Axonal regeneration proceeds through specific axonal fusion in transected C. elegans neurons

Functional neuronal recovery following injury arises when severed axons reconnect with their targets. In Caenorhabditis elegans following laser‐induced axotomy, the axon still attached to the cell body is able to regrow and reconnect with its separated distal fragment. Here we show that reconnection of separated axon fragments during regeneration of C. elegans mechanosensory neurons occurs through a mechanism of axonal fusion, which prevents Wallerian degeneration of the distal fragment. Through electron microscopy analysis and imaging with the photoconvertible fluorescent protein Kaede, we show that the fusion process re‐establishes membrane continuity and repristinates anterograde and retrograde cytoplasmic diffusion. We also provide evidence that axonal fusion occurs with a remarkable level of accuracy, with the proximal re‐growing axon recognizing its own separated distal fragment. Thus, efficient axonal regeneration can occur by selective reconnection and fusion of separated axonal fragments beyond an injury site, with restoration of the damaged neuronal tract. Developmental Dynamics 240:1365–1372, 2011. © 2011 Wiley‐Liss, Inc.     

Axonal cytoskeletal changes after non-disruptive axonal injury

In animal models of human diffuse axonal injury, axonal swellings leading to secondary axotomy occur between 2 and 6 h after injury. But, analysis of cytoskeletal changes associated with secondary axotomy has not been undertaken. We have carried out a quantitative analysis of cytoskeletal changes in a model of diffuse axonal injury 4 h after stretch-injury to adult guinea-pig optic nerves. The major site of axonal damage was the middle portion of the nerve. There was a statistically significant increase in the proportion of small axons with a diameter of 0.5 m and smaller in which there was compaction of neurofilaments. Axons with a diameter greater than 2.0 m demonstrated an increased spacing between cytoskeletal elements throughout the length of the nerve. However, in the middle segment of the nerve these larger axons demonstrated two different types of response. Either, where periaxonal spaces occurred, there was a reduction in axonal calibre, compaction of neurofilaments but no change in their number, and a loss of microtubules. Or, where intramyelinic spaces occurred there was an increased spacing between neurofilaments and microtubules with a significant loss in the number of both. Longitudinal sections showed foci of compaction of neurofilaments interspersed between regions where axonal structure was apparently normal. Neurofilament compaction was correlated with disruption of the axolemma at these foci present some hours after injury. We suggest that the time course of these axonal cytoskeletal changes after stretch-injury to central axons is shorter than those changes documented to occur during Wallerian degeneration.     

Retrograde degeneration of myelinated axons and re-organization in the optic nerves of adult frogs (Xenopus laevis) following nerve injury or tectal ablation

Summary The optic nerve proximal to the lesion (toward the retina) was examined by light and electron microscopy in adultXenopus laevis after various types of injury to optic nerve fibres. Intraorbital resection, transection or crush of the optic nerve or ablation of the contralateral optic tectum all resulted in marked alterations in the myelinated axon population and in the overall appearance of the nerve proximal to the site of injury. Examination of the nerves from 3 days to 6 months postoperatively indicated that a progressive, retrograde degeneration of myelin and loss of large-diameter axons occurred throughout the retinal nerve stump regardless of the type of injury or distance of the injury from the retina. The retinal stump of nerves receiving resection or transection showed a nearly complete loss of myelin and large-diameter axons while the degree of degeneration was subtotal in nerves receiving crush injury or after lesions farther from the retina (i.e. tectal ablation). In addition, the entire retinal nerve stump after all types of injury was characterized by the appearance of an actively growing axon population situated circumferentially under the glia limitans. The latter fibres are believed to represent regrowing axons which are being added onto the nerve, external to the original axon population and are suspected to modify actively the glial terrain and glia limitans.     

Focal axonal injury: the early axonal response to stretch

Summary The development of a model for axonal injury in the optic nerve of the guinea pig has allowed analysis of early morphological changes within damaged axons. We provide evidence that the initial site of damage after stretch is the nodes of Ranvier, some of which develop nodal blebs. The development of nodal blebs is correlated with the loss of subaxolemmal density, disruption of the neurofilament cytoskeleton and aggregation of membranous profiles of smooth endoplasmic reticulum. Nodal blebs are numerous 15 min after injury but less so at later survivals. The glial-axonal junction is intact at early survivals in damaged nodes. Marked accumulation of membranous organelles occurs in the paranodal and internodal regions adjacent to damaged nodes between two and six hours and is correlated with disruption of the myelin sheath. Axotomy and the formation of degeneration bulbs occurs between 24 and 72 h. The area of axonal injury is invaded by phagocytic cells by 72 h and large numbers of myelin figures occur within the neuropil until 14 days.The results are compared with those of other studies of diffuse axonal injury and other neuropathies. The time course of axonal changes is more rapid than during Wallerian degeneration. Our data from longer surviving animals is exactly comparable with published data. We are confident that the principal site of axonal injury is the node of Ranvier. We suggest that damage at the node results in disruption of axonal transport, which in turn leads to a cascade of events, culminating in axotomy between 24 and 72 h after the initial insult.     

Identified septohippocampal neurons survive axotomy: a fine-structural analysis in the rat.

Previous studies have indicated that interruption of the connections between the medial septum and hippocampus by cutting the axons results in degeneration and death of the projecting septal neurons. However, in these studies cell death has been inferred primarily from the loss of immunoreactivity for transmitter-specific enzymes. In the present study, we labeled septohippocampal projection neurons by retrograde tracing and then cut their axons. Subsequent intracellular injection of prelabeled cells revealed the morphology of the soma and dendrites and allowed us to examine the ultrastructure of these neurons. A large number of septohippocampal neurons survived even 10 weeks after axotomy, suggesting that axotomized septohippocampal neurons survive for considerable periods beyond the time at which they stop expressing transmitter-specific immunoreactivity. Survival of axotomized neurons is a prerequisite for pharmacological interference aimed at reactivating transmitter expression, axonal re-growth, and the eventual reintegration into functionally relevant circuitries.     

Rescuing axons from degeneration does not affect retinal ganglion cell death

After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage.     

Axon Reactive B Cells Clonally Expanded in the Cerebrospinal Fluid of Patients with Multiple Sclerosis

Demyelination and axonal loss have been described as the histological hallmarks of inflammatory lesions of multiple sclerosis (MS) and are the pathological correlates of persistent disability. However, the immune mechanisms underlying axonal damage in MS remain unknown. Here, we report the use of single chain-variable domain fragments (scFv) from clonally expanded cerebrospinal fluid (CSF) B cells to show the role of an anti-axon immune response in the central nervous system (CNS) in MS. The cellular and subcellular distribution of the antigen(s) recognized by these CSF-derived clonal scFv antibodies (CSFC-scFv Abs) was studied by immunochemical staining of brain tissues obtained at autopsy from patients with MS. Immunochemistry showed specific binding of CSFC-scFv Abs to axons in acute MS lesions. The stained axons showed three major types of axonal pathological changes: 1) linear axons, axonal ovoid formation, and axonal transection were seen in the myelinated white matter adjacent to the lesion; 2) accumulation of axonal ovoid formations and Wallerian degeneration were seen at the border between demyelinated lesions and the adjacent white matter; and 3) Wallerian degeneration occurred at the center and edge of acute demyelinated lesions. These findings suggest a B cell axonal specific immune response in the CNS in MS.     

Cytoskeletal changes correlated with the loss of neuronal polarity in axotomized lamprey central neurons

Axotomy within 500 m of the soma (close axotomy) causes identified neurons (anterior bulbar cells or ABCs) in the lamprey hindbrain to lose their normal polarity and regenerate axons ectopically from dendritic tips, while axotomy at more distal sites (distant axotomy) results in orthotopic axonal regeneration from the axon stump. We performed immunocytochemical, electron microscopic and in situ hybridization analyses comparing ABCs subjected to close and distant axotomy to elucidate the mechanism by which neuronal polarity is lost. We show that polarity loss in ABCs is selectively and invariably preceded and accompained by the following cellular changes: (1) a loss of many dendritic microtubules and their replacement with neurofilaments, (2) a loss of immunostaining for acetylated tubulin in the soma and proximal dendrites, and (3) an increase of immunostaining for phosphorylated neurofilaments in the distal dendrites. We also show that these changes do not depend on either the upregulation or spatial redistribution of neurofilament message, and thus must involve changes in the routing of neurofilament protein within axotomized ABCs. We conclude that close axotomy causes dendrites to undergo axonlike changes in the mechanisms that govern the somatofugal transport of neurofilament protein, and suggest that these changes require the reorganization of dendritic microtubules. We also suggest that the bulbous morphology and lack of f-actin in the tips of all regenerating sprouts supports the possibility that axonal regeneration in the lamprey CNS does not involve actin-mediated "pulling" of growth cones, but depends instead on the generation of internal extrusive forces.     

Early and Sustained Activation of Autophagy in Degenerating Axons after Spinal Cord Injury

Axonal degeneration is one of the initial steps in many neurological disorders and has been associated with increased autophagic activity. Although there are increasing data on the regulation of autophagy proteins in the neuronal soma after spinal cord injury (SCI), their characterization in the axon is scarce. Here, we examined the regulation of autophagy during axonal degeneration in a rat model of SCI following a lesion at Th 8. We analyzed the morphological and ultrastructural changes in injured axons by immunohistochemical evaluation of autophagy‐related proteins and electron microscopy at different time points following SCI. The expression of ULK1, Atg7 and Atg5 in damaged axons was rapidly upregulated within hours after SCI. The number of axonal LC3‐positive autophagosomes was also rapidly increased after SCI and remained at an increased level for up to 6 weeks. Ultrastructural analysis showed early signs of axonal degeneration and increased autophagy. In conclusion, we show that autophagy is increased early and for a sustained period in degenerating axons after SCI and that it might be an important executive step involved in axonal degeneration. Therefore, autophagy may represent a promising target for future therapeutic interventions in the treatment of axonal degeneration in traumatic central nervous system disorders.     

Neurites undergoing Wallerian degeneration show an apoptotic-like process with Annexin V positive staining and loss of mitochondrial membrane potential

Wallerian degeneration, the disintegration of the distal part of an injured axon, is an important event in many neurodegenerative diseases. We studied Wallerian degeneration in dorsal root ganglion (DRG) explants in culture by separating neurites from their cell bodies with a scalpel. The severed neurites showed Annexin V positive staining, that spreads distally with a rate comparable to that of slow axonal transport in intact neurons in vivo. Moreover, the injured neurites showed loss of mitochondrial membrane potential. These features resemble those seen when cells undergo apoptosis. These data contribute to a new understanding of the mechanism of axonal degeneration, have implications for the response of stromal cells in central nervous system (CNS) and raise the prospect of new pharmacological treatments for those neurodegenerative pathologies where the protection of the cell body alone does not alleviate the disease.     

Emergence of highly neurofilament-immunoreactive zipper-like axon segments at the transection site in scalpel-cordotomized adult rats

Following transection of the spinal cord, severed axonal ends retract from the lesion site and attempt regeneration within 24 h of injury. Molecular mechanisms underlying such rapid axonal reactions after severance are not fully characterized so far. To better understand the early axonal degenerating and regenerating processes, we examined the immunohistological expression of axonal cytoskeletal proteins from 5 min to 48 h after scalpel-transection of adult rat spinal cord white matter. Within 30 min of transection, expression of neurofilament (NF)- and peripherin-like immunoreactivity (-IR) was enhanced in severed axonal ends, which conversely lost beta-III-tubulin-IR expression, indicating differential expression of beta-III-tubulin-IR and NF/peripherin-IR. During the next few hours, the strongly-NF/peripherin-IR-positive severed axonal ends adhered to each other and these cytoskeletal alterations expanded bi-directionally (rostro-caudally) 100-300 microm away from the transection point. Within 6 h of transection, secondary axotomy occurred at about 300 microm-rostral and -caudal to the primary transection point, which finally formed strongly-NF/peripherin-IR-positive zipper-like axon segments at the transection site. Notably, sprouting of secondarily severed axons was observed within 6 h of injury. The regenerative axons, which extended toward the transection site, could not traverse the transection site where the zipper-like axon segments resided. The zipper-like axon segments showed abnormal axolemmal permeability through the leakage of an axonal tracer. Western blot analysis revealed a slight increase in peripherin content in transected spinal cord. Local treatment with cycloheximide suppressed the axotomy-induced peripherin-IR-enhancement in severed ends, suggesting the occurrence of intra-axonal peripherin synthesis in vivo. Treatment with calpain inhibitors frequently formed abnormally swollen microtubule-free ends, which suggests that calpain-activation is critical for functional growth cone formation in adult rat spinal cord. These observations indicate that adult rat cordotomy with a scalpel results in the rapid formation of intensely NF-IR-positive zipper-like axon segments at the transection site, which are similar to "preserved fibers" reported by Ramon y Cajal [Ramon y Cajal S (1928) Degeneration and regeneration in the nervous system. New York: Hafner]. On the other hand, axonal regenerative responses start within 6 h of injury, which may be supported by calpain-activation and intra-axonal protein synthesis.     

Re-expression of p75NTR by adult motor neurons after axotomy is triggered by retrograde transport of a positive signal from axons regrowing through damaged or denervated peripheral nerve tissue.

To investigate different types of potential signalling mechanisms that regulate neuronal reactions to axotomizing injury, we compared the re-expression of the low-affinity neurotrophin receptor, p75NTR, and the down-regulation of choline acetyltransferase expression, after various combinations of axotomy, crush injury and blockade of axonal transport in adult hypoglossal motor neurons in the rat. We found that pure axotomy in the absence of crush injury down-regulated choline acetyltransferase, but did not induce p75NTR re-expression. Blockade of axonal transport with colchicine had an identical effect. In contrast, both a crush injury on its own, or a crush injury proximal to a complete axotomy, induced p75NTR re-expression and down-regulated expression of choline acetyltransferase. Blockade of axonal transport with colchicine or tight ligation proximal to a crush prevented the crush injury-induced re-expression of p75NTR. Infusion of vehicle, nerve growth factor or ciliary neurotrophic factor induced low levels of p75NTR re-expression that were not significantly different from each other and were substantially lower than crush-induced levels. These findings confirm previous suggestions that the loss of choline acetyltransferase expression is due to the interruption of a constitutive retrograde signal, and show that the re-expression of p75NTR by adult motor neurons after axotomy is triggered by the retrograde transport of a positive signal derived from axons that are regrowing through damaged or denervated peripheral nerve tissue. The precise source and nature of this signal are not yet clear.     

Spatiotemporal localization of injury potentials in DRG neurons during vincristine-induced axonal degeneration

The distal to proximal degeneration of axons, or "dying back" is a common pattern of neuropathology in many diseases of the PNS and CNS. A long-standing debate has centered on whether this pattern of neurodegeneration is due to an insult to the cell body or to the axon itself, although it is likely that mechanisms are different for specific disease entities. We have addressed this question in a model system of vincristine-induced axonal degeneration. Here, we created a novel experimental apparatus combining a microfluidic divider with a multielectrode array substrate to allow for independent monitoring of injury-induced electrical activity from dorsal root ganglion (DRG) cell bodies and axons while isolating them into their own culture microenvironments. At specified doses, exposure of the cell body to vincristine caused neither morphological neurodegeneration nor persistent hyperexcitability. In comparison, exposure of the distal axon to the same dose of vincristine first caused a decrease in the excitability of the axon and then axonal degeneration in a dying back pattern. Additionally, exposure of axons to vincristine caused an initial period of hyperexcitability in the cell bodies, suggesting that a signal is transmitted from the distal axon to the soma during the progression of vincristine-induced axonal degeneration. These data support the proposition that vincristine has a direct neurotoxic effect on the axon.     

大脑锥体束华勒氏变性影像学研究进展

原发性脑损伤可导致病灶远端的锥体束发生华勒氏病变,基于磁共振技术(magnetic resonance imaging,MRI)的弥散加权成像(diffusion weighted imaging,DWI)、弥散张量成像(diffusion tensor imaging,DTI)等成像技术对于华勒氏变性的检测有不同的特点和优势,本文针对华勒氏变性各个阶段不同的影像学表现进行了综述,并对临床检测早期华勒氏变性做出了指导。     

A qualitative and quantitative analysis of the response of the retinal ganglion cell soma after stretch injury to the adult guinea-pig optic nerve

Summary The development of a model for focal axonal injury in the optic nerve of the adult guinea-pig has allowed a qualitative and quantitative analysis of the response of the retinal ganglion cell soma to this type of injury. Large and medium sized retinal ganglion cells show classic central chromatolysis in about 30% of ganglion cells between three and seven days after injury, a high proportion of which undergo degeneration between seven and 14 days. Small ganglion cells and small neurons do not demonstrate any morphological response to stretch injury of the optic nerve. However, a small number of larger ganglion cells demonstrate enlargement of the cell soma and nucleolus together with reconstitution of the rough endoplasmic reticulum between seven and 14 days after stretch injury. We suggest that these cells are either recovering from or regenerating after a non-disruptive lesion to their axons. We suggest that some of these morphological changes parallel documented regenerative responses in peripheral/extrinsic neurons after injury to their axons. We conclude that the time course of the axon reaction after stretch injury to axons is longer than that obtained after crush or transection. We provide good morphological evidence that the level of injury after application of non-disruptive mechanical strain to axons is less severe than in the former two models of axonal injury and that a proportion of damaged neurons do not die but rather demonstrate either/or recovery or a regenerative response.     

Wallerian Degeneration: A Major Component of Early Axonal Pathology in Multiple Sclerosis

Axonal loss is a major component of the pathology of multiple sclerosis (MS) and the morphological basis of permanent clinical disability. It occurs in demyelinating plaques but also in the so‐called normal‐appearing white matter (NAWM). However, the contribution of Wallerian degeneration to axonal pathology is not known. Here, we analyzed the extent of Wallerian degeneration and axonal pathology in periplaque white matter (PPWM) and lesions in early multiple sclerosis biopsy tissue from 63 MS patients. Wallerian degeneration was visualized using an antibody against the neuropeptide Y receptor Y1 (NPY‐Y1R). The number of SMI‐32‐positive axons with non‐phosphorylated neurofilaments was significantly higher in both PPWM and plaques compared to control white matter. APP‐positive, acutely damaged axons were found in significantly higher numbers in plaques compared to PPWM. Strikingly, the number of NPY‐Y1R‐positive axons undergoing Wallerian degeneration was significantly higher in PPWM and plaques than in control WM. NPY‐Y1R‐positive axons in PPWM were strongly correlated to those in the lesions. Our results show that Wallerian degeneration is a major component of axonal pathology in the periplaque white matter in early MS. It may contribute to radiological changes observed in early MS and most likely plays a major role in the development of disability.     

A morphometric study of the retinal ganglion cell response to optic nerve severance in the frog Rana pipiens

Summary This study examines the cell body response to axotomy of retinal ganglion cells in the frogRana pipiens. Cell soma sizes were measured in carefully matched regions of Nissl-stained wholemounted retinae after either nerve crush, nerve cut with stump separation, nerve crush with intraocular nerve growth factor (NGF) or nerve cut with NGF applied to the proximal stump. The state of axonal regeneration was also assessed in each case by anterograde transport of HRP.Following nerve crush axons crossed the lesion by 7 days, reached the chiasma by 14 days and entered the tectum around 20–30 days. The normally evenly stained ganglion cells exhibited granular Nissl staining at 7 and 10 days but very little change in soma size. From 10 to 28 days the mean retinal ganglion cell area increased by 102% and maintained this size until at least 75 days. By 102 days soma size had nearly returned to normal. A population of displaced amacrine cells retained a normal appearance and soma size throughout regeneration.Following nerve cut and stump separation the retinal ganglion cells were slightly more reactive in appearance at 7 days after crush but otherwise the soma reaction developed in a similar manner. Axon tracing revealed no extension beyond the lesion site in these animals and therefore the state of axonal growth did not affect the early soma response.NGF applied at the time of the lesion had no detectable effect on the soma reaction.Although many retinal ganglion cells re-establish contact with visual centres after axotomy in the frog, a considerable proportion die. This contrasts with both the goldfish, where all cells regenerate successfully, and various mammals, where none do so and all retinal ganglion cells die. All retinal ganglion cells in the frog undergo reactive changes similar to those of goldfish and there is no sign of the cell shrinkage seen in mammals. Therefore the cell death in frog would appear to be different from that in mammalian retina but similar to that of mammalian peripheral nerve in which chromatolysis generally preceeds death.     

Specificity of peripheral nerve regeneration: interactions at the axon level

Peripheral nerves injuries result in paralysis, anesthesia and lack of autonomic control of the affected body areas. After injury, axons distal to the lesion are disconnected from the neuronal body and degenerate, leading to denervation of the peripheral organs. Wallerian degeneration creates a microenvironment distal to the injury site that supports axonal regrowth, while the neuron body changes in phenotype to promote axonal regeneration. The significance of axonal regeneration is to replace the degenerated distal nerve segment, and achieve reinnervation of target organs and restitution of their functions. However, axonal regeneration does not always allows for adequate functional recovery, so that after a peripheral nerve injury, patients do not recover normal motor control and fine sensibility. The lack of specificity of nerve regeneration, in terms of motor and sensory axons regrowth, pathfinding and target reinnervation, is one the main shortcomings for recovery. Key factors for successful axonal regeneration include the intrinsic changes that neurons suffer to switch their transmitter state to a pro-regenerative state and the environment that the axons find distal to the lesion site. The molecular mechanisms implicated in axonal regeneration and pathfinding after injury are complex, and take into account the cross-talk between axons and glial cells, neurotrophic factors, extracellular matrix molecules and their receptors. The aim of this review is to look at those interactions, trying to understand if some of these molecular factors are specific for motor and sensory neuron growth, and provide the basic knowledge for potential strategies to enhance and guide axonal regeneration and reinnervation of adequate target organs.     

Soluble complement receptor 1 protects the peripheral nerve from early axon loss after injury

Complement activation is a crucial early event in Wallerian degeneration. In this study we show that treatment of rats with soluble complement receptor 1 (sCR1), an inhibitor of all complement pathways, blocked both systemic and local complement activation after crush injury of the sciatic nerve. Deposition of membrane attack complex (MAC) in the nerve was inhibited, the nerve was protected from axonal and myelin breakdown at 3 days after injury, and macrophage infiltration and activation was strongly reduced. We show that both classical and alternative complement pathways are activated after acute nerve trauma. Inhibition of the classical pathway by C1 inhibitor (Cetor) diminished, but did not completely block, MAC deposition in the injured nerve, blocked myelin breakdown, inhibited macrophage infiltration, and prevented macrophage activation at 3 days after injury. However, in contrast to sCR1 treatment, early signs of axonal degradation were visible in the nerve, linking MAC deposition to axonal damage. We conclude that sCR1 protects the nerve from early axon loss after injury and propose complement inhibition as a potential therapy for the treatment of diseases in which axon loss is the main cause of disabilities.     

Neurofilamentous hypertrophy of intramedullary axonal arbors in intact spinal motoneurons undergoing peripheral sprouting

An incomplete motor nerve injury or a partial loss of motoneurons leads to a partial denervation of skeletal muscle. As part of a compensatory response, the remaining intact motoneurons undergo peripheral sprouting and increase their motor unit size. Our knowledge about the responses in the more proximal parts of these sprouting motoneurons is sparse, however. We investigated the effects of an incomplete transection of the medial gastrocnemius (MG) nerve in the adult cat on the morphology of the intramedullary axon and axon collateral systems of the remaining intact MG motoneurons. At twelve weeks following the partial transection of the MG nerve, intracellular recording and labeling techniques were used to deposit horseradish peroxidase into single intact MG motoneurons for detailed morphological studies. The light microscopic appearance and caliber of the intramedullary stem motor axons of the intact MG motoneurons were indistinguishable from controls. The number and size of the intramedullary motoraxon collateral systems were also unchanged. However, frequent and marked hypertrophy of the distal portions of the motoraxon collaterals was encountered. Electron microscopic studies of the hypertrophied collaterals demonstrated abnormal accumulations of disorganized neurofilaments arranged in bundles or whorls. The morphological changes were indistinguishable from the neurofilamentous hypertrophy that has previously been reported in Wallerian degeneration, in experimental and human motor neuron disease and in some regenerating axonal processes of spinal motoneurons. We conclude that, neurofilamentous hypertrophy of the intramedullary arbors of motor axons may also be part of a reactive and non-degenerative response in intact motoneurons undergoing compensatory peripheral sprouting.