and (ii) stimulating the proximal stump of a cut nerve- which would excite both effective and ineffective axon branches -should produce ARs distributed randomly throughout the limb. Because the present results do not support either prediction, repressed synapses, if present at all, must last less than the recovery period (5 months or more) between nerve transection and examination or else be very rare. These experiments show that axon branches innervate muscles selectively in the axolotl. They do not make it clear, however, whether this selectivity results from a preestablished affinity between a given motoneuron and a given muscle, or is the result of an interaction between the axon’s branches at the time they reinnervate the muscles. They show that the selectivity is not perfect, however, because ARs between dissimilar muscles do occur occasionally. REFERENCES I. CASS, D. T., T. J. SUTTON, AND R. F. MARK. 1973. Competition between nerves for functional connections with axolotl muscles. Nature (London) 243: 201-203. 2. CZI?H, G., AND G. SZBKELY. 1971. Muscle activities recorded simultaneously from normal and supernumerary forelimbs in Ambystoma. Acta Physiol. Acad. Sci. Hung. 40: 287-301. 3. ELUL, R., R. MILEDI, AND E. STEFANI. 1970. Neural control of contracture in slow muscle fibers of the frog. Acta Physiok Lat. Am. 20: 194-226. 4. FANGBONER, R. F., AND J. W. VANABLE. 1974. Formation and regression of inappropriate nerve sprouts during trochlear nerve regeneration inXenopus laevis. J. Comp. Neural. 157: 391-406. 5. FRANCIS, E. T. B. 1934. The Anatomy ofthe Salamander. Oxford Univ. Press, London. 6. FRANK, E., AND J. K. S. JANSEN. 1976. Interaction between foreign and original nerves innervating gill muscles in fish. J. Neurophysiol. 39: 84-90. 7. GAZE, R. M. 1970. The Formation of Nerve Connections. Academic Press, New York and London. 8. GRIMM, L. 1971. An evaluation of myotypic respecification in axolot1s.J. Exp. Zoo/. 178: 479-496. 9. HOWE, H. A., S. S. TOWER, AND A. B. DUEL. 1937. Facial tic in relation to injury of the facial nerve. Arch. Neural. Psychiat. 38: 1190-1198. 10. LANGLEY, J. N., ANDH. K. ANDERSON. 1894. Onreflexactionfrom sympatheticganglia. J. Physiol. (London) 16: 410-440. 11. LEHOUELLEUR, J., AND A. CHATELAIN. 1974. Analysis of electrical responses of newt skeletal muscle fibers in response to direct and indirect stimulation. J. Physiol. (Paris) 68: 615-632. 12. LITWILLER, R. 1938. Quantitative studies on nerve regeneration in amphibia. I. Factors controlling nerve regeneration in adult limbs. J. Comp. Neural. 69: 427-447. 13. MARK, R. F. 1965. Fin movement after regeneration of neuromuscular connections: an investigation of myotypic specificity. Exp. Neural. 12: 292-302. 14. MARK, R. F., L. R. MAROTTE, AND J. R. JOHNSTONE. 1970. Reinnervatedeye musclesdo not respond to impulses in foreign nerves. Science 170: 193- 194.
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L. R., AND R. F. MARK. 1970. The mechanism of selective reinnervation of fish eye muscle. I. Evidence from muscle function during recovery. Brain Res. 19: 41-51. 16. PEARSON, K. G., AND A. B. BRADLEY. 1972. Specific regeneration of excitatory motoneurons to leg muscles in the cockroach. Brain Res. 47: 492-496. 17. PIATT, .I. 1957. Studies on the problem of nerve pattern. II. Innervation of the intact forelimb by different parts of the central nervous system in Ambystoma. J. Exp. Zool. 134: 103-125. 18. SCOTT, S. A. 1975. Persistence of foreign innervation on reinnervated goldfish extraocular muscles. Science 189: 644-646. 19. SHAWE, G. D. H. 1955. On the number of branches formed by regenerating nerve-fibers. 15.
MAROTTE,
20.
SPERRY,
Br. 1. Surg.
42: 474-488.
25.
R. W. 1945. The problem of central nervous reorganization after nerve regeneration and muscle transposition. Q. Rev. Biol. 20: 311-369. SPERRY, R. W., AND H. L. ARORA. 1965. Selectivity in regeneration of the oculomotor nerve in the cichlid fish,Astronotus ocellatus. J. Embryol. Exp. Morphol. 14: 307-3 17. STEPHENSON, R. S. 1973. Branching of regenerated motoneuron axons in axolotl limbs. Doctoral dissertation, Massachusetts Institute of Technology, Cambridge. STEPHENSON, R. S. 1978. A bipolar electrode for electromyography of limb muscles of small animals. Am. J. Physiol. 234: C198-C200. VERZAR, F., AND P. WEISS. 1930. Untersuchungen tiber des Phanomen der identischen Bewegungsfunktion mehrfacher benachbarter Extremitaten. Zugleich: Direkte Vorfuhrung von Eigenreflexen. PJligers Arch. 223: 671-684. WEISS, P. 1922. Die Funktion transplantierten Amphibienextremititen. Anz. AX-ad. Wiss.
26.
WEISS,
21. 22. 23. 24.
Vienna
59: 199-201.
P. 1931. Das Resonanzprinzip der Nerventatigkeit, dargestellt in Funktionspriifungen an transplantierten iiberzahhgen Muskeln. Pfliigers Arch. 226: 600-658. 27. WEISS, P. 193 1. Die Nervenversorgung der iiberzahligen Extremitaten an den von Verzar und Weiss in Bd. 223 dieser Zeitschrift beschriebenden hypermelen Frosch. PJIiigers Arch.
228: 486-497.
28. WEISS, P. 1936. Selectivity controlling the central-peripheral relations in the nervous system. Biol. Ret’. Cambridge Phil. Sot. 11: 494-531. 29. WEISS, P. 1937. Further experimental investigations on the phenomenon of homologous response in transplanted amphibian limbs. I. Functional observations. J. Comp. Neural. 66: 181-209. 30. WEISS, P. 1937. Further experimental investigations on the phenomenon of homologous response in transplanted amphibian limbs. II. Nerve regeneration and the innervation of transplanted limbs. J. Camp. Neural. 66: 481-535. 3 1. WEISS, P. 1937. Further experimental investigations on the phenomenon of homologous response in transplanted amphibian limbs. III. Homologous response in the absence of sensory innervation. J. Comp. Neural. 66: 537-548. 32. WEISS, P. 1937. Further experimental investigations on the phenomenon of homologous response in transplanted amphibian limbs. IV. Reverse locomotion after the interchange of right and left limbs. J. Comp. Neurol. 67: 269-315. 33. YIP, .I. W., AND M. J. DENNIS. 1976. Supression of transmission at foreign synapses in adult newt muscle involves reduction in quanta1 content. Nature (London) 260: 350-352.
NEUROLOGY
64, 174- 189 (1979)
Axon Reflexes in Axolotl Limbs: Evidence that Branched Motor Axons Reinnervate Muscles Selectively ROBERT Research
Laboratory
of Electronics,
S.
STEPHENSON’
Massachusetts Institute Massachusetts 02139
Received
October
of Technology,
Cambridge,
12. 1978
Numerous axon reflexes were found in grafted forelimbs, regenerated forelimbs, and forelimbs with regenerated nerves in the axolotlAmbys?oma mexicanurn. They were not seen in normal limbs. Each reflex was due to a single motor axon which branched, generally at a point central to the limb. In most cases several consecutive electrical stimuli were necessary to cause a visible contraction or a muscle action potential in the motor units concerned. I observed a total of 13 axon reflexes between the supernumerary limb and the ipsilateral host limb. All but one could be stimulated in either direction. In other animals I demonstrated 13 axon reflexes by cutting a small nerve in the distal limb and stimulating its proximal stump. In at least five, and possibly as many as 14 of these 26 axon reflexes, the two muscles involved were synonymous. In 20 of the 26, the muscles involved were situated in the same limb region and were synergistic or synonymous. Only in five axon reflexes were the muscles in widely different parts of the limb and clearly unrelated in function. Random innervation or mechanical guidance alone cannot account for these muscle-specific axon reflexes. Axon branches innervated muscles selectively, although the mechanism remains unclear. The selectivity was not perfect, though, because axon reflexes between dissimilar muscles did occur occasionally. Although it has been proposed that an axon may remain in an inappropriate muscle but be functionally “repressed,” no evidence of such repressed synapses was found. Abbreviations: AR-axon reflex, SNL-supernumerary limb, HL-host limb, MAPmuscle action potential. ’ Supported in part by NIH grants 5TOlGM0155 and EY07008 and by a grant from the Bell Telephone Co. The author is indebted to Dr. J. Y. Lettvin for his advice and support, to Dr. V. R. Stirling for participating in one of the experiments, and to Drs. P. A. Weiss, W. L. Pak, J. R. Vanable, and J. E. Mittenthal for their valuable comments. I also thank R. Graham, J. Cook, M. Ben Chakri, W. Boyle, L. Winchester, and Drs. R. R. Humphrey, P. Modell, and C. Dalton. The present address of the author is Department of Biological Sciences, Purdue University, West Lafayette, IN 47906. 174 0014-4886/79/040174-16$02.00/O Copyright All rights
Q 1979 by Academic Press, Inc. of reproduction in any form reserved
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INTRODUCTION The nervous system of amphibia and fishes, unlike that of higher vertebrates, has an extraordinary capacity to regenerate itself with return of function (7, 20). In the motor system of lower vertebrates, where this regenerative ability has been much studied, it is not clear to what extent functional recovery after interruption of peripheral nerves depends on the restoration of specific motoneuron-muscle connections. Much of the evidence that it does not comes from the comprehensive experiments of Weiss concerning the “homologous response” of a supernumerary limb (SNL) transplanted to the limb region of a salamander (25, 29-32). The SNL, when innervated, appears to duplicate muscle for muscle the movements of the host limb (HL) beside it (24, 26, 29), although the precision of this duplication has been questioned (2). Weiss concluded that the homologous response depended not on any morphological selectivity in the reinnervation of the transplant (30), but rather on a readjustment or “modulation” occurring more centrally (28). On the other hand, experiments involving reinnervation of the external eye muscles (4, IS, 21) and fin muscles (13) of fish, as well as the reinnervation of frog (3) and salamander (1, 8, 33) limb muscles, suggest that a muscle is preferentially innervated by its original nerve, and foreign nerve fibers are driven out or repressed. Similar experiments on fish yielded conflicting results, however (6, 18). Evidence for specificity at a grosser level is that the distribution pattern of the forelimb nerves is normal only when the limb is innervated by the normal segments of the spinal cord, i.e., the third, fourth, and fifth (17). The apparent inconsistency of these different studies has not been explained. Another experimental approach to the question of selective regrowth of peripheral nerves is to consider the fate of branched axons. It was found that the addition of a SNL to the flank of an amphibian does not cause a significant increase in the number of nerve fibers leaving the spinal cord (27, 30). On the other hand, those nerve fibers branch as they grow peripherally so that each limb receives approximately the normal number of fibers, in proportion to its size (12,27,30). Thus the total number of limb nerve fibers is greater when counted at the bases of the HL and SNL than at their exit from the spinal cord. If the outgrowth of the branched motor fibers is unspecific, as Weiss suggested (28, 30), then the majority of these should innervate two or more unrelated muscles in the periphery. When a branched nerve fiber innervates two different muscles A and B, stimulating muscle A electrically should evoke a contraction in B and vice versa, even after removal of the spinal cord. Such a contraction, which was termed an axon reflex (AR) (lo), provides a test of Weiss’s claim of unspecific
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innervation. In examining his grafted animals, Weiss (30) found only three ARs, apparently between dissimilar muscles, but it was unclear why he didn’t see more. The present study will show that ARs are common in axolotls with regenerated motor nerves, and that ARs between unrelated muscles are exceptional. METHODS Supernumerary forelimbs were grafted onto the shoulder region of axolotls (Ambystoma mexicanum) 3 to 5 months old, according to the procedure of Weiss (29). The fourth or fifth spinal nerve was cut at the time of the grafting, and this provided the major nerve supply to the graft, as shown by stimulation. Ten such grafts were successful, and the SNL moved homologously with the HL in all cases. In other animals I simply amputated a forelimb and allowed it to regenerate. After 3 to 22 months’ recovery, the animal was anesthetized briefly with tricaine methanesulfonate, the medulla was transected, and the animal was decerebrated. 1 exposed the brachial spinal cord by laminectomy, transected all brachial dorsal roots, and skinned the HL and SNL. The preparation was maintained at about 15°C and 100% relative humidity. ARs were evoked electrically with a small bipolar electrode (23)) using trains of five pulses 25 ms apart, repeated once a second. This type of electrode was also used for recording the muscle action potentials (MAPS). To locate ARs I stimulated one forelimb and looked with a dissecting microscope for contraction in a more central muscle or in the other, ipsilateral forelimb. Then I stimulated the muscle which had contracted reflexly and watched for a contraction in the region originally stimulated. By repeatedly stimulating in one direction and then in the other with diminishing stimulus intensity it was possible to isolate each AR present and identify the most sensitive region for stimulating it in each direction. RESULTS Properties of Axon Reflexes. The reflexes so obtained were ARs because (i) they were entirely peripheral, unaffected by ventral root transection and removal of the spinal cord; (ii) they were bidirectional, i.e., they could be stimulated in both directions; (iii) they were persistent, lasting in general as long as the circulation was functional6 days in several cases; and (iv) they disappeared when I cut the limb nerves at a level central to the two muscles involved. The site of the axonal bifurcation-as determined by sectioning more and more distally until the reflex disappeared-was usually central to the SNL and HL and near the region where the brachial nerves had been cut or damaged during grafting or amputation. This is
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consistent with other studies on regenerated nerve (9, 19, 30). Each AR, moreover, involved only one branched axon because (i) as the stimulus intensity increased or decreased continuously the reflex appeared or disappeared in all-or-none fashion; (ii) only one MAP was present and arose from a single end-plate region- usually corresponding to the most sensitive spot for stimulating the reflex-as shown by displacing the recording electrode along the muscle surface (23); finally, (iii) the reflex contraction occurred only in a small group of contiguous muscle fibers. All ARs seemed to involve only two muscles, although one might expect to find axons with three or more peripheral branches as well. This may have been because I grafted only one SNL on each animal and thus did not overload the limb nerve supply excessively. In 80% of the ARs studied, a single stimulus pulse caused neither MAP nor visible contraction. The response facilitated, and when several stimuli followed each other at a 25ms interval, the second and subsequent stimuli evoked MAPS of progressively shorter latency (23). On two occasions I was able to record directly from the branched axon with suction electrodes, and it conducted impulses without failure to a rate of about 60/s with no significant change in latency. Thus, the failure and facilitation of the MAPS seem to be properties of axolotl end-plates, as reported for those of Triturus (1 I). ARs were numerous in grafted limbs, regenerated limbs, and limbs whose nerves had been cut and allowed to regenerate; but none was present in 10 normal limbs examined. Stimulation as described above typically revealed several ARs between different muscles in a regenerated or grafted limb. This procedure could not demonstrate any axon reflexes operating between two parts of a single muscle or between two adjacent muscles, however, because of the difficulty of distinguishing direct from reflex stimulation. To decide how often the latter type of AR occurred it was necessary to look for ARs operating between the HL and SNL. These were present in small numbers in half the grafted animals examined. Such a case is shown schematically in Fig. 1. A study of such interlimb ARs showed that the proportion between synonymous or synergistic muscles was greater than could be due to chance alone. Interlimb Axon Reflexes. I observed a total of 13 ARs between HL and SNL in four different animals. These are shown schematically in Fig. 2. For each reflex the motor units involved are indicated in black on a map of the skeleton: HL at left, SNL at right, dorsal view above, ventral view below. For convenience I will refer hereafter to the group of muscle fibers innervated by one branch of an axon as a motor unit, although strictly speaking they constitute only part of it. The size of the motor units, which in some cases involved as few as six to eight muscle fibers, has been exaggerated for clarity. In the case of AR D4-1, synonymous muscles were
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FIG. 1. A schematic drawing of an axon branching to both the host limb (lower) and the supernumerary limb (upper), giving rise to an interlimb axon reflex.
involved in the HL and SNL: the anconaeus scapular-is mediali+ which is a dorsal division of the extensor of the elbow. Figure 3A shows the response of this axon reflex recorded in the host limb when the SNL was stimulated. The reverse is shown in B. In C a rootlet of the fifth ventral root was stimulated while the recording electrode was placed over the unit in the HL. This excited the same characteristic MAP as in A as well as an earlier, unrelated one. The fifth spinal nerve had been cut at the time the SNL was grafted, and when this nerve was crushed in the vertebral foramen at the conclusion of the experiment the AR disappeared. Trace D was recorded under the same conditions as A but 5 days later, demonstrating the persistence of the ARs. The axon reflex D4-2 in the same animal ran from the anconaeus * I adopt the terminology of Francis (5) from Salamandra salumandra. For its applicability to the axolotl, as well as systematic description of the forelimb muscles in the latter species see
(22).
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coracoideus (a ventral division of the elbow extensor) in the HL to a unit on the ventral side of the anconaeus in the SNL. In both this and the preceding AR the motor units were too weak to produce any gross movement of the limb. It was clear, nevertheless, exactly where the contracting unit lay because (i) it produced a local distortion of the anconaeus, (ii) at 25x I could see a slippage of the active muscle fibers relative to their inactive neighbors, and (iii) the MAP was localized at the spot. A movie showing these two ARs is available from the author. AR D7-1 caused a very slight flexion of digits III and IV (counting from the radial or medial side of the forearm) of the HL when the SNL was stimulated, and when the HL was stimulated, digits I and II of the SNL flexed. The unit in the HL inserted on the overlying palmar tendon, which in turn inserts on the phalanges of each digit, The unit in the SNL inserted on the metacarpals of digits I and II. Because the origins of these units were concealed beneath the palmar tendon, it was not possible to tell which of several small finger or wrist flexors were involved, and therefore I have marked their location on Fig. 2 with an open outline. In animal D8 there were six different ARs between the HL and SNL, all but one involving hand or forearm flexors in both limbs. D8-1 involved a deep flexor of digit II of the HL, and in the SNL the flexor brevis superficialis of digit II. A deep flexor of digit I of the SNL also contracted intermittently when this AR was stimulated. D8-2 involved a unit on the ulnar side of the flexor primordialis communis (the common flexor of the digits and wrist, inserted on the palmar tendon) in the HL, and a wrist flexor on the ulnar side of the SNL forearm. AR D8-3 was between the flexor accessorius lateralis of the HL and the flexor antebrachii et carpi ulnaris of the SNL. The former unit arose from the end of the ulna and inserted on the palmar tendon. The latter arose from the distal end of the humerus and inserted on the ulnar carpus, just distal to the origin of the former. In the case of this and some of the following ARs it was necessary to dissect away the flexor primordialis to expose the active units. D8-4 was a counterpart of D8-3, but situated more toward the radial side of both limbs. In the HL the flexor accessorius medialis contracted, and in the SNL the flexor antebrachii et carpi radialis contracted. Once again, the latter unit inserted just beneath the former on the radial carpus. Reflex DS-5 involved the flexor brevis superficialis of digit IV of the HL, and a flexor of the SNL wrist. Reflex D8-6 was between a central division of the extensor of the HL elbow and an unseen deep muscle on the flexor side of the SNL forearm. The latter produced a slight twisting of the SNL forearm. The remaining four interlimb ARs occurred in animal K7, which had both a transplanted limb and an extra, regenerated limb. I examined K7 only 3 months after grafting, whereas the other animals had a much longer
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FIG. 3. Muscle action potentials due to axon reflex D4-1. Each trace is a superposition of the responses to several consecutive stimuli (indicated by the rectangle beneath each trace) 25 ms apart. A-the recording electrode was positioned over the anconaeus scapularis medialis (a division of the elbow extensor) in the host limb, and the anconaeus of the supernumerary limb was stimulated. B-the reverse; the recording electrode was over the anconaeus scapularis medialis of the supernumerary limb, and the stimulus was delivered to the anconaeus of the host limb. The elevation at the start ofthis trace is an unusually large stimulus artifact. C-the recording electrode was as in A, but a rootlet of the ipsilateral fifth ventral root was stimulated with a suction electrode. This rootiet contained the axons of two units in the anconaeus scapularis medialis, but note that the later muscle action potential has the same characteristic shape as, and a slightly smaller latency than that in A. D-same as A, but recorded 5 days later. Differences in latency between A and B or A and D are due to differences in electrode position. The bars at the lower right represent 50 PV and 2 ms and apply to all traces.
recovery before examination. Except for the small size of his limbs the results were the same. In the HL, K7-1 involved a caudal part of the pectoralis (a shoulder muscle which depresses and retracts the humerus), and in the transplant a rudimentary muscle on the coracoid portion of the shoulder girdle, I was unable to identify the latter muscle as it appeared to be attached to the coracoid only and was without effect on the limb. It was probably a shoulder muscle whose other attachment had been severed at the time of grafting. It had roughly the same position relative to the transplanted limb that the unit in the pectoralis had to the HL, but it should
FIG. 2. (opposite) Maps showing location of motor units relative to the forelimb skeleton for each interlimb axon reflex. Each rectangle represents one axon reflex, with the host limb at left and the supernumerary limb at right. Dorsal and ventral views of the limb are shown above and below the horizontal midline, respectively. Extensors of the forearm and hand appear on the upper outline, flexors on the lower. For clarity each darkened area is larger than the motor unit it represents and corresponds in most cases to the entire muscle involved. Where the origin or insertion of a motor unit was unknown an open outline marks the unit’s approximate location. When a motor unit is inserted on the palmar tendon the latter is indicated by stippling. Projection onto a standard outline required distortion in some cases. The regenerated limb of K7 was so abnormal as to be represented individually. Only its dorsal side was visible. Dimensions of the host limb are typically 15 to 20 mm elbow to fingertip; the supernumerary limb was two to three times smaller.
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be noted that although the pectoralis overlies the coracoid it is not attached to it. Reflex K7-3 involved the flexor primordialis communis of the HL, and in the transplant a muscle which ran the length of the forearm and flexed the digits. I could not see the latter unit because the digits of the regenerated limb overlay the forearm of the transplant. Its position and action suggest strongly that it, too, was the flexor primordialis communis. K7-5 involved the anconaeus, the extensor of the elbow, in both HL and transplant. In the transplant, however, the tendon of insertion of the extensor had slipped off the elbow, reversing the action of this muscle and causing the elbow to assume a sharp angle. This happened to the SNLs of several animals. This AR could not be stimulated in the transplant because of the overlying regenerated limb. The last AR, K7-2, was between the HL and the regenerated limb, which was only partially developed as shown in Fig. 2. The unit in the HL was an extensor on the radial side of the forearm. The unit in the regenerate arose from the middle of the humerus and inserted on the distal end of the radius, toward the extensor side. In spite of its rudimentary appearance it was most probably part of the extensor antebrachii et carpi radialis, which in a mature arm arises from the end of the humerus and inserts on the distal end of the radius, as well as on the ulnar carpus. In conclusion, of these 13 interlimb ARs, four--4-1, D4-2, Da-l, and K7-S-involved synonymous muscles in both limbs, and in two additional cases, K7-2 and K7-3, this is likely but not certain. I treat as synonymous all the divisions of the anconaeus, and similarly all the small flexors of a particular digit. With these two conventions, 21 different muscles may be distinguished in the axolotl forearm, and seven different muscles in the shoulder. But there is even more pattern to the distribution of the ARs than this alone would indicate. If one divides all limb muscles into two general categories, flexor-retractors and extensor-protractors, 11 of the ARs respected this partition, and only one, D8-6, involved muscles of both types. The case of K7-2 was unclear. If one makes a dichotomy at right angles to the preceding division, between ventral and dorsal in the shoulder and upper arm, and between radial and ulnar in the forearm, then eight ARs respected this new partition, and only one, D7-1, clearly did not. Cases D8-5, D8-6, K7-3, and K7-5 were unclear. Finally, if one divides limb muscles along a proximal-distal axis into shoulder, upper arm, forearmwrist, and finger categories; nine ARs were confined to muscles of a single category. Two, D8-5 and D8-6, confused two adjacent categories. The cases of D7-1 and K7-2 were unclear. Taken together, the preceding divisions categorized the limb musculature into 16 compartments. Seven of the ARs (D4-1, D4-2, Da-l, D8-2, D8-3, D8-4, and K7-1) involved two motor units within a single such compartment. ARs D7-1, DS-5, and Da-6 did not respect the compartmental boundaries, and the three remaining
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cases were uncertain. Only one AR, D8-6, linked muscles differing widely in location and function. Intralimb Axon Reflexes. The preceding results indicate that, in the case of motor axons branching to both the HL and the SNL, the two branches have a marked tendency to innervate similar, if not synonymous, muscles. Presumably the same is true of branches regenerating to the same limb. This presumption was tested in the following way. I exposed the forearm flexor or extensor nerves by removing the flexor primordialis communis or extensor digitorum communis, respectively, after having skinned the limb and removed the spinal cord. A nerve trunk that supplied only one or two muscles was dissected free for a few millimeters and cut. While stimulating the proximal stump with a suction electrode I examined the limb for ARs. Then I reversed the suction pipet and stimulated the distal stump to see which muscles were innervated by that nerve. This is shown schematically in Fig. 4, where the cross marks the point at which the nerve was cut. This technique could detect ARs between two parts of a single muscle, providing only that the two branches of the axon in question reached the muscle via different nerve trunks. Almost every nerve trunk cut yielded at least one apparent AR, except on the exterior surface of the muscles where most nerves are purely sensory. To rule out the possibility that an apparent AR might really be due to local stimulus spread or to fibers growing centripetally in the nerve, I tied a fine silk thread at or slightly below the shoulder and tightened it until it cut to the bone. If this abolished the contraction when the proximal stump was stimulated, and if stimulating the distal stump showed that the muscle had not failed due to ischemia (this never proved to be the case), the proximal contraction was accepted as a genuine AR. Thus, in Fig. 4 a ligature at the level of the solid line would eliminate the AR. This procedure revealed 13 ARs in five different limbs, shown in Fig. 5. In case E5-A a small extensor nerve, the dorsalis manus intermedius, was cut in the middle of a regenerated forearm. Stimulating the distal stump caused an MAP in the adductor digiti I, shown in Figs. 6A and C. Stimulating the proximal stump caused an AR in the same muscle, Fig. 6B, which disappeared, Fig. 6D, when the arm was ligated at the shoulder. In case E5-B I cut the ulnar nerve just distal to the elbow in the other, regenerated forelimb of the same animal. Stimulating the distal end excited the flexors of digits III, II, and possibly I. Stimulating the proximal end caused ARs in a flexor of digit II, flexor of digit III, in the flexor accessorius lateralis (a flexor of digits II and III), and in the humero-antebrachialis (flexor of the elbow). These were four distinct ARs as they were separable by threshold. In case K3-B the interosseus nerve was cut near its origin just proximal to the elbow in a SNL. Distal stimulation excited the flexor antebrachii et carpi radialis and extensor antebrachii et carpi radialis in the
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FIG. 4. A schematic drawing of an intralimb axon reflex. The nerve trunk is transected at the “X.” Stimulating the proximal stump will cause an axon reflex in the more distal muscle. A tight ligature about the upper arm at the level of the solid line will eliminate this reflex, although ligation at a more proximal level (dotted lines) is without effect. The bipolar electrode is drawn to correct scale over the active muscle.
forearm, whereas proximal stimulation caused ARs in the anconaeus (elbow extensor) and in a trunk muscle. In case K3-D I cut the ulnar nerve of a regenerated limb distal to the elbow. When the distal stump was stimulated the flexor accessorius lateralis contracted. Proximal stimulation gave ARs in the closely related flexor accessorius medialis and in the more proximal flexor antebrachii et carpi radialis. In case KS-A the ulnar nerve of a SNL was cut just distal to the elbow. Stimulating the distal stump caused the flexor antebrachii et carpi ulnaris and flexor antebrachii et cat-pi radialis to contract. Proximal stimulation caused ARs in the former muscle, as well as in the pronator profundus and the anconaeus. There were at least two distinct ARs in the flexor antebrachii et carpi ulnaris which were separable by threshold. Of the 13 ARs elicited by stimulating the proximal stump of a cut nerve,
K5-A
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REFLEX E5-B
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FIG. 5. Axon reflexes within single limbs, demonstrated by stimulating forearm nerves with a suction electrode. Within each rectangle the same limb is shown twice. On left-those muscles excited by direct (i.e., orthodromic) stimulation applied to the distal nerve stump. On rightmuscles excited via an axon reflex when the proximal stump was stimulated. The location of the stimulating pipet is shown schematically. Dorsal and ventral views as in Fig. 1.
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FIG. 6. Muscle action potentials recorded from the adductor digiti I in case E5-A. Astimulating the distal stump of the cut nerve trunk shows that it innervates many units in the adductor digiti I. B-stimulating the proximal stump of the same nerve trunk produces an axon reflex in the adductor digiti I. The arm was then ligated tightly at the shoulder level and traces C and D repeat A and B, respectively, showing that the axon reflex was abolished by this ligation (D) whereas the response of the muscle to orthodromic stimulation (C) was unaffected. As in Fig. 3, each trace is a superposition of responses to five consecutive stimuli (indicated by the rectangle beneath each trace) 20 ms apart. The calibration bars represent 20 PV and 2 ms.
seven occurred in the same muscle or muscles innervated by the distal stump (where I consider the flexor accessorius medialis and lateralis as divisions of a single muscle), whereas only four occurred in muscles remote from those innervated by the distal stump. These results, like those of the previous section, give clear evidence of selective innervation. The surgery in these cases was done when the animals were 5 to 21 months old, and they were examined for ARs about 6 months later. These results were in marked contrast to the case of an animal whose limb nerves were cut at 35 months, and examined 5 months later. In the two limbs of this animal I cut a total of five nerves and found eight ARs, none of which involved two parts of a single muscle and seven of which were between muscles widely separated in the limb. It is tempting to speculate that the crucial difference between this case and the others was the age of the animal at the time of surgery, because it is well known that the ability of amphibia to recover function after nerve section declines with age. DISCUSSION As mentioned above, Weiss (30) found a total of three ARs and concluded that they operated between different muscles. Because he did not succeed in stimulating these ARs in both directions and identified the muscles involved on the basis of their actions alone, this conclusion may have been in error. In any case, in the light of the results presented above, it is likely that he observed only a small fraction of the ARs actually present, as he himself surmised (30). It may be relevant to recall here that the majority of the ARs studied here gave no visible response to a single
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stimulus. Weiss concluded that many motor axons were connected to dissimilar muscles, and this posed a serious paradox for his modulation theory (28,30). On the other hand, the present results show clearly that this happens only exceptionally, and so, in salamanders, nerve branching does not present the obstacle to coordination that it does in mammals (9, 20). Mechanical factors alone are clearly insufficient to account for the selectivity demonstrated here. In the case of axons going to both HL and SNL, the branches grew in different directions for a centimeter or more before innervating similar muscles. The SNLs, moreover, were oriented in different directions in different animals with no obvious effect on the selectivity. The results presented here can be explained only by some form of selective affinity between nerve branches and muscles, but the nature of this selectivity is not clear. On the one hand it is possible that the first branch innervates any muscle unselectively, but subsequent branches of the same axon seek out muscles of similar specificity. Alternatively, it is possible that axons which establish themselves on muscles of differing specificity tend to degenerate and be replaced. Although such theories can explain the muscle-specific ARs described above, they cannot explain the homologous response of the SNL. This is because branches between the HL and SNL were rare and were completely absent in two animals whose limbs moved homologously nevertheless. Branching within the spinal cord was excluded also as there were no ARs present in these cases even with the ventral roots and spinal cord intact. The alternative explanation of the selective ARs described above is that each motoneuron is specified ab initio to innervate a particular muscle. This may be the case, for example, in the cockroach (16). Such a theory could account for the homologous response as well. Weiss’s principal argument against such selectivity was that he observed ARs only between dissimilar muscles, but this is invalidated by the present results. Other evidence in support of this hypothesis was cited in the introduction. The modulation theory (28), on the other hand, rests now on two crucial experiments: the homologous movement of SNLs innervated by distal flexor nerves taken from the HL (30) and the homologous response of single muscles innervated by various foreign nerves (26). These experiments deserve to be repeated. Mark et al. (14) proposed that motor axons innervate muscles competitively on the basis of preestablished affinities, and that a synapse between a muscle fiber and the “wrong” axon is “repressed” when the “right” axon arrives. “Repressed” synapses are enduring and morphologically normal, but ineffective. There have been reports that such synaptic repression occurs in axolotl limb muscles (1, 33), although its occurrence has been disputed in other animals (4,6, 18). Such a theory implies that, after nerve regeneration, a great many ineffective axon branches remain in
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“wrong” muscles. It predicts that (i) many ARs (those between dissimilar muscles) should operate in one direction only, and (ii) stimulating the proximal stump of a cut nerve- which would excite both effective and ineffective axon branches -should produce ARs distributed randomly throughout the limb. Because the present results do not support either prediction, repressed synapses, if present at all, must last less than the recovery period (5 months or more) between nerve transection and examination or else be very rare. These experiments show that axon branches innervate muscles selectively in the axolotl. They do not make it clear, however, whether this selectivity results from a preestablished affinity between a given motoneuron and a given muscle, or is the result of an interaction between the axon’s branches at the time they reinnervate the muscles. They show that the selectivity is not perfect, however, because ARs between dissimilar muscles do occur occasionally. REFERENCES I. CASS, D. T., T. J. SUTTON, AND R. F. MARK. 1973. Competition between nerves for functional connections with axolotl muscles. Nature (London) 243: 201-203. 2. CZI?H, G., AND G. SZBKELY. 1971. Muscle activities recorded simultaneously from normal and supernumerary forelimbs in Ambystoma. Acta Physiol. Acad. Sci. Hung. 40: 287-301. 3. ELUL, R., R. MILEDI, AND E. STEFANI. 1970. Neural control of contracture in slow muscle fibers of the frog. Acta Physiok Lat. Am. 20: 194-226. 4. FANGBONER, R. F., AND J. W. VANABLE. 1974. Formation and regression of inappropriate nerve sprouts during trochlear nerve regeneration inXenopus laevis. J. Comp. Neural. 157: 391-406. 5. FRANCIS, E. T. B. 1934. The Anatomy ofthe Salamander. Oxford Univ. Press, London. 6. FRANK, E., AND J. K. S. JANSEN. 1976. Interaction between foreign and original nerves innervating gill muscles in fish. J. Neurophysiol. 39: 84-90. 7. GAZE, R. M. 1970. The Formation of Nerve Connections. Academic Press, New York and London. 8. GRIMM, L. 1971. An evaluation of myotypic respecification in axolot1s.J. Exp. Zoo/. 178: 479-496. 9. HOWE, H. A., S. S. TOWER, AND A. B. DUEL. 1937. Facial tic in relation to injury of the facial nerve. Arch. Neural. Psychiat. 38: 1190-1198. 10. LANGLEY, J. N., ANDH. K. ANDERSON. 1894. Onreflexactionfrom sympatheticganglia. J. Physiol. (London) 16: 410-440. 11. LEHOUELLEUR, J., AND A. CHATELAIN. 1974. Analysis of electrical responses of newt skeletal muscle fibers in response to direct and indirect stimulation. J. Physiol. (Paris) 68: 615-632. 12. LITWILLER, R. 1938. Quantitative studies on nerve regeneration in amphibia. I. Factors controlling nerve regeneration in adult limbs. J. Comp. Neural. 69: 427-447. 13. MARK, R. F. 1965. Fin movement after regeneration of neuromuscular connections: an investigation of myotypic specificity. Exp. Neural. 12: 292-302. 14. MARK, R. F., L. R. MAROTTE, AND J. R. JOHNSTONE. 1970. Reinnervatedeye musclesdo not respond to impulses in foreign nerves. Science 170: 193- 194.
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L. R., AND R. F. MARK. 1970. The mechanism of selective reinnervation of fish eye muscle. I. Evidence from muscle function during recovery. Brain Res. 19: 41-51. 16. PEARSON, K. G., AND A. B. BRADLEY. 1972. Specific regeneration of excitatory motoneurons to leg muscles in the cockroach. Brain Res. 47: 492-496. 17. PIATT, .I. 1957. Studies on the problem of nerve pattern. II. Innervation of the intact forelimb by different parts of the central nervous system in Ambystoma. J. Exp. Zool. 134: 103-125. 18. SCOTT, S. A. 1975. Persistence of foreign innervation on reinnervated goldfish extraocular muscles. Science 189: 644-646. 19. SHAWE, G. D. H. 1955. On the number of branches formed by regenerating nerve-fibers. 15.
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R. W. 1945. The problem of central nervous reorganization after nerve regeneration and muscle transposition. Q. Rev. Biol. 20: 311-369. SPERRY, R. W., AND H. L. ARORA. 1965. Selectivity in regeneration of the oculomotor nerve in the cichlid fish,Astronotus ocellatus. J. Embryol. Exp. Morphol. 14: 307-3 17. STEPHENSON, R. S. 1973. Branching of regenerated motoneuron axons in axolotl limbs. Doctoral dissertation, Massachusetts Institute of Technology, Cambridge. STEPHENSON, R. S. 1978. A bipolar electrode for electromyography of limb muscles of small animals. Am. J. Physiol. 234: C198-C200. VERZAR, F., AND P. WEISS. 1930. Untersuchungen tiber des Phanomen der identischen Bewegungsfunktion mehrfacher benachbarter Extremitaten. Zugleich: Direkte Vorfuhrung von Eigenreflexen. PJligers Arch. 223: 671-684. WEISS, P. 1922. Die Funktion transplantierten Amphibienextremititen. Anz. AX-ad. Wiss.
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28. WEISS, P. 1936. Selectivity controlling the central-peripheral relations in the nervous system. Biol. Ret’. Cambridge Phil. Sot. 11: 494-531. 29. WEISS, P. 1937. Further experimental investigations on the phenomenon of homologous response in transplanted amphibian limbs. I. Functional observations. J. Comp. Neural. 66: 181-209. 30. WEISS, P. 1937. Further experimental investigations on the phenomenon of homologous response in transplanted amphibian limbs. II. Nerve regeneration and the innervation of transplanted limbs. J. Camp. Neural. 66: 481-535. 3 1. WEISS, P. 1937. Further experimental investigations on the phenomenon of homologous response in transplanted amphibian limbs. III. Homologous response in the absence of sensory innervation. J. Comp. Neural. 66: 537-548. 32. WEISS, P. 1937. Further experimental investigations on the phenomenon of homologous response in transplanted amphibian limbs. IV. Reverse locomotion after the interchange of right and left limbs. J. Comp. Neurol. 67: 269-315. 33. YIP, .I. W., AND M. J. DENNIS. 1976. Supression of transmission at foreign synapses in adult newt muscle involves reduction in quanta1 content. Nature (London) 260: 350-352.