Formation of functional endplates by spinal axons regenerating through a peripheral nerve graft. A study in the adult rat

Brain Research Bulk-tin, Vol. 22, pp. 103-I 14. B Pergamon Press plc, 1989. Printed in the U.S.A.

0361.9230/89 $3.00 + .OO

Formation of Functional Endplates by Spinal Axons Regenerating Through a Peripheral Nerve Graft. A Study in the Adult Rat J. C. HORVAT,*f’ J. C. MIRAtS

M. PECOT-DECHAVASSINE,$ AND Y. DAVARPANAH*

*Laboratoire Biologie- VertPbrPs, Universite’ Paris Sud, F-91405 Orsay TLaboratoire de Neurobiologie, Universite’ Rent! Descartes, 45 rue des Saints-Pkres, F-75006 Paris $DPpartement de Cytologie, Institut des Neurosciences du CNRS Universite’ Pierre et Marie Curie, 7 Quai Saint-Bernard, F-75005 Paris

HORVAT, J. C., M. PECOT-DECHAVASSINE, J. C. MIRA AND Y. DAVARPANAH. Formation of functional endplates by spinal axons regenerating through a peripheral nerve graft. A study in the adult rat. BRAIN RES BULL 22(l) 103-l 14, 1989. -Peripheral nerve (PN) autografts were used in the adult rat to join the midcervical spinal cord to a nearby denervated skeletal muscle. Retrograde tracing, morphological and electrophysiological studies indicated the following: 1) a great number of neurons, located bilaterally, between C3 and C7, in most laminae of the grey matter, extended axons into the PN grafts, 2) a lesser number of neurons regenerated up to the reconnected muscle, but most of them were typical motoneurons, 3) neuromuscular junctions were formed in ectopic locations, around the tip of the grafted nerve, and at the sites of original endplates, 4) these junctions were functional and formed by axons that had regenerated into the PN bridges, as muscle contraction was obtained by electrical stimulation of the grafted nerves, 5) they were proved to be cholinergic since endplate potentials, evoked by stimulating the PN graft, were suppressed by curare. These results strongly suggest that spinal neurons, and especially motoneurons, are involved in the formation, through PN bridges, of new functional cholinergic connections with denervated skeletal muscles. Spinal neurons Retrograde axonal

Transplantation of peripheral Electrophysiological tracing

nerve Axonal recordings

regeneration

Motor

endplates

IN adult mammals, spontaneous lengthy regrowth of cut axons is known to occur in the peripheral nervous system and thus may lead to restoration of peripheral connectivity and function. In contrast, regeneration of axons injured in the central nervous system (CNS) is generally abortive and usually fails to reestablish synaptic contacts with target neurons. Yet, most CNS neurons of the adult rat were shown to have the actual capability to regrow lengthy axons along “blindended” peripheral nerve (PN) grafts, thus providing evidence of the determining influence of the local environment on axonal elongation (1). This beneficial effect of the nonneuronal components of PN grafts on axonal extension was more likely to be observed for short distances between neuronal somata and the site of injury and grafting (29). In this way, PN autografts to the spinal cord were reinnervated by numerous axons originating from spinal segments adjacent to the grafted nerve

and not by cortico-spinal axons, for instance, whose neuronal somata, located in the motor cortex, were probably too remote (30). Using PN segments whose both ends were left “open,” axonal regrowth from central neurons could be directed, through these “bridges,” to other parts of the CNS (12) and lead to the formation of synaptic connections with target neurons, as was recently demonstrated for retinal ganglion cells reconnected, in this way, to the superior colliculus (35). Anticipating that, under similar experimental conditions, the formation of synaptic contacts with peripheral targets would be likely to occur, PN bridges were used in the present experiments to join the cervical spinal cord of the adult rat to a nearby skeletal muscle which was denervated at the time of PN grafting. Blind-ended grafts, similarly implanted into the spinal cord, were also used to compare axonal regrowth in connected and unconnected PN conduits.

‘Requests Saints-Peres,

Universite

for reprints should F-75006 Paris.

be addressed

to Dr. Jean-Claude

Horvat,

103

RenC Descartes,

Laboratoire

de Neurobiologie,

45 rue des

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ET AL.

Thus, the main objectives of these studies were 1) to reevaluate the regrowth capabilities of spinal neurons, and especially of motoneurons of the ventral horn, into PN bridges connected to peripheral targets, such as those offered by a denervated muscle, comparatively to blind-ended PN grafts, 2) to investigate the ability of the axons, which were expected to regenerate into the PN bridges, to establish functional synaptic connections with the denervated muscle and, 3) to determine which neuronal populations are involved in the restoration of motor function, if actually observed. In addition, since cortico-spinal axons were likely to be injured by the grafting procedure, its possible, though unexpected (30), participation to the reinnervation of the PN grafts was checked in a few animals. Parts of this work have been presented as short accounts in preliminary notes (19,20). METHOD

Fifty-one young female Sprague-Dawley rats, weighing 200250 g, were used in the present experiments. Prior to any surgical procedure, the animals were anaesthetized with a 7% solution of chloral hydrate (0.6 ml intraperitoneal/lOO g body weight). Selection of Spinal and Muscular Grafting Sites To anticipate an optimal axonal regrowth from spinal neurons, and especially from motoneurons of the ventral horn, the PN graft was inserted into the cervical enlargement which contains a great number of these neurons (Fig. lb). The longissimus atlantis (LA) muscle was chosen both for its proximity to the spinal grafting site, in relation with the length of the PN graft, and for its suitability for electrophysiological studies, as it is a thin muscle composed of parallel fibres. This muscle is part of a deep layer of the dorsal musculature and stretches from the atlas to the first thoracic vertebrae. Surgical Procedures The dorsal musculature was widely exposed in the cervical and upper thoracic regions. Nerve grafting (Fig. 2a, 6, c), After laminectomy of vertebrae C4 or C.5, one end of a 25-30 mm segment of the common peroneal nerve, removed from the leg, was introduced into the cervical spinal cord through a small incision of the meninges, 0.5-l mm lateral to the dorsal midline, mostly on the right side. This end of the nerve was pushed l-l.5 mm deep with the help of a glass rod, towards the ventral horn of the spinal grey matter. It was secured to the dura with a 10.0 suture. The other end was either tightly ligatured and anchored at the same time to nearby muscles with a 5.0 suture (blind-ended grafts: group A, 5 rats), or driven through several layers of the dorsal musculature, then inserted into a transversal groove made in an aneural region of the caudal part of the LA muscle, close to the myotendinous line. The wound in the muscle was closed around the nerve end with two 10.0 sutures, one of them stitching up the perineurium at the same time (PN bridges: group B, 27 rats). Denervation of the LA muscle. Just prior to PN grafting, the LA muscle was carefully denervated by ligaturing each of its two intrinsic nerves (Fig. la) with three successive 7.0 sutures and transecting them between the two more distal ones. This procedure was adopted in the experimental series as it proved fully efficient in 7 control rats examined from 3 to 28 weeks later: under these conditions, deep muscular atrophy and degeneration of motor endplates were consistently observed.

FIG. I. Schematic representation of the cervical spinal cord (SC) before (a) and after (b) denervation and PN grafting. a: Intrinsic nerves (N) to the rostra1 (r) and caudal (c) parts of the LA muscle (M). b: PN graft (G) joining the cervical spinal cord to the caudal part of the LA muscle. Retrograde labelling from transected intrinsic nerves (in a) or PN graft (in b) led to neuronal labelling within the black or grey areas, respectively. Cl, C5: cervical levels of the spinal cord.

Examination of the Grafted Animals. The animals were examined from 6 weeks to 5 months (group A) and from 2 to 7 months (group B) postoperatively. No apparent sensorimotor impairment was observed during the survival period. Preliminary test (group B). In the anaesthetized animal, the PN bridge was dissected along its course up to the LA muscle and cleared from scar tissue. In response to an electrical stimulus (2-10 volts; 0.2-l msec) applied to the PN graft, full or partial contraction of the LA muscle was searched for under the dissecting microscope. A positive response was a prerequisite for further studies. In the subgroup Bl (23 rats), the PN bridge was then transected 15-20 mm away from its spinal insertion. Its distal part together with the LA muscle to which it remained connected were removed and transferred in a physiological solution. In 8 of these animals, the proximal stump of the PN graft was used for retrograde labelling studies (Fig. 2b). In subgroup B2 (4 animals), the PN bridge was left intact and anatomical tracers were injected into the LA muscle (Fig. 2~). Retrograde labelling studies. I. HRP application to the proximal stump of transected nerves or PN grafts. The following sequence was applied to retransected blind-ended PN grafts (group A) (Fig. 2a) or PN bridges (8 animals in subgroup Bl), as well as to transected intrinsic nerves of the LA muscle in 2 control rats. The cut end of the nerves was placed on a parafilm sheet and surrounded with petroleum jelly to avoid tracer contamination of the neighbouring tissues. Then, a small gelfoam (Upjohn) pledget, soaked in a 30% HRP (Sigma type VI) solution, was left in contact with the tip of the nerve or of the PN graft for one hour (3). After that, the exposed area was washed out several times with saline. 48 hr later, the animals were perfused through the heart with saline, then with a 3% glutaraldehyde solution in 0.1 M phosphate buffer.

SPINAL AXON REGENERATION

Enzyme activity (27) was searched for in 40 micrometres cryostat cross sections of the brain (group A and 4 rats in subgroup Bl to check for a possible, though unlikely, labelling in the motor cortex). of the whole cervical suinal cord (grotto A. group B and two control animals) and, in a few instances, of the spinal ganglia adjacent to the grafting site. All sections were counterstained with neutral red. 2. HRP or Fast Blue (FB) injection into the reconnected LA muscle (Fig. 2~). The tracer (30% HRP or 3% FB solutions) was injected into the LA muscle (5x3 ~1) around the site of graft implantation (subgroup B2). When HRP was used (3 animals), further processing was similar to that described above. 3. Double labelling study. When FB was injected into the muscle (1 animal), the nerve bridge was transected one week later and a 3% Nuclear Yellow (NY) solution was applied for 45 minutes to its proximal stump with the same precautions as those taken for HRP application. Forty to forty-eight hours later, the rat was perfused with chilled solutions: 0.9% saline followed by 10% paraformaldehyde in 0.1 M phosphate buffer, then by 10% buffered paraformaldehyde in 10% sucrose. Thirty micrometres thick cryostat sections of the cervical spinal cord, mounted with di-n-butylphtalate xylene (DPX), were examined and photographed under epifluorescent illumination at 360 nm (4, 21, 24, 32). Electrophysiological

Recordings

In subgroup Bl, the PN graft-muscle preparation was removed from the animal, transferred in a chamber and bathed in an oxygenated physiological solution (25). The nerve graft was stimulated, by means of a glass suction electrode, with current pulses of varying intensity (2-10 volts) and duration (0.21 msec). Intracellular recordings of the variations in muscle membrane potentials (muscle action potentials, endplate potentials and miniature endplate potentials) were made with conventional electrophysiological techniques using microelectrodes filled with a 3M KC1 solution (7-10 MB). When endplate potentials were recorded, curare (2-8. lo-’ M) was added to the bathing physiological solution. Morphology

of the Neuromuscular

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Junctions

The neuromuscular junctions were identified and localized by light microscopy in LA muscles fixed with a 10% formaldehyde solution and stained for cholinesterase activity (10,22). Their ultrastructure was revealed in E.M. after fixation with a 3% glutaraldehyde solution in 0.2 M cacodylate buffer, postfixation with buffered 2% osmium tetroxide and embedding in Spurr or araldite resins. The recycling of synaptic vesicles, which attests the actual neurosecretory activity of nerve terminals (17), was documented by looking for HRP incorporation into synaptic vesicles under long-lasting stimulation of the PN graft. For this purpose, the muscles were soaked in a HRP solution (10 mg/ml). After 30 minutes rest, they were stimulated at 2 Hz during one hour. The muscles were then fixed and dissected into thin blocks containing endplates. HRP activity was revealed with 3-3’-diaminobenzidine in tris-HCl buffer at pH 7.6 (15). Further processing was as described above. Histology of the PN Grafts In some rats of group A and subgroup Bl, a short segment of the distal stump of the PN graft, which was retransected at

the time of tracer application, was removed and processed for E.M. according to conventional techniques. RESULTS

Gross Anatomical Findings in Grafted Animals Following dissection in anaesthetized rats (and/or at autopsy), the grafted nerves were seen to have remained firmly attached to the dorsal aspect of the cervical spinal cord, with which they were in gross continuity (groups A and B), as well as to the LA muscle (group B). With rare exceptions, the PN bridges had a healthy appearance along their entire course from the cervical spinal cord to the reconnected LA muscle. Retrograde Labelling Studies Origin of the intrinsic innervation of the LA muscle (Fig. la). Dissection of the intrinsic nerves of the LA muscle up to the spinal cord (one rat) indicated that they arise from spinal segments Cl and C2 and innervate respectively the cranial and the caudal portions of the LA muscle which are separated by a myotendinous line. HRP application (two rats) to the proximal stump of these nerves, which were transected close to their muscular targets, led to ipsilateral labelling of motoneurons (Fig. 5c) located at the very tip of the ventral horn and arranged in a narrow and continuous band, about 4 mm long, extending rostro-caudally along spinal segments Cl and C2. Fifty-seven labelled motoneurons were counted in one animal, 43 in the other. Origin of the axons which had regrown into the PN grafts. In bright field examination of serial cross sections of the spinal cord, made in the region of implantation, the tip of the PN graft was found to lie into the dorsal half of the operated side, namely into the dorsal horn of the grey matter. Cysts and cavitation of the spinal tissue were inconsistently seen in the vicinity of the grafted nerves. I. Blind-ended grafts (group A, 5 animals). This preliminary study was aimed at determining the localization, distribution and number of spinal neurons, and possibly of cortico-spinal neurons, which were expected to extend axons into blind-ended grafts (which, as a direct consequence, lacked peripheral connections). The distribution and number of neurons retrogradely labelled after application of HRP to the extraspinal portion of the grafts were as follows: none in the motor cortex and 985 (average: meankSEM=197+91, range 52 to 503) in the cervical spinal cord. Their approximate position with regard to the central canal appears in the diagram (Fig. 2d). They are distribute i on both sides of the grey matter, and lie in most laminae of Rexed (28), with the possible exception for more distal laminae I and II. The greater labelling density is observed in the intermediate area of the grey matter. The rostro-caudal distribution of these HRP labelled neurons is shown in Fig. 3a. Their number falls with increasing distance from the implantation site, but labelled neurons have been encountered as far as 5 mm rostrally and 6 mm caudally. 2. PN bridges. Subgroup BI. The HRP labelling studies performed in 8 rats of this subgroup were made for comparison, not only with group A, but also with subgroup B2 in which the tracer was injected into the reconnected LA muscle. Here, HRP was applied to the proximal stump of the transected PN bridge (Fig. 2b). No labelled neurons were encountered in the motor cortex. A total of 1818 HRP labelled cells (average: 227+62, range 26 to 415) were counted in the spinal grey matter. The distribution

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FIG. 2. Diagrammatic representation of retrograde labelling studies. a, b, c: tracer application in experimental animals of groups A, Bl and B2, respectively. d, e, f: diagrams of neuronal labelling in group A (985 cells), Bl (1818 cells) and B2 (374 cells), respectively. Each labelled neuron is represented by a dot whose approximate position is defined with respect to the central canal. For each group, all labelled cells encountered in serial cross sections of the cervical spinal cord of all animals

are collected on the same diagram. HRP: horseradish peroxidase, FB: Fast Blue, G: peripheral

nerve graft,

SC: spinal cord, M: LA muscle.

ET AL.

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C

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of these cells was roughly similar to that observed in group A (Fig. 2e). However, the highest density of labelled neurons was observed somewhat more ventrally, probably in relation with a slight shift in the positioning of the intraspinal tip of the graft in this group. In addition, most cells were observed very close to the transplanted nerve (62% were within 1 mm from the center of the graft), many of them appearing scattered on the contralateral side, between the central canal and the lower part of the ventral horn. Yet, some labelled neurons could be seen as far as 8 mm from the graft, both rostrally and caudally (Fig. 3b). Subgroup 82. In the 4 rats of this subgroup, the tracer (HRP, 3 animals or FB, 1 animal) was injected into the reconnected LA muscle, around the site of implantation of the PN grafts. Thus, the tracer was picked up by terminals of axons which had actually regenerated up to the muscle and not by cut axons in the PN graft as in subgroup Bl. A total of 374 cells (average: 93549, range 27 to 210), either labelled with HRP or with FB, were counted. As shown in the diagram (Fig. 2f), labelled neurons are widely distributed throughout the grey matter, but the highest density is clearly seen in the ventralmost part of the ventral horn (54% of the labelled neurons). Moreover, in two animals in which HRP was used, labelling was strictly confined to the ventral horn and most, if not all, of the labelled cells (32 and 105 respectively)

FIG. 3. Rostro-caudal distribution of total numbers of labelled neurons in groups A (a), Bl (b) and B2 (c). Negative or positive values correspond to rostra1 (R) or caudal (C) parts of the cervical spinal cord with regard to the centre of the implanted PN tip. Ordinate: number of labelled neurons: Abscissa: millimetres from the graft.

could be identified as typical motoneurons (Fig. 4). nearly 96% of the 374 labelled cells counted in the 4 rats were located within 3 mm apart from the injury site, but a few labelled neurons could be seen as far as 7 mm rostrally and 4 mm caudally (Fig. 3~). In the only animal of this subgroup in which FB was used, 28 of the 210 FB labelled neurons were also stained with NY which had been applied to the transected PN bridge one week following FB injection into the LA muscle (Fig. 5e). They were scattered among the whole FB singly labelled neuronal population. In addition, 13 other neurons, singly labelled with NY, were also encountered, but they were not seen close to motoneurons of the ventral horn; these cells do not appear in the diagram (Fig. 2f) and are not taken into account in the histogram (Fig. 3~). When it was checked (a few instances in both experimental groups A and B), HRP labelled neurons were also observed in the spinal ganglia adjacent to the site of injury and grafting.

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FIG. 4. Diagrams of neuronal labelling after intramuscular injection of Fast Blue (a) or HRP (b, c, d) in each of the 4 animals of group B2. For additional explanations, refer to Fig. 2. Numerous labelled neurons are located either in the ipsilateral or in the contralateral ventral horn with regard to the implantation site (here, on the right). In c and d, no labelling is seen outside this region of the grey matter.

Labelling of individual neurons. In group A and subgroup Bl, individual HRP labelled neurons appeared generally heavily loaded with chromogen granules filling the perikaryal cytoplasm as well as proximal segments of dendrites and axons. Large HRP labelled neurons of the ventral horn could be clearly identified as motoneurons, owing to their location, size and shape which were similar to that of nearby unquestionable motoneurons stained with neutral red only (Fig. 5a, d). From them, one and sometimes two neuronal projections, filled with chromogen granules, could occasionally be traced to the grafted nerve (Fig. Sa). Labelled neuronal projections were often seen close to the central canal, crossing from the contralateral side (Fig. 5b). HRP neuronal labelling from the reconnected muscle was, on the whole, weaker than that resulting from application of the tracer to the transected PN graft and was generally observable as scattered minute granules. However, similar labelling procedure with FB led to brillant and strong staining of both neuronal somata and projections. Histology of the PN Grafts The overall appearance of cross semithin sections of blindended grafts as well as of PN bridges was that of typical peripheral nerves, well revascularized, in the process of regeneration (Fig. 6a). Thin sections observed in E.M. showed numerous myelinated and unmyelinated axons ensheathed by Schwann cells (Fig. 6b). Electrophysioiogical and Morphological Studies of the Connections Between the PN Graft and the LA Muscle Determination of the onset of reinnervation. The chronology of reinnervation was searched for in a special series of 9 grafted rats examined from postoperative days 20 to 33. The earliest signs of contraction and/or the first muscle fibre potentials, evoked by electrical stimulation of the PN graft, were detected during the fifth postoperative week. Consequently, the shortest delay for examining the experimental rats, fixed at 2

months, was considered as long enough to ensure a widespread pattern of reinnervation throughout the muscle. Functional activity of the new motor endplates. A perceptible contraction of the reconnected LA muscle in response to an adequate electrical stimulus applied, in situ, to the intact PN bridge, was a prerequisite for further studies. Indeed, a positive response was obtained in 24 out of 27 rats checked from 2 to 7 months after grafting, by stimulating any point of the PN bridge as well as the ventral part of the spinal cord close to the tip of the grafted nerve. Failure to respond to electrical stimulus in 3 rats was thought to be related either to an illlooking PN graft (1 rat) or to mishandling (2 rats). In isolated PN graft-muscle preparations, action potentials, evoked by electrical stimulation of the graft, were recorded in most muscle fibres (Fig. 7b). Typical spontaneous miniature endplate potentials were obtained at focal sites, either around the region of nerve implantation or in the region of original endplates (Fig. 7a). Endplate potentials, evoked by stimulating the PN graft, were also recorded in both areas after adding curare (2.10-’ M) to the medium (Fig. 7~). The cholinergic nature of the transmission was further demonstrated by the gradual disappearance of the endplate potentials with increasing concentrations of curare from 2 to 8. lop7 M (Fig. 7~). In addition, the functional capacity of the new endplates could also be documented by the capability of the nerve endings to recycle synaptic vesicles. This was tested by the actual labelling of some of these vesicles with HRP, after long stimulation of the graft-muscle preparation which was immersed into the enzyme solution (Fig. 8). Distribution and appearance of the neuromuscular junctions. In whole mount preparations of the normal LA muscle, processed for cholinesterase activity, the endplates appeared distributed along two narrow transverse bands: one was located in the rostra1 part, the other in the caudal part of the LA muscle and were both roughly parallel to the medial line of myotendinous junctions (Fig. 9a). In the reconnected muscles, the new motor endplates were not only located in the regions of original innervation, but also around the site of PN grafting and at places in between (Fig. 9b). Some of them had the typical appearance of normal endplates, displaying continuous and ramified, “pretzel-like,” synaptic gutters and subneural lamellae (Fig. 10a). Others were more elongated and made of a series of separated cupules (Fig. lob). These atypical neuromuscular junctions looked like ectopic endplates (23) or endplates which had been remodeled consecutively to a muscle injury (9). Both types of endplates were observed at any place of the whole reinnervated area. Two main types of neuromuscular junctions were also found in E.M. In some of them, the synaptic gutter was deep and displayed numerous and well developed synaptic folds, characteristic of a normal subneural apparatus (Fig. 10~). In some others, the synaptic gutter was shallow and folds were either lacking or poorly developed (Fig. 10d). In most instances, both types of subsynaptic structures were in contact with normal looking axon terminals where usual organelles, and namely clear synaptic vesicles, were present. Yet, a number of them were either deprived of any kind of presynaptic structure (Fig. llb, c) or partially occupied with small atypical nerve endings (Fig. 1la). DISCUSSION

Our overall results provide additional evidence that various neuronal populations of the injured CNS of adult mammals have the actual capability to initiate and sustain axonal re-

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FIG. 5. Retrograde neuronal labelling in the cervical spinal cord. Upper part of photographs is dorsal with regard to the lower part. a: Site of injury and grafting. HRP labelled (thick arrows) and neutral red stained (arrowheads) motoneurons in the ipsilateral ventral horn. Labelled projections, extending from a motoneuron or crossing from the contralateral side (thin arrows), close to the central canal, are seen converging towards the nerve graft (G). Bar: 100 micrometres. b: Labelled axons (arrows), likely originating from nearby labelled neurons in the contralateral side, are seen crossing by the flattened central canal. Bar: 50 micrometres. c: HRP labelled motoneurons after application of the tracer to the intrinsic nerves of the LA muscle. Bar: 20 micrometres. d: HRP labelled motoneurons after injection of the tracer into the reconnected LA muscle. Bar: 20 micrometres. e: A double labelled motoneuron after intramuscular injection of Fast Blue followed, one week later, by application of Nuclear Yellow to the retransected PN bridge. Bar: 20 micrometres.

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FIG. 6. Cross sections of the distal part of the PN graft, 5 months after grafting. a: Semithin section. The overall appearance is that of a tvnical regenerating neriuheral nerve. Toluidine Blue. Bar: 10 micrometres. b: Electron micrograph revealing myelinated and unmyelinated nerve fibres. Bar: 1 micrometre.

growth into peripheral conduits (1) and to establish synaptic contacts with specific targets (35). The following discussion will be conducted along four main lines: 1) axonal regrowth into blind-ended and bridging PN grafts, 2) formation of neuromuscular junctions by ingrown axons, 3) identification of the neurons involved in the return of motor function, and 4) neuronal damage and regeneration. Axonal Regrowth into Blind-Ended and Bridging PN Grafts Cortico-spinal neurons. Our unsuccessful1 attempts at labelling cortico-spinal neurons from cervical PN blind-ended grafts as well as from PN bridging grafts, are consistent with earlier experiments which emphasized the role of the distance between the neuronal somata and the size of injury and grafting (29). Indeed, these neurons have been shown to reinnervate PN grafts inserted close to their somata, that means directly into the motor cortex (18), but failed to extend axons into PN grafts to the thoracic spinal cord (30). Spinal neurons. The main results of our labelling studies indicate that PN grafts probably act in a nonspecific way on the general axonal regrowth of a great variety of spinal neurons, as they appear to favour nearby neurons rather than particular neuronal classes. These data are in good agreement with previous investigations showing that, after injury to the cord, spinal neurons could regrow lengthy axons into nearby PN conduits such as a ventral root (31), reimplanted dorsal (7) or ventral roots (8), a blind-ended PN graft (29) or a PN bridge (12,13). Besides, comparative examination of diagrams and histograms corresponding to blind-ended (group A) and bridging grafts (subgroup Bl) reveals a striking similarity between both labelling patterns in the spinal grey matter, despite minor differences. In addition, the individual counts of labelled neurons in both groups are within the same range. This suggests that the reconnected skeletal muscle has probably little influence, if any, on the recruitment of neurons which are likely to extend axons into the PN grafts.

Formation of Functional Neuromuscular Junctions by Ingrown Axons At the time of PN grafting, the indigenous nerve fibres of the PN segments are ineluctably bound to degenerate, since they are separated from their neuronal somata. Consequently, axons which were encountered in the PN grafts a few weeks or months following transplantation were regenerated axons. Thus, contraction of the LA muscle under electrical stimulation either of the PN bridge in situ or of the distal nerve stump which remained connected to the muscle in in vitro preparations, necessarily implies that at least some of the regenerated axons in the PN graft had actually established functional neuromuscular junctions. In fact, both electrophysiological and morphological data provide abundant documentation upon the reality of such functional contacts. In this respect, the presence of numerous ectopic endplates which appeared scattered around the tip of the grafted nerve strongly suggests a morphological continuity between regenerated axons in the PN graft and synaptic structures within the muscle. The appearance of these neuromuscular junctions was that of newly formed ectopic endplates (23). These endplates were proved to be functional for at least two reasons: 1) endplate potentials evoked by stimulating the PN graft could be recorded in this primitively aneural region, and 2) some of the original endplates, though deprived of any presynaptic element, displayed a typical subsynaptic organization which is known to persist when the corresponding muscle fibres are reinnervated at ectopic sites (5, 26, 36). Morphological and functional connectivity between regenerated axons in the PN graft and synaptic structures within the muscle was also documented in the region of original endplates. Here, endplate potentials could be recorded in response to PN graft stimulation and reinnervated endplates were observed in E.M. Acetylcholinesterase activity revealed that endplates had either the typical “pretzel-like” appearance of normal endplates or that of deeply modified junctions with numerous cupules spreading along the muscle fibres. These atypical features likely resulted from the remodeling of the original endplates consecu-

HORVAT

ET AL.

FIG. 7. Electrophysiological recordings. a: Miniature endplate potentials recorded in continuous sweep. Calibrations: 0.5 mV-5 msec. b: Muscle fibre action potential evoked by stimulating the nerve graft. Calibrations: 20 mV-2 msec. c: Superimposed endplate potentials recorded in the same site as the miniature endplate potentials in a after addition of curare to the medium. The amplitude is gradually reduced with increasing concentrations of curare from 2 to 8. lo-’ M. Calibrations: 2 mV-2 msec.

tively to the lesion of the muscle fibres that occurred at the time of grafting (9). The functional capacity of ectopic and reinnervated endplates to release cholinergic transmitter was further demonstrated, firstly, by the actual recycling of synaptic vesicles in nerve terminals under PN graft stimulation and, secondly, by the gradual disappearance of endplate potentials evoked by stimulating the nerve graft with increasing concentrations of curare.

Identification of the Neurons Involved in the Return of Motor Function In a general way, PN autografts to the adult mammalian CNS are known to be repopulated not only by intrinsic “central” axons, but also by a great number of “peripheral” axons from nearby meninges, blood vessels or spinal cord (1). Thus, contraction of the reconnected muscle under electrical stimu-

lation of the PN graft could result, at least in part, from the actual recruitment of such peripheral nerve fibres. Conversely, application of tracers to the proximal stump of the retransected PN bridges led to the retrograde labelling of spinal neurons which had extended axons at least to the site of tracer application, and likely farther. However, these labelling studies from the grafted nerves do not provide absolute evidence that this axonal regrowth from the CNS has actually resulted in the formation of these neuromuscular junctions revealed by electrophysiological and morphological observations. Absolute proof of the muscle reinnervation by intrinsic neurons of the injured spinal cord would be produced by documenting the morphological and functional continuity between a given neuronal soma and a particular endplate. Adequate methodology for such demonstration was not brought into play in the present investigation. Yet, a number of converging arguments obtained from our experimental data considered together, strongly suggest the participation of intrinsic spinal neurons,

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*_

mt

.-

.c

‘_

C

FIG. 8. Nerve terminal in a reconnected LA muscle after stimulation of the PN bridge at 1 Hz for 60 minutes in presence of HRP (10 mg/ml). Some synaptic vesicles containing HRP reaction product (arrow). Bar: 0.1 micrometre.

especially of large motoneurons of the ventral horn nuclei, to the reformed peripheral connectivity. 1) Except for the stimulation of the PN graft, muscle contraction could be obtained by electrical stimulation of the sole ventral region of the spinal cord close to the tip of the grafted nerve. In addition, subsequent mechanical disruption of this region abolished all kinds of response to renewed stimulation of this damaged spinal tissue, and led within two days to a degenerating process of some motor endplates. 2) Intramuscular injection of the tracers (HRP or FB) led to the labelling of a smaller number of neurons than that obtained after tracer application to retransected PN bridges, but most of them were located in the ventralmost part of the spinal grey matter and could be identified as typical motoneurons. This observation suggests that these particular neurons were more likely to grow axons up to the muscle than the other spinal neurons. HRP is known to be avidly picked up by axon terminals in the CNS (34) as well as in the periphery, especially at the endplate level (37) and also, though probably to a lesser extent, by undamaged axons along their entire course in the CNS (6) or in the periphery (2). In this respect, it can be assumed that, in our labelling experiments from the reconnected muscle, HRP was picked up mainly by axon terminals issued from motoneurons, and, to a lesser degree, by unconnected axons along their intramuscular course. Yet, this assumption does not exclude that a) axons which have extended from other labelled neurons and/or axons of peripheral origin, might also participate in the formation of new motor endplates, b) some motoneurons might grow axons to the muscle, but fail to establish synaptic contacts. In addition, double labelling studies indicate that most, if

FIG. 9. Schematic representation of the distribution of the endplates in the LA muscle (visualization by cholinesterase activity). a: Control muscle. The endplates are distributed along two transverse bands roughly parallel to the medial line of myotendinous junctions (mt). b: Reconnected muscle. The endplates are located not only at the sites of original innervation, but also around the tip of the grafted nerve (G), especially in the caudal part of the muscle where the graft was inserted. Many endplates display an elongated shape.

not all, of the axons which had reached the muscle actually coursed through the PN bridge. 3) The gradual disappearance of endplate potentials with increasing concentrations of curare demonstrates the cholinergic nature of the transmission at the new synaptic sites. This is in favour of the actual participation of the motoneurons in the muscle reinnervation; however, it cannot be excluded that other spinal cholinergic neurons might also participate in the reinnervation process. 4) In those rats where labelling studies were performed, contraction of the LA muscle under electrical stimulation of the PN bridge was always correlated with the presence of labelled neurons in ipsilateral and/or contralateral ventral horns. Taken together, these arguments strongly suggest that most of the motoneurons which have regrown axons into the graft have, in addition, established synaptic contacts with the reconnected muscle. Whether other spinal neurons, which were also labelled in the intermediate or dorsal grey matter as well as in spinal ganglia, are endowed of the same capability to make peripheral connections, still remains to be documented. In our experimental model, the somata of those motoneurons which were proved to grow axons into the PN bridges, and most likely to make neuromuscular connections, were located in spinal segments (C3 to C7) different from those (Cl and C2) which contained the motoneurons giving rise to the intrinsic innervation of the LA muscle (Fig. 1). This absence of overlapping is in favour of a lack of segmental specificity

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a 0,

FIG. 10. Endplates of the reconnected LA muscle. a, b: Light microscopy of cholinesterase activity. Bar: 10 micrometres. a: Normal endplate with a typical “pretzel-like” appearance. b: Atypical endplate, elongated and made of separate cupules. c, d: Thin cross sections in E.M. In c, the endplate displays numerous and deep subsynaptic folds that are not seen in the other one (d). Bar: 0.5 micrometre.

between muscles,

particular motoneurons and corresponding at least within the cervical spinal cord.

skeletal

Neuronal Damage and Regeneration A recent experimentation has provided evidence that axonal regrowth into PN grafts to the adult mammalian CNS is more likely to result from the extension of damaged main axons or damaged collateral branches rather than from uninjured neurons (14,33). In reason of their obvious interest in this study, discussion of this point will be restricted to spinal motoneurons. In our study, the original stem motoraxon was probably left undamaged in most motoneurons that extended axons into the PN grafts as 1) the corresponding ventral roots were left intact, 2) the PN graft was inserted through a dorsal approach into the dorsal horn of the spinal cord, 3) a great number of these neu-

rons were seen regenerating from the contralateral side and crossing by the central canal, 4) some motoneurons were labelled as far as several millimetres from the site of injury and grafting, and 5) intrinsic axotomized motoneurons of the reinnervated muscle were not labelled. However, if terminal regeneration from the main axon that courses ventrally towards the adjoining ventral root was, thus unlikely to occur, axonal regrowth might have originated from damaged intraspinal collateral branches similar to those observed in the cat (11). An additional possibility is that supernumerary axons, originating from the cell body (16), might have developed and grown into the PN graft. This is actually suggested by some of our observations in the region of the PN graft insertion where one, occasionally two projections were seen coursing from the motoneuronal soma to the intraspinal tip of the grafted nerve. Whether such axonal development needed damage to axonal

SPINAL AXON REGENERATION

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INTO PN GRAFTS

FIG. 11. Thin cross sections of endplates at the site of original innervation. The endplates display a typical subsynaptic organization but, in a, the synaptic gutter is partially occupied by a small nerve terminal (arrow), while in b and c, it is deprived of any presynaptic element. In b, a cell process, probably issued from a Schwann cell, is in close contact with the postsynaptic membrane and occupies a part of the gutter. Bar: 0.5 micrometre.

and/or collateral systems (16) is likely but difficult to establish. Whatever process may be involved in the axonal regrowth, the question whether some motoneurons could both make functional connections with the experimental muscle and, at the same time, retain their original control on the skeletal muscles they normally innervate, is exciting to consider but difficult to answer.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Ministere de la Recherche et de la Technologie (MRT 85-C-l 193). We thank Jorge Diaz and Marie-Odile Brouard (Departement de Cytologie, Universite Pierre et Marie Curie), Josette Legagneux (Laboratoire de Microchirurgie des Hopitaux de Paris) and Marie-Odile Aubert (Laboratoire d’Entomologie, Universite Paris Sud) for helpful assistance.

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