Regenerating corticospinal fibers in the Marmoset (Callitrix jacchus) after spinal cord lesion and treatment with the anti-Nogo-A antibody IN-1

Neutralizing the myelin-associated growth inhibitor Nogo-A in adult spinal cord-injured rats can promote regeneration of injured and compensatory sprouting of uninjured axons. Nogo-A is present in humans, making its neutralization a possible novel treatment option for paraplegic patients. In this study we examined the effects of an extensively used anti-Nogo-A antibody (mAb IN-1) on the regenerative capabilities of lesioned corticospinal tract (CST) axons in a primate, the Marmoset monkey. Unilateral thoracic lesions of the CST were performed in six adult Marmosets, followed by the application of mAb IN-1 into the cerebrospinal fluid circulation by a graft of hybridoma cells. A unilateral injection of biotin dextran amine into the motor cortex was performed to analyse sprouting and regeneration of the lesioned axons. In the control antibody-treated animal CST fibers stopped rostral to the lesion site and often showed retraction bulbs. In contrast, in four out of five mAb IN-1-treated animals fine labeled neurites had grown into, through and around the lesion site. Thus, this study provides first anatomical evidence that in primates, the neutralization of the myelin-associated inhibitor Nogo-A results in increased regenerative sprouting and growth of lesioned spinal cord axons.     

The fate of severed corticospinal axons

The potential for regeneration of severed corticospinal axons was examined by labeling these axons with horseradish peroxidase following thoracic spinal cord transections in mice. Shortly after severance, the proximal ends of corticospinal axons formed terminal bulbs that persisted for weeks and were associated with axonal retraction. There were few signs of corticospinal axonal sprouting or elongation. By 2 months after injury, corticospinal axons near the transection site showed an increased number of probable labeled terminals in the adjacent gray matter. These new terminals may contribute to the persistence of many corticospinal axons near the injury site long after a spinal cord transection.     

Regeneration and Sprouting of Chronically Injured Corticospinal Tract Fibers in Adult Rats Promoted by NT-3 and the mAb IN-1, Which Neutralizes Myelin-Associated Neurite Growth Inhibitors

Myelin-associated inhibitors of neurite growth play an important role in the regenerative failure after injury in the adult mammalian CNS. The application of the mAb IN-1, which efficiently neutralizes the NI-250/35 inhibitory proteins, alone or in combination with neurotrophin-3 (NT-3), has been shown to promote axonal regeneration when applied in acute injury models. To test whether IN-1 application can induce axonal growth also in a chronic injury model, we treated rats with IN-1 and NT-3 starting 2 or 8 weeks after injury. Rats underwent bilateral dorsal hemisection of the spinal cord at the age of 5–6 weeks. Regeneration of corticospinal (CST) fibers into the caudal spinal cord was observed in three of eight of those animals with a 2-week delay between lesion and treatment. CST fibers regenerated for 2–11.4 mm. In the control group sprouting occurred rostral to the lesion but no long-distance regeneration occurred. In animals where treatment started at 8 weeks after injury the longest fibers observed grew up to 2 mm into the caudal spinal cord. The results show that transected corticospinal axons retain the ability to regenerate at least for a few weeks after injury. Functional analysis of these animals showed a slight improvement of functional recovery.     

Degeneration and sprouting of identified descending supraspinal axons after contusive spinal cord injury in the rat

Contusive spinal cord injury (SCI) results in the formation of a chronic lesion cavity surrounded by a rim of spared fibers. Tissue bridges containing axons extend from the spared rim into the cavity dividing it into chambers. Whether descending axons can grow into these trabeculae or whether fibers within the trabeculae are spared fibers remains unclear. The purposes of the present study were (1) to describe the initial axonal response to contusion injury in an identified axonal population, (2) to determine whether and when sprouts grow in the face of the expanding contusion cavity, and (3) in the long term, to see whether any of these sprouts might contribute to the axonal bundles that have been seen within the chronic contusion lesion cavity. The design of the experiment also allowed us to further characterize the development of the lesion cavity after injury. The corticospinal tract (CST) underwent extensive dieback after contusive SCI, with retraction bulbs present from 1 day to 8 months postinjury. CST sprouting occurred between 3 weeks and 3 months, with penetration of CST axons into the lesion matrix occurring over an even longer time course. Collateralization and penetration of reticulospinal fibers were observed at 3 months and were more extensive at later time points. This suggests that these two descending systems show a delayed regenerative response and do extend axons into the lesion cavity and that the endogenous repair can continue for a very long time after SCI.     

Differential effect of aging on axon sprouting and regenerative growth in spinal cord injury

The demographics of acute spinal cord injury (SCI) are changing with an increased incidence in older age. However, the influence of aging on the regenerative growth potential of central nervous system (CNS) axons following SCI is not known. We investigated axonal sprouting along with the efficiency of the infusion of the stromal cell-derived growth factor-1 (SDF-1/CXCL12) and regenerative growth along with the anti-scarring treatment (AST) in young (2–3 months) and geriatric (22–28 months) female rats following SCI. AST included local injection of iron chelator (2,2′-dipyridine-5,5′-dicarboxylic acid) and 8-bromo-cyclic adenosine monophosphate solution into the lesion core. Axon outgrowth was investigated by immunohistological methods at 5 weeks after a partial dorsal hemisection at thoracic level T8. We found that aging significantly reduces spontaneous axon sprouting of corticospinal (CST), serotonergic (5-HT) raphespinal and catecholaminergic (TH) coerulospinal tracts in distinct regions of the spinal cord rostral to the lesion. However, impairment of axon sprouting could be markedly attenuated in geriatric animals by local infusion of SDF-1. Unexpectedly and in contrast to rostral sprouting, aging does not diminish the regenerative growth capacity of 5-HT-, TH- and calcitonin gene-related peptide (CGRP)-immunoreactive axons at 5 weeks after SCI. Moreover, 5-HT and TH axons maintain the ability to react upon AST with significantly enhanced regeneration in aged animals. These data are the first to demonstrate, that old age compromises axonal plasticity, but not regenerative growth, after SCI in a fiber tract-specific manner. Furthermore, AST and SDF-1 infusions remain efficient, which implicates that therapy in elderly patients is still feasible.     

Brain-derived neurotrophic factor applied to the motor cortex promotes sprouting of corticospinal fibers but not regeneration into a peripheral nerve transplant

Previous experiments from our laboratory have shown that application of brain-derived neurotrophic factor (BDNF) to the red nucleus or the motor cortex stimulates an increase in the expression of regeneration-associated genes in rubrospinal and corticospinal neurons. Furthermore, we have previously shown that BDNF application stimulates regeneration of rubrospinal axons into a peripheral graft after a thoracic injury. The current study investigates whether application of BDNF to the motor cortex will facilitate regeneration of corticospinal neurons into a peripheral nerve graft placed into the thoracic spinal cord. In adult Sprague Dawley rats, the dorsal columns and the corticospinal tract between T9 and T10 were ablated by suction, and a 5-mm-long segment of predegenerated tibial nerve was autograft implanted into the lesion. With an osmotic pump, BDNF was infused directly into the parenchyma of the motor cortex for 14 days. Growth of the corticospinal tract into the nerve graft was then evaluated by transport of an anterograde tracer. Anterogradely labeled corticospinal fibers were not observed in the peripheral nerve graft in animals treated with saline or BDNF. Serotinergic and noradrenergic fibers, as well as peripheral sensory afferents, were observed to penetrate the graft, indicating the viability of the peripheral nerve graft as a permissive growth substrate for these specific fiber types. Although treatment of the corticospinal fibers with BDNF failed to produce regeneration into the graft, there was a distinct increase in the number of axonal sprouts rostral to the injury site. This indicates that treatment of corticospinal neurons with neurotrophins, e.g., BDNF, can be used to enhance sprouting of corticospinal axons within the spinal cord. Whether such sprouting leads to functional recovery after spinal cord injury is currently under investigation.     

PlexinA2 limits recovery from corticospinal axotomy by mediating oligodendrocyte-derived Sema6A growth inhibition

Axonal growth from both intact and severed fibers is limited after adult mammalian CNS injury. Myelin proteins contribute to inhibition of axonal growth. Semaphorin6A protein inhibits the extension of developing axons and is highly expressed in adult oligodendrocytes. This expression pattern suggests that a developmental axon guidance cue contributes to the restriction of adult CNS growth. Here, we assessed the role of a Sema6A receptor, PlexinA2, in recovery from adult trauma. Adult sensory neuron inhibition by Sema6A requires PlexinA2, with complete protection in PlexinA2-/- cultures. Mice lacking another myelin inhibitor receptor, NgR1, are known to exhibit greater axonal sprouting and functional recovery after lesions of the corticospinal tract at the medullary pyramid, so we investigated PlexinA2 in this lesion. Without injury, the corticofugal projection into the cervical spinal cord is normal in adult PlexinA2 null mice. After unilateral pyramidotomy, unlesioned PlexinA2-/- corticospinal fibers sprout across the midline to innervate the contralateral gray matter of the spinal cord to a significantly greater extent than do fibers in wild type mice. Sprouted fibers display frequent synaptophysin-positive synaptic puncta. The increased axonal growth in PlexinA2-/- mice after injury is accompanied by improved behavioral recovery in a pellet retrieval task using the impaired forelimb, and in a tape removal task. Thus, PlexinA2, as a receptor for oligodendrocyte-derived Sema6A and for secreted class 3 Semaphorins, plays a role in limiting adult axon growth and recovery after trauma.     

Combined with anti‐Nogo‐A antibody treatment,BDNF did not compensate the extra deleterious motor effect caused by large size cervical cord hemisection in adult macaques

In spinal cord injured adult mammals, neutralizing the neurite growth inhibitor Nogo‐A with antibodies promotes axonal regeneration and functional recovery, although axonal regeneration is limited in length. Neurotrophic factors such as BDNF stimulate neurite outgrowth and protect axotomized neurons. Can the effects obtained by neutralizing Nogo‐A, inducing an environment favorable for axonal sprouting, be strengthened by adding BDNF? A unilateral incomplete hemicord lesion at C7 level interrupted the main corticospinal component in three groups of adult macaque monkeys: control monkeys (n = 6), anti‐Nogo‐A antibody‐treated monkeys (n = 7), and anti‐Nogo‐A antibody and BDNF‐treated monkeys (n = 5). The functional recovery of manual dexterity was significantly different between the 3 groups of monkeys, the lowest in the control group. Whereas the anti‐Nogo‐A antibody‐treated animals returned to manual dexterity performances close to prelesion ones, irrespective of lesion size, both the control and the anti‐Nogo‐A/BDNF animals presented a limited functional recovery. In the control group, the limited spontaneous functional recovery depended on lesion size, a dependence absent in the combined treatment group (anti‐Nogo‐A antibody and BDNF). The functional recovery in the latter group was significantly lower than in anti‐Nogo‐A antibody‐treated monkeys, although the lesion was larger in three out of the five monkeys in the combined treatment group.     

Astrocyte progenitor transplantation promotes regeneration of bulbospinal respiratory axons,recovery of diaphragm function,and a reduced macrophage response following cervical spinal cord injury

Stem/progenitor cell transplantation delivery of astrocytes is a potentially powerful strategy for spinal cord injury (SCI). Axon extension into SCI lesions that occur spontaneously or in response to experimental manipulations is often observed along endogenous astrocyte “bridges,” suggesting that augmenting this response via astrocyte lineage transplantation can enhance axon regrowth. Given the importance of respiratory dysfunction post-SCI, we transplanted glial-restricted precursors (GRPs)—a class of lineage-restricted astrocyte progenitors—into the C2 hemisection model and evaluated effects on diaphragm function and the growth response of descending rostral ventral respiratory group (rVRG) axons that innervate phrenic motor neurons (PhMNs). GRPs survived long term and efficiently differentiated into astrocytes in injured spinal cord. GRPs promoted significant recovery of diaphragm electromyography amplitudes and stimulated robust regeneration of injured rVRG axons. Although rVRG fibers extended across the lesion, no regrowing axons re-entered caudal spinal cord to reinnervate PhMNs, suggesting that this regeneration response—although impressive—was not responsible for recovery. Within ipsilateral C3-5 ventral horn (PhMN location), GRPs induced substantial sprouting of spared fibers originating in contralateral rVRG and 5-HT axons that are important for regulating PhMN excitability; this sprouting was likely involved in functional effects of GRPs. Finally, GRPs reduced the macrophage response (which plays a key role in inducing axon retraction and limiting regrowth) both within the hemisection and at intact caudal spinal cord surrounding PhMNs. These findings demonstrate that astrocyte progenitor transplantation promotes significant plasticity of rVRG-PhMN circuitry and restoration of diaphragm function and suggest that these effects may be in part through immunomodulation.     

Chronically injured corticospinal axons do not cross large spinal lesion gaps after a multifactorial transplantation strategy using olfactory ensheathing cell/olfactory nerve fibroblast-biomatrix bridges

Transplantation of mixed cultures containing olfactory ensheathing cell (OEC) and olfactory nerve fibroblasts (ONF) has been shown to stimulate regrowth of both acutely and chronically injured corticospinal (CS) axons across small spinal cord lesion gaps. Here, we used a multifactorial transplantation strategy to stimulate regrowth of chronically injured CS axons across large spinal cord lesion gaps. This strategy combined the transplantation of aligned OEC/ONF-biomatrix complexes, as described previously (Deumens et al. [2004] Neuroscience 125:591-604), within the lesion gap with additional OEC/ONF injections rostral and caudal to the lesion site. We show an enhanced presence of injured CS axons directly rostral to the lesion gap, with no effects on injured CS axons at or caudal to the lesion gap. Furthermore, injured CS axons did not penetrate the OEC/ONF-biomatrix complex within the lesion gap. The enhanced presence of CS axons rostral to the lesion gap was not accompanied by any recovery of behavioral parameters assessed with the BBB locomotor rating scale or CatWalk gait analysis. We conclude that our multifactorial transplantation strategy should be optimized to create an OEC/ONF continuum in the injured spinal cord and thereby stimulate regrowth of injured CS axons across large spinal lesion gaps.     

Morphological and electrophysiological study of distal motor nerve fiber degeneration and sprouting after irreversible cholinesterase inhibition

A single subcutaneous injection of a sublethal dose of the irreversible organophosphate sarin (0.08 mg/kg) in rats induced a non-Wallerian-type axonal degeneration of the neuromuscular synapse in the slow twitch, soleus muscle. These alterations of the endplate region were more obvious in the soleus than in the fast extensor digitorum longus muscle and were slowly reversible, complete recovery requiring about 10 days. Silver-cholinesterase staining and electrophysiological techniques were used to define the spatiotemporal evolution of prejunctional abnormalities. The non-Wallerian-type axonal degeneration of the neuromuscular synapse was characterized by bead or balloon-like varicosities of the focal, distal, and terminal nerve fibers and a retraction of terminal axons. Axonal degeneration was accompanied by junctional and extrajunctional membrane depolarization and was followed by nerve sprouting at focal, distal, and terminal nerve fibers. Transients similar to miniature endplate potentials were recorded along the muscle fiber at distances of 800-2500 microns away from the parent endplate. New ectopic endings, originating from the same endplate, were discovered adjacent to the terminal axon and also distant from the parent endplate. Very elaborate terminal arborization and occasional multibranching arose from a progressive growth sprout. The new sprouting may have served to compensate for the loss of synaptic contact caused by sarin. Thus the present study demonstrates a direct cytotoxic effect of sarin and indicates that this organophosphate agent may be an important neurotoxicological tool to understand the mechanisms involved in nerve sprouting.     

Nogo-A antibody improves regeneration and locomotion of spinal cord-injured rats

Spinal cord trauma leads to loss of motor, sensory and autonomic functions below the lesion. Recovery is very restricted, due in part to neurite growth inhibitory myelin proteins, in particular Nogo-A. Two neutralizing antibodies against Nogo-A were used to study recovery and axonal regeneration after spinal cord lesions. Three months old Lewis rats were tested in sensory-motor tasks (open field locomotion, crossing of ladder rungs and narrow beams, the CatWalk(R) runway, reactions to heat and von Frey hairs). A T-shaped lesion was made at T8, and an intrathecal catheter delivered highly purified anti-Nogo-A monoclonal IgGs or unspecific IgGs for 2 weeks. A better outcome in motor behavior was obtained as early as two weeks after lesion in the animals receiving the Nogo-A antibodies. Withdrawal responses to heat and mechanical stimuli were not different between the groups. Histology showed enhanced regeneration of corticospinal axons in the anti-Nogo-A antibody groups. fMRI revealed significant cortical responses to stimulation of the hindpaw exclusively in anti-Nogo-A animals. These results demonstrate that neutralization of the neurite growth inhibitor Nogo-A by intrathecal antibodies leads to enhanced regeneration and reorganization of the injured CNS, resulting in improved recovery of compromised functions in the absence of dysfunctions.     

The pattern of dendritic sprouting and retraction induced by axotomy of lamprey central neurons

We have investigated some of the factors controlling the distribution of axonal and dendritic sprouting following axotomy of a subset of Muller giant interneurons (anterior bulbar cells or ABCs) in the hindbrain of the larval sea lamprey (Petromyzon marinus). Sprouts originated from different sites in the cell depending on the distance of the axonal lesion from the soma. When the axon was cut close to the soma (within 500 microns), the dendritic tips sprouted profusely, whereas the proximal axon stump showed few sprouts and frequently disappeared entirely. Axotomy further from the soma (1000-1400 microns) resulted in less sprouting from the dendrites and more from the axon stump, with the total amount of dendritic plus axonal sprouting remaining constant. Axotomy at sites distant from the soma (1 cm or more) did not result in dendritic sprouting. No sprouts were ever observed emerging from the soma proper or from the axon stump except at the lesion site. Neuritic sprouts from dendrites and axon were similar in their gross morphology. Sprouts resembled axons rather than dendrites whatever their sites of origin; they followed linear, rostrocaudally oriented paths in the "basal plate" region of the hindbrain. Dendritic and axonal sprouts grew both rostrally and caudally within the brain. Either "close" or "distant" axotomy resulted in the retraction of the dendritic tree and of both dendritic and axonal sprouts by several months postaxotomy. Reaxotomy close to the soma 30 d after a distant axotomy accelerated the onset of this evoked dendritic retraction. Reaxotomy close to the soma also induced sprouting significantly sooner than did close axotomy alone. These results suggest that axotomy close to the soma causes axonal regeneration to be shunted into ectopic locations at the dendritic tips. The emerging sprouts then follow guidance cues appropriate for regenerating ABC axons.     

Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons

Several diseases and injuries of the central nervous system could potentially be treated by delivery of an enzyme, which might most effectively be achieved by gene therapy. In particular, the bacterial enzyme chondroitinase ABC is beneficial in animal models of spinal cord injury. We have adapted the chondroitinase gene so that it can direct secretion of active chondroitinase from mammalian cells, and inserted it into lentiviral vectors. When injected into adult rat brain, these vectors lead to extensive secretion of chondroitinase, both locally and from long-distance axon projections, with activity persisting for more than 4 weeks. In animals which received a simultaneous lesion of the corticospinal tract, the vector reduced axonal die-back and promoted sprouting and short-range regeneration of corticospinal axons. The same beneficial effects on damaged corticospinal axons were observed in animals which received the chondroitinase lentiviral vector directly into the vicinity of a spinal cord lesion.     

Ectopic expression of the neural cell adhesion molecule L1 in astrocytes leads to changes in the development of the corticospinal tract

The cell recognition molecule L1, of the immunoglobulin superfamily, participates in the formation of the nervous system and has been shown to enhance cell migration and neurite outgrowth in vitro. To test whether ectopic expression of L1 would influence axonal outgrowth in vivo, we studied the development of the corticospinal tract in transgenic mice expressing L1 in astrocytes under the control of the GFAP-promoter. Corticospinal axons innervate their targets by extending collateral branches interstitially along the axon shaft following a precise spatio-temporal pattern. Using DiI as an anterograde tracer, we found that in the transgenic animals, corticospinal axons appear to be defasciculated, reach their targets sooner and form collateral branches innervating the basilar pons at earlier developmental stages and more diffusely than in wild type littermates. Collateral branches in the transgenic mice did not start out as distinct rostral and caudal sets, but they branched from the axon segments in a continuous rostrocaudal direction across the entire region of the corticospinal tract overlying the basilar pons. The ectopic branches are transient and no longer present at postnatal day 22. The earlier outgrowth and altered branching pattern of corticospinal axons in the transgenics is accompanied by an earlier differentiation of astrocytes. Taken together, our observations provide evidence that the ectopic expression of L1 on astrocytes causes an earlier differentiation of these cells, results in faster progression of corticospinal axons and influences the branching pattern of corticospinal axons innervating the basilar pons.     

Modification of the regenerative response of dorsal column axons by olfactory ensheathing cells or peripheral axotomy in adult rat

The regeneration of sciatic-dorsal column (DC) axons following DC crush injury and treatment with olfactory ensheathing cells (OECs) and/or sciatic axotomy ("conditioning lesion") was evaluated. Sciatic-DC axons were examined with a transganglionic tracer, cholera toxin conjugated to horseradish peroxidase, and evaluated at chronic time points, 2-26 weeks post-lesion. With DC injury alone (n = 7), sciatic-DC axons were localized to the caudal border of the lesion terminating in reactive end bulbs with no indication of growth into the lesion. In contrast, treatment with either a heterogeneous population of OECs (equal numbers of p75- and fibronectin-positive OECs) (n = 9) or an enriched population of OECs (75% p75-positive OECs) (n = 6) injected either directly into the lesion or 1-mm rostral and caudal to the injury, stimulated DC axon growth into the lesion. A similar regenerative response was observed with a conditioning lesion either concurrent to (n = 4) or 1 week before (n = 4) the DC injury. In either of the latter two paradigms, some DC axons grew across the injury, but no axons grew into the rostral intact spinal cord. Upon combining OEC treatment with the conditioning lesion (n = 21), the result was additive, increasing DC axon growth beyond the rostral border of the lesion in best cases. Additional factors that may limit DC regeneration were tested including formation of the glial scar (immunoreactivity to glial fibrillary acidic protein in astrocytes and to chondroitin sulfate proteoglycans), which remained similar between treated and untreated groups.     

Olfactory ensheathing cells, olfactory nerve fibroblasts and biomatrices to promote long-distance axon regrowth and functional recovery in the dorsally hemisected adult rat spinal cord

Cellular transplantation, including olfactory ensheathing cells (OEC) and olfactory nerve fibroblasts (ONF), after experimental spinal cord injury in the rat has previously resulted in regrowth of severed corticospinal (CS) axons across small lesion gaps and partial functional recovery. In order to stimulate CS axon regrowth across large lesion gaps, we used a multifactorial transplantation strategy to create an OEC/ONF continuum in spinal cords with a 2-mm-long dorsal hemisection lesion gap. This strategy involved the use of aligned OEC/ONF-poly(D,L)-lactide biomatrix bridges within the lesion gap and OEC/ONF injections at 1 mm rostral and caudal to the lesion gap. In order to test the effects of this complete strategy, control animals only received injections with culture medium rostral and caudal to the lesion gap. Anatomically, our multifactorial intervention resulted in an enhanced presence of injured CS axons directly rostral to the lesion gap (65.0 +/- 12.8% in transplanted animals versus 13.1 +/- 3.9% in control animals). No regrowth of these axons was observed through the lesion site, which may be related to a lack of OEC/ONF survival on the biomatrices. Furthermore, a 10-fold increase of neurofilament-positive axon ingrowth into the lesion site as compared to untreated control animals was observed. With the use of quantitative gait analysis, a modest recovery in stride length and swing speed of the hind limbs was observed. Although multifactorial strategies may be needed to stimulate repair of large spinal lesion gaps, we conclude that the combined use of OEC/ONF and poly(D,L)-lactide biomatrices is rather limited.     

Collateral sprouting in the electrosensory lateral line lobe of weakly electric teleosts (Gymnotiformes) following ricin ablation

Sprouted collateral axons were observed in the electrosensory lateral line lobe (ELL) of gymnotiform teleosts (Apteronotus leptorhynchus:) following the ablation of the supraorbital branch of the anterior lateral line nerve. Ablation was accomplished by using microinjections of the toxic lectin ricin. Sprouted axons were followed for up to 26 weeks postablation. Ricin exposure severely reduced axonal numbers and the peripheral electroreceptors in the region innervated by these fibers. To visualize sprouted fibers, intact lateral line afferent nerve branches were anterogradely labelled with the neuronal tract tracers horseradish peroxidase or cobalt chloride, or the monoclonal antibody Q26A3. Within the four somatotopically organized ELL segments, sprouted collaterals were first observed two weeks after ricin injection in the medial and centromedial segments, and four weeks postinjection in the centrolateral and lateral segments. Sprouting involved intrasegmental, horizontally directed axons from adjacent nerve branch terminal fields, and mixed intra-and extrasegmental, dorsally directed axons from the ELL deep fiber layer. The sprouting response was robust but variable in its timing, peaking between 6 and 12 weeks. Subsequently, the intrasegmental, horizontally directed fibers were retained but the mixed dorsally directed fibers, including all extrasegmental axons, were retracted. Therefore, this sprouting response appears to consist of a collateral overproduction followed by a selective axonal retraction. In our view, the most likely explanation for this axonal retraction is that the descending inputs from the isthmus and the cerebellum, as well as commissural fibers from the contralateral ELL, maintain established somatotopic relationships by eliminating somatotopically mismatched sprouted collaterals. © 1993 Wiley-Liss, Inc.