The pattern of innervation in serially duplicated axolotl limbs: further evidence for the existence of local pathway cues?

The innervation of the biceps muscle was examined in regenerated and vitamin A-induced serially duplicated axolotl forelimbs using retrograde transport of horseradish peroxidase. The regenerated biceps muscle becomes innervated by motor neurones in the same position in the spinal cord as the normal biceps motor pool. In previous experiments in which the innervation of a second copy of a proximal limb muscle was examined in serially duplicated limbs (Stephens, Holder & Maden, 1985), the duplicate muscle was found to become innervated by motor neurones that would normally have innervated distal muscles. In the present study, the innervation of the second copy of biceps was examined under conditions designed to encourage nerve sprouting from 'correct' biceps axons. Following either partial limb denervation or denervation coupled with removal of the proximal biceps, the second copy of the muscle was still innervated by inappropriate motor neurones, which again would normally innervate distal limb muscles. These results are interpreted as evidence for the necessity for an appropriate local environment for axonal growth to allow reformation of a correct pattern of motor innervation in the regenerated limb.     

Reformation of specific neuromuscular connections during axolotl limb regeneration: evidence that the first contacts are correct

Retrograde neuronal tracing with horseradish peroxidase was used to determine the position in the spinal cord of the motor neurone pools of a proximal (biceps) and a distal (extensor digitorum) limb muscle at various times during axolotl limb regeneration. It was found that from the earliest stages of muscle redifferentiation (as judged by light and electron microscopic analysis) the vast majority of axons innervating the regenerating muscles came from cells within the bounds of the normal motor neurone pool for each muscle. A few incorrect projections were noted in that the regenerating proximal muscle was sometimes innervated by some cells caudal to its normal motor neurone pool. The results are discussed in terms of mechanisms that may be operating in the regenerating limb to ensure that specific neuromuscular connections are made.     

The distribution of motoneurons supplying hind limb muscles in the clawed toad, Xenopus laevis

The distribution of motoneurons in the lumbar spinal cord (spinal segments 8-10) of the clawed toad, Xenopus laevis, was studied with the horseradish peroxidase technique. In a total of 13 different hind limb muscles this tracer was applied in a slow-release gel. Motoneurons innervating a particular hind limb muscle were clustered in longitudinally arranged motor pools. Motor pools of different muscles did show considerable overlap both in the rostrocaudal and transverse plane. But, the various motor pools clearly show a somatotopic organization of motoneurons even in such a condensed lumbar spinal cord as in Xenopus laevis. Motoneurons innervating more distally positioned muscles are generally found in more caudal segments, while proximal muscles (with the exception of the m. adductor magnus) are supplied by motoneurons more or less throughout the lumbar enlargement. Flexor muscles usually are innervated by motoneurons situated ventrolaterally in the ventral horn, extensor muscles by dorsomedially found motoneurons. This pattern is particularly apparent for proximal (thigh) muscles, less so for more distal (shank and foot) muscles. The present data are in keeping with those obtained with the retrograde cell degeneration technique in ranid frogs and are consistent with observations in other tetrapods, although a more clear separation of motor pools is evident in "higher" vertebrates such as birds and mammals.     

Target-specific nerve regeneration through a nerve guide in the rat

Nerve regeneration across a gap in peripheral nerve has been achieved through various nonneural nerve guides in both lower and primate species. This technique can only be useful if the regenerated nerve cable grows specifically to and reinnervates the appropriate distal target. In this study, the proximal peroneal fascicle of rat sciatic nerve was inserted into the proximal limb of a Y-shaped nerve guide. Distal peroneal and tibial fascicles were placed within the two distal limbs of the same Y. The proximal peroneal nerve grew preferentially by a 2:1 ratio to the appropriate distal peroneal fascicle suggesting that target-specific reinnervation is possible through a nerve guide.     

Regeneration of monoamine-containing axons in the developing and adult spinal cord of the rat following intraspinal 6-OH-dopamine injections or transections

Summary Growth of descending noradrenaline (NA) and 5-hydroxytryptamine (5-HT) axons in the rat spinal cord during ontogenesis and following mechanical or chemical, 6-hydroxydopamine (6-OH-DA) induced, axotomy, was studied with the Falck-Hillarp histochemical fluorescence method for monoamines.The major NA and 5-HT axon bundles and terminal innervation areas are present already at birth and an essentially mature pattern of innervation is reached after two weeks.Complete degeneration of both 5-HT and NA nerves in the distal segment is obtained by a transection of the spinal cord. Sprouting of the cut monoamine fibers into the necrotic zone and scar tissue is vigorous in both immature and mature animals, but regeneration into the distal segment is very poor.Selective degeneration of the descending NA axons and terminals is obtained by a localized intraspinal 6-OH-DA injection. Thus, the 5-HT fiber systems as well as all other parts of the spinal cord are left intact. The method should therefore prove useful for evaluating the exact functional role of the NA and 5-HT neuron systems in the spinal cord.Reinnervation of the distal part of the spinal cord by new NA fibers following 6-OH-DA induced denervation is described. This process is faster in younger animals but takes place also in adult animals. The present evidence suggests that reinnervation mainly is the result of downgrowth of the axotomized fibers, but growth in the form of collateral sprouting from a few possibly surviving fibers in the distal region may also contribute. Reinnervation lead to a normal innervation pattern within 1–2 months in the various age groups.It is suggested that the poor regeneration of many spinal nerve tracts often reported in the literature following transection of the spinal cord is due to extraneuronal factors such as scar tissue and impaired circulation rather than to the nerves per se since reinnervation by NA nerves was very poor following mechanical transection but good following chemical, 6-OH-DA-induced axotomy.     

Spinal cord atrophy and reorganization of motoneuron connections following long-standing limb loss in primates

Primates with long-standing therapeutic amputations of a limb at a young age were used to investigate the possibility that deefferented motor nerves sprout to new muscle targets. Injections of anatomical tracers into the muscles proximal to the amputated stump labeled a larger extent of motoneurons than matched injections on the intact side or in normal animals, including motoneurons that would normally supply only the missing limb muscles. Although the total numbers of distal limb motoneurons remained normal, some distal limb motoneurons on the amputated side were smaller in size and simpler in form. These results suggest that deprived motoneurons survive and retain function by reinnervating new muscle targets. The sprouted motor efferents may account for some of the reorganization of primary motor cortex that follows long-standing amputation.     

Transient inability of neonatal rat motoneurons to reinnervate muscle

We studied the reinnervation of internal intercostal muscles of newborn rats. The distal halves were denervated by nerve section at various ages between birth and 6 weeks. Regardless of the age at denervation, neither evoked nor spontaneous nerve-muscle transmission reappeared until the animals were at least 3 weeks old. Older rats recovered a substantial degree of function within 7 days of nerve section. Normally the motor units in this muscle are narrowly distributed, so most axotomized motoneurons lost their entire synaptic periphery. Reinnervation was by axons which had been sectioned, and regenerated motor units were of normal size and number. There was no collateral sprouting from end plates left intact. Motoneurons axotomized at birth did regenerate axons the full length of the muscle within 7 days of operation. Their failure to reinnervate the muscle was due to delay in forming functional end plates. Nerve section in animals aged 1 month or older resulted in an abnormal pattern of reinnervation; reinnervated motor units were diffusely spread through large portions of the muscle, although they still did not overlap with the region left intact. This indicates that thoracic motoneurons respond to axotomy differently in neonatal rats than they do in adults.     

The effects of long-standing limb loss on anatomical reorganization of the somatosensory afferents in the brainstem and spinal cord

We examined the terminations of sensory afferents in the brainstem and spinal cord of squirrel monkeys and prosimian galagos 4-8 years after a therapeutic forelimb or hindlimb amputation within 2 months of birth. In each animal, the distributions of labeled sensory afferent terminations from remaining body parts proximal to the limb stump were much more extensive than in normal animals. These sprouted afferents extended into the portions of the dorsal horn of the spinal cord as well as the cuneate and external cuneate nuclei of the brainstem (forelimb amputees) or spinal Clarke's column (hindlimb amputee) related to the amputated limb. Such reorganization in sensory afferents along with reorganization of the motor efferents to muscles (Wu and Kaas, J Neurosci 19: 7679-7697, 1999, Neuron 28: 967-978, 2000) may provide a basis for mislocated phantom sensations of missing forelimb movements accompanying actual shoulder movements during cortical stimulation or movement imagery in patients with amputations.     

The effects of long-standing limb loss on anatomical reorganization of the somatosensory afferents in the brainstem and spinal cord

We examined the terminations of sensory afferents in the brainstem and spinal cord of squirrel monkeys and prosimian galagos 4-8 years after a therapeutic forelimb or hindlimb amputation within 2 months of birth. In each animal, the distributions of labeled sensory afferent terminations from remaining body parts proximal to the limb stump were much more extensive than in normal animals. These sprouted afferents extended into the portions of the dorsal horn of the spinal cord as well as the cuneate and external cuneate nuclei of the brainstem (forelimb amputees) or spinal Clarke's column (hindlimb amputee) related to the amputated limb. Such reorganization in sensory afferents along with reorganization of the motor efferents to muscles (Wu and Kaas, J Neurosci 19 : 7679-7697, 1999, Neuron 28 : 967-978, 2000) may provide a basis for mislocated phantom sensations of missing forelimb movements accompanying actual shoulder movements during cortical stimulation or movement imagery in patients with amputations.     

The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.     

Body wall morphogenesis: Limb-genesis interferes with body wall-genesis via its influence on the abaxial somite derivatives

The vertebrate body wall is regionalized into thoracic and lumbosacral/abdominal regions that differ in their morphology and developmental origin. The thoracic body wall has ribs and intercostal muscles, which develops from thoracic somites, whereas the abdominal wall has abdominal muscles, which develops from lumbosacral somites without ribs cage. To examine whether limb-genesis interferes with body wall-genesis, and to test the possibility that limb generation leads to the regional differentiation, an ectopic limb was induced in the thoracic region by transplanting prospective limb somatopleural mesoderm of Japanese quail between the ectoderm and somatopleural mesoderm of the chick prospective thoracic region. This ectopic limb generation induced the somitic cells to migrate into the ectopic limb mesenchyme to become its muscles and caused the loss of distal thoracic body wall (sterno-distal rib and distal intercostal muscle), without causing any significant effect on the more proximal region (proximal rib, vertebro-distal rib and proximal intercostal muscle). According to a new primaxial–abaxial classification, the proximal region is classified as primaxial and the distal region, as well as limb, is classified as abaxial. We demonstrated that ectopic limb development interfered with body wall development via its influence on the abaxial somite derivatives. The present study supports the idea that the somitic cells give rise to the primaxial derivatives keeping their own identity and fate, whereas they produce the abaxial derivatives responding to the lateral plate mesoderm.     

Anatomical specificity of regenerated muscle sensory afferents in the spinal cord of the bullfrog

The specificity of central projections made by regenerated muscle sensory fibers in the brachial spinal cord was studied with anatomical tracing methods. Sensory fibers were interrupted by freezing dorsal roots in postmetamorphic bullfrogs. After several months, regenerated sensory fibers were labeled with horseradish peroxidase applied to the triceps brachii muscle nerve, and their arborizations within the spinal cord were reconstructed from serial cross sections. Most of the regenerated projections from triceps muscle sensory afferents ended in or near their normal terminal field. A few branched and appeared to terminate more dorsally than normal, however, sometimes within the region where cutaneous afferents normally terminate. In contrast to the normal pathway followed by muscle afferents within the spinal cord, many regenerated afferents grew along the circumference of the spinal cord, just under the pial surface, and then turned abruptly toward the midline and into their appropriate terminal region. This suggests that regenerating afferents may actively seek out their appropriate targets and are not simply passively guided to them.     

Msx1‐2 immunolocalization in the regenerating tail of a lizard but not in the scarring limb suggests its involvement in the process of regeneration

The immunolocalization of the muscle segmental homoeobox protein Msx1‐2 of 27–34 kDa in the regenerating tail blastema of a lizard shows prevalent localization in the apical ependyma of the regenerating spinal cord and less intense labelling in the wound epidermis, in the apical epidermal peg (AEP), and in the regenerating segmental muscles. The AEP is a micro‐region of the regenerating epidermis located at the tail tip of the blastema, likely corresponding to the AEC of the amphibian blastema. No immunolabelling is present in the wound epidermis and scarring blastema of the limb at 18–21 days of regeneration, except for sparse repairing muscles. The presence of a proximal–distal gradient of Msx1‐2 protein, generated from the apical ependyma, is suggested by the intensity of immunolabelling. The AEP and the ependyma are believed to induce and maintain tail regeneration, and this study suggests that Msx1‐2 proteins are components of the signalling system that maintains active growth of the tail blastema. The lack of activation and production of Msx1‐2 protein in the limb are likely due to the intense inflammatory reaction following amputation. This study confirms that, like during regeneration in fishes and amphibians, also the blastema of lizards utilizes common signalling pathways for maintaining regeneration.     

Morphology of regenerated spinal cord in Sternarchus albifrons

Summary The tail of the gymnotid Sternarchus albifrons, including the spinal cord, regenerates following amputation. Regenerated spinal cord shows a rostro-caudal gradient of differentiation. Cross sections of the most distal regenerated cord show radially enlarged ependymal cells, relatively undifferentiated cells, and numerous blood vessels. More anterior sections contain well differentiated electromotor neurons, glial cells, and myelinated axons. The number of electromotor-neuron cell bodies in cross sections of regenerated spinal cord is three to six times the number in nonregenerated cord. Distinct tracts of axons, easily identifiable in normal cord, are not distinguishable in cross sections of regenerated cord. Some reorganization of the spinal cord also appears to take place anterior to the site of transection.Individual electromotor neurons in the regenerated spinal cord have morphologies largely similar to those of normal electrocytes, i.e., cell bodies are rounded, lack dendrites, have synapses characterized by gap junctions with presynaptic axons, and lack an unmyelinated initial segment. The presence of electromotor neurons with normal morphology in regenerated spinal cord correlates with the re-establishment of relatively normal electrocyte axonSchwann cell relationships in the regenerating electric organ of this sternarchid.Supported in part by the Medical Research Service, Veterans Administration and by a grant from the National Institutes of Health. We also thank the Paralyzed Veterans of America for their support. We thank Mary E. Smith and Susan Cameron for excellent technical support     

Neuroanatomical and functional analysis of neural tube formation in notochordless Xenopus embryos; laterality of the ventral spinal cord is lost.

Notochordless Xenopus embryos were produced by u.v. irradiation of the uncleaved fertilized egg. The spinal cords were examined using intermediate filament staining for glial cells, retrograde HRP staining for neuronal morphology and an anti-glycinergic antibody to reveal commissural cells and axons. The floorplate cells of the normal cord appear to be absent and their position along the ventral midline of the cord is occupied by motor neurones, Kolmer-Agduhr cells, radial glial cells and a ventrally placed marginal zone containing the longitudinal axons. Motor neurone number is reduced to 15% of control values, and the sensory extramedullary cell number is increased twentyfold. Commissural axons are still able to cross the ventral cord but do so at abnormal angles and some commissural axons continue to grow circumferentially up the contralateral side of the cord rather than turning to grow longitudinally. Extracellular electrophysiological recordings from motor axons reveal that the normal alternation of locomotor activity on the left and right side of the embryo is lost in notochordless animals. These results suggest that the notochord and/or the normal floor plate structure are important for the development of the laterality of spinal cord connections and may influence motor neurone proliferation or differentiation.     

The role of skin afferent in regulation of locomotion evoked by electrical epidural stimulation of spinal cord in decerebrated cats

The role of hindpaw skin afferent input in the locomotor pattern formation induced by epidural spinal cord stimulation was investigated in decerebrated cats. Locomotor activity was evoked by continuous 3-5Hz stimulation of dorsal surface of L4-L5 spinal segments. Kinematic and electromyographic activity (EMG) of m. Quadriceps, m. Semitendinosus, m. Tibialis anterior an m. Gastrocnemius lateralis before and after blocking of skin receptors in one hind limb were recorded. In addition, reflex responses in the hind limb muscles to epidural stimulation with frequency 0.5 Hz were analysed. Blocking of skin receptors of the foot with chlorothane paw irrigation or 2 % lidocaine administrated into the hind paw was performed. After blocking of skin receptors of the foot the stepping pattern changed. Stepping with dorsal foot placement and dragging during swing phase was observed. Duration of stance phase significantly decreased. Inhibition of polysynaptic activity of proximal and distal extensor muscles and distal flexor muscles of hind paw during locomotion was found. Monosynaptic responses after blocking of skin receptors of the foot changed insignificantly.     

Patterns of spinal sensory-motor connectivity prescribed by a dorsoventral positional template

Sensory-motor circuits in the spinal cord are constructed with a fine specificity that coordinates motor behavior, but the mechanisms that direct sensory connections with their motor neuron partners remain unclear. The dorsoventral settling position of motor pools in the spinal cord is known to match the distal-to-proximal position of their muscle targets in the limb, but the significance of invariant motor neuron positioning is unknown. An analysis of sensory-motor connectivity patterns in FoxP1 mutant mice, where motor neuron position has been scrambled, shows that the final pattern of sensory-motor connections is initiated by the projection of sensory axons to discrete dorsoventral domains of the spinal cord without regard for motor neuron subtype or, indeed, the presence of motor neurons. By implication, the clustering and dorsoventral settling position of motor neuron pools serve as a determinant of the pattern of sensory input specificity and thus motor coordination.     

The growth of motor axons in the spinal cord of Xenopus embryos

The innervation of the myotomal muscles in the trunk region of Xenopus embryos has been examined to see how the path taken by motoneurons within the spinal cord is formed. The growth of motor axons has been studied by retrograde labeling with horseradish peroxidase and the growth of the spinal cord and myotomes has been studied by labeling with fluorescent beads. Results show that motoneurons initially innervate the nearest muscles. Then through a process of differential growth whereby the muscles elongate more than the spinal cord, the axonal terminals in the muscles become displaced caudally relative to their cell bodies. In this manner the central pathway taken by the motor axons develops after initial innervation of their peripheral targets.     

Regulation after proximal or distal transposition of limb regeneration blastemas and determination of the proximal boundary of the regenerate.

When blastemas of several stages of differentiation were grafted in normal orientation to stump levels proximal or distal to their level of origin, normal limbs regenerated. Histological and autoradiographic studies of the development of these regulated limbs showed that the grafted blastemas formed only structures normally distal to their level of origin. In the case of a blastema transplanted proximally, regulation occurred by intercalary regeneration from the stump, whereas, when blastemas were transplanted distally, regulation appeared to take place within the blastema itself by a distal shift in its pattern of organization. The results suggest that the proximal limit of the limb regenerate is determined by level-specific properties of the limb cells but that these properties allow for interactions leading to regulation when different levels of stump and blastema are brought together.     

Functional and morphometric evaluation of end-to-side neurorrhaphy for muscle reinnervation

This study was undertaken to quantify the effect of motor collateral sprouting in an end-to-side repair model allowing end organ contact. Besides documentation of the functional outcome of muscle reinnervation by end-to-side neurorrhaphy, this experimental work was performed to determine possible downgrading effects to the donor nerve at end organ level. In 24 female New Zealand White rabbits, the motor nerve branch to the rectus femoris muscle of the right hindlimb was dissected, cut, and sutured end-to-side to the motor branch to the vastus medialis muscle after creating an epineural window. The 24 rabbits were divided into two groups of 12 each, with the second group receiving additional crush injury of the vastus branch. After a period of 8 months, maximum tetanic tension in the reinnervated rectus femoris and the vastus medialis muscles was determined. The contralateral healthy side served as control. The reinnervated rectus femoris muscle showed an average maximum tetanic force of 24.9 N (control 26.2 N, p = 0.7827), and the donor- vastus medialis muscle 11.0 N (control 7.3 N, p = 0.0223). There were no statistically significant differences between the two experimental groups (p = 0.9914). The average number of regenerated myelinated nerve fibers in the rectus femoris motor branch was 1,185 +/- 342 (control, 806 +/- 166), and the mean diameter was 4.6 +/- 0.6 microm (control, 9.4 +/- 1.0 microm). In the motor branch to the vastus medialis muscle, the mean fiber number proximal to the coaptation site was 1227 (+/-441), and decreased distal to the coaptation site to 795 (+/-270). The average difference of axon counts in the donor nerve proximal to distal regarding the repair site was 483.7 +/- 264.2. In the contralateral motor branch to the vastus medialis muscle, 540 (+/- 175) myelinated nerve fibers were counted. In nearly all cross-section specimens of the motor branch to the vastus medialis muscle, altered nerve fibers could be identified in one fascicle distal and proximal to the repair site. The results show a relevant functional reinnervation by end-to-side neurorrhaphy without functional impairment of the donor muscle. It seems to be evident that most axons in the attached segment were derived from collateral sprouts. Nonetheless, the present study confirms that end-to-side neurorrhaphy is a reliable method of reconstruction for damaged nerves, which should be applied clinically in a more extended manner.