Axonal Remodeling Research Articles

INJURY AND RECOVERY OF FAST AND SLOW MUSCLE FIBERS AFFECTED BY A SNAKE VENOM MYOTOXIN 375

To study the response of individual muscle fiber types to snake venom PLA2 myotoxins, we have tested the effect of ACLMT (ACL myotoxin fromAgkistrodon contortrix laticinctus) on soleus andgastrocnemius mice muscles. All animals received 5 mg of ACLMT/kg into the subcutaneous lateral region of the right hind limb, near the Achilles tendon. The contralateral muscles were used as control. Frozen muscles were cut at medial region in Cryostat and alternate serial cross-sections stained with Toluidine Blue and for Acid Phosphatase, Myofibrillar ATPase activity, Succinate Dehydrogenase and Acetilcholinesterase. Signs of fiber injury were identified, three hours after ACLMT injection, in both muscles. Three days after, these muscles showed clusters of recovered muscle fibers. Twenty-one days after soleus and gastrocnemius presented only chronic signs of damage as split fibers and centralized nucleus. By m-ATPase reactions was possible to identify that both muscle fiber types I and II were injured in both muscles by ACLMT. Significative increased number of fiber type IIC (17.37 ± 4.11versus 1.0 ± 0.6; p=0.008, paired Student t-test) and decrease of type II (60.7 ± 8.8 versus 76.8 ± 10.1; p=0.01, paired Student t-test), suggest muscle fiber type change from type II to type I through type IIC. Although ACLMT is known by its myotoxic activity, the results presented here showed that it can also be used as model to induce axonal remodeling and muscle fiber type change in both fast and slow muscle fibers of mice.

Maintenance of sympathetic innervation into the hippocampal formation requires a continuous local availability of nerve growth factor.

The sprouting of peripheral sympathetic fibers into the septally denervated hippocampal formation is a well-characterized model of lesion-induced plasticity. While various studies have demonstrated the importance of nerve growth factor for evoking sympathetic sprouting, little is known concerning whether nerve growth factor continues to be required for maintaining innervation once it has occurred. In the present study we have addressed this point by (i) investigating the consequences of withdrawing exogenous nerve growth factor support from rats in which sympathetic innervation was enhanced by a nerve growth factor infusion and (ii) using blocking antibodies to interfere with the actions of endogenous nerve growth factor. The results of this investigation clearly indicate that a continuous supply of nerve growth factor (either exogenous or endogenous) is required to maintain sympathetic innervation within the hippocampal formation. Evidence is also provided demonstrating that the nerve growth factor must be made available locally within a given region to evoke and maintain the sympathetic innervation within this location. Axonal rearrangement within the developing and adult brain is believed to be an important mechanism underlying learning and memory is crucial for lesion-related plasticity. In various experimental paradigms, nerve growth factor has been shown to be an important cue for initiating axonal remodeling. In the current study, we have demonstrated that once such rearrangements have taken place, nerve growth factor may also be required to maintain them.

Increase of B-50/GAP-43 immunoreactivity in uninjured muscle nerves of mdx mice

Lack of dystrophin in mdx mice leads to muscle fibre degeneration followed by the formation of new myofibres. This degeneration—regeneration event occurs in clusters. It is accompanied by inflammation and remodelling of the intramuscular terminal nerve fibres. Since the growth-associated protein B-50/GAP-43 has been shown to be involved in axonal outgrowth and synaptic remodelling following neuronal injury, we have investigated the presence of B-50 in gastrocnemius and quadriceps muscles of mdx mice. Using immunocytochemistry we demonstrate increased presence of B-50 in terminal nerve branches at motor endplates of mdx mice, particularly in the clusters of de- and regenerating myofibres. In comparison, the control mice displayed no B-50 immunoreactivity in nerve fibres contacting motor endplates. Our findings indicate that during axonal remodelling and collateral sprouting the B-50 level in the terminal axon arbours is increased although there is no direct injury to the motoneurons. We suggest that the degenerating target and/or the inflammatory reaction induces the increased B-50 level in the motoaxons. The increased B-50 may be important for sprouting of the nerve fibres and re-establishment of synaptic contacts, and in addition, for maturation and survival of the newly formed myofibres.

Effects of brain-derived neurotrophic factor on optic axon branching and remodelling in vivo.

Neurotrophins are thought to be important for the survival and differentiation of vertebrate neurons. Roles have been suggested for target-derived neurotrophins, based both on their expression in target tissues at the time of neuron innervation, and on their effects on axonal sprouting. However, direct in vivo evidence of their involvement in axon arborization has remained elusive. We have used in vivo microscopy to follow individual optic axons over time, and have examined the role of the neurotrophin brain-derived neurotrophic factor (BDNF) in their development. Here we show that injection of BDNF into the optic tectum of live Xenopus laevis tadpoles increased the branching and complexity of optic axon terminal arbors. In contrast, injection of specific neutralizing antibodies to BDNF reduced axon arborization and complexity. The onset of these effects was rapid (within 2 hours) and persisted throughout the 24-hour observation period. Other neurotrophins had little or no significant effects. These results demonstrate the involvement of neurotrophins in the dynamic elaboration of axon terminals, and suggest a direct role for target-derived BDNF during synaptic patterning in the developing central nervous system.

Morphogenetic effect of kainate on adult hippocampal neurons associated with a prolonged expression of brain-derived neurotrophic factor.

Intraperitoneal or intrahippocampal injections of kainate induce both hippocampal cell death and axonal remodeling of the dentate gyrus granular neurons. We report here that injection of kainate into the dorsal hippocampus of adult mice may also trigger a conspicuous and long-lasting global trophic response of granule cells. Morphological changes include somatic and dendritic growth and increased nuclear volume with ultrastructural features characteristic of neuronal development. The trophic response is correlated with a specific overexpression of brain-derived neurotrophic factor that is maintained for at least six months. This shows that plasticity in adult neurons can, in addition to axonal remodeling, extend to generalized cell growth. Our results further suggest that brain-derived neurotrophic factor could be involved in the activation and/or maintenance of this phenomenon.

Single thalamocortical axons diverge to multiple patches in neonatal auditory cortex

Thalamic afferents originating in the ventral division of the medial geniculate body (vMGB) terminate in patches within lamina III/IV of the primary auditory cortex in adult rabbits. Focal iontophoretic injections of the anterograde tracer, biocytin, were made into the vMGB of neonatal rabbits to examine the morphological organization of auditory thalamocortical (TC) afferents prior to hearing onset. TC afferents terminated in distinct patches as early as postnatal day 1 (PD-0 = day of birth), 6 days before the behavioral onset of hearing. In contrast to TC afferents in adults, the terminal arbors of neonatal vMGB axons occupied the entire depth of the cortical plate and lamina I. Serial section reconstructions revealed that single vMGB axons in neonates branched to form multiple patches within the cortical plate. Collaterals also extended to lamina I where they coursed tangentially for several millimeters. An unusual feature of the neonatal TC patches was the contribution of descending collaterals from axons coursing in lamina I. The presence of distinct patches prior to hearing onset indicates that the segregation of auditory TC axons occurs in the absence of acoustically driven activity. The extensive postnatal remodeling of TC axons, however, may indicate activity-dependent refinement of arbor size.

Localization of differentially phosphorylated isoforms of microtubule-associated protein 1B in cultured rat hippocampal neurons

The development and plasticity of axons and dendrites in mammalian neurons may depend on the presence and phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins. One of these proteins, microtubule-associated protein 1B, is modified by different protein kinases, which give rise to two major types of phosphorylated isoforms. The distribution of these isoforms in cultured hippocampal neurons has been studied using antibodies to specific phosphorylation-sensitive epitopes. Mode I-phosphorylated MAP1B is largely restricted to developing axonal processes, particularly at their distal regions including their growth cones where no mode I-dephosphorylated MAP1B is present. Axonal maturation is accompanied by dephosphorylation of MAP1B at mode I sites. Thus, mode I-phosphorylated MAP1B may be a marker for axonal growth. In contrast, mode II-phosphorylated MAP1B is abundant in the axonal and somatodendritic compartments, and no increased dephosphorylation occurs during maturation. These results are compatible with a role for the mode I phosphorylation of M AP1B (which might be catalysed by proline-directed protein kinases) in supporting a rapid axonal-specific growth mechanism and a more general role for the mode II phosphorylation of MAP1B (which seems to be catalysed by casein kinase II) in controlling axonal and dendritic growth and remodeling.

Regulation of neurotrophin receptors during the maturation of the mammalian visual system

Cell division, cell death, and remodeling of connections are major features of the construction of the mammalian CNS. We have begun to address the role of neurotrophins in these events through characterization of the expression of their receptors in the developing ferret visual system. By use of chemical cross-linking of iodinated neurotrophins, proteins corresponding to trkB, trkC, and p75 were identified as receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) throughout development. BDNF was also cross-linked to a truncated form of trkB that lacks the tyrosine kinase domain (trkB. T1) in retinal target tissues and in cortex. At the earliest developmental age examined (E24), the ratio of full-length to truncated trkB is > > 1 in the retinal target tissues, LGN and superior colliculus. During the ensuing period of retinal ganglion cell death and segregation into eye-specific layers, the amount of truncated trkB increases markedly relative to full-length trkB. By P27, truncated trkB is the predominant receptor for BDNF in the retinal target tissues and this pattern is maintained into adulthood. Within all subdivisions of visual cortex including the ventricular zone (VZ), intermediate zone (IZ), and cortical plate (CP), similar profiles of bands are observed. The developmental increase in abundance of truncated trkB relative to full-length occurs earliest in the VZ, with a major increase between E30 and P3. In the IZ, this shift to a predominance of truncated trkB occurs between P15 and P30, while in the CP the shift is even further delayed, not occurring until after P30. Within each subdivision of cortex, the shift to a predominance of truncated trkB occurs at times that correlate with the onset of cell death and maturation of axonal connections. This study demonstrates that members of the trk family, previously identified in the CNS on the basis of mRNA transcripts, are present as receptors with specific binding affinities for BDNF and NT-3. Moreover, the correspondence between the developmental shift from full-length to truncated trkB and the critical periods for cell fate determination, cell death, and axonal remodeling suggests an important role for neurotrophic factors in the development of the visual system.

Long-term growth and remodeling of regenerated retino-collicular connections in adult hamsters

The capacity of regenerating axons for long-term growth and synaptic plasticity was investigated in the visual system of adult hamsters. Four to six and 8-10 months after the eye and the superior colliculus (SC) were linked by a peripheral nerve (PN) graft, the retinal ganglion cell (RGC) axons that had regrown into the SC were examined ultrastructurally. Together with the data from hamsters with similar PN grafts for 2 months (Carter et al., 1989), this study spans most of the life of these animals. The overall findings indicate that (1) the RGC axons extended twice as far into the SC and the number of RGC terminals increased 30-fold between 2 and 4-6 months. These parameters did not change thereafter. The highest density of regenerated RGC terminals observed in the SC was 11.5% of controls. (2) The new RGC terminals acquired most of their normal ultrastructural characteristics by 2 months. (3) The mean size of the terminals was larger than in controls but decreased gradually, and there was a small increase in the size of the regenerated synapses. (4) At all times, the RGC terminals remained confined to the layers of the SC that normally receive retinal inputs, and their synapses were formed in normal proportions with the dendritic shafts and spines of SC neurons. Thus, there is a protracted long-term growth and remodeling of the RGC axons that have regenerated into the SC of these adult mammals.(ABSTRACT TRUNCATED AT 250 WORDS)

Double immunolocalization of B-50 and A4 protein in Alzheimer's disease: A.B. Oestreicher, W.H. Gispen and J.M. Rozemuller ∗. Rudolf Magnus Institute, Medical Faculty, University of Utrecht and ∗Dept. of Neuropathology, Medical Faculty, Free University, Amsterdam, The Netherlands

Lack of dystrophin in mdx mice leads to muscle fibre degeneration followed by the formation of new myofibres. This degeneration—regeneration event occurs in clusters. It is accompanied by inflammation and remodelling of the intramuscular terminal nerve fibres. Since the growth-associated protein B-50/GAP-43 has been shown to be involved in axonal outgrowth and synaptic remodelling following neuronal injury, we have investigated the presence of B-50 in gastrocnemius and quadriceps muscles of mdx mice. Using immunocytochemistry we demonstrate increased presence of B-50 in terminal nerve branches at motor endplates of mdx mice, particularly in the clusters of de- and regenerating myofibres. In comparison, the control mice displayed no B-50 immunoreactivity in nerve fibres contacting motor endplates. Our findings indicate that during axonal remodelling and collateral sprouting the B-50 level in the terminal axon arbours is increased although there is no direct injury to the motoneurons.We suggest that the degenerating target and/or the inflammatory reaction induces the increased B-50 level in the motoaxons. The increased B-50 may be important for sprouting of the nerve fibres and re-establishment of synaptic contacts, and in addition, for maturation and survival of the newly formed myofibres.

Remodeling of retinal ganglion cell dendrites in the absence of action potential activity.

The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.

Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order

The retinotectal projection is organized in a precise retinotopic manner. We find, though, that during development the growth and arborization of temporal retinal axons within the optic tectum of chick embryos is initially imprecise. Axonal targeting errors occur along the rostral-caudal and medial-lateral tectal axes, and arbors are formed at topographically inappropriate positions. Subsequent course corrections along both tectal axes and large-scale axonal remodeling lead to the retinotopic ordering of terminal arborizations characteristic of the mature projection. The trajectories and branching patterns of temporal retinal axons labeled with Dil or DiO were determined in whole mounts of retina and tectum from chicks ranging in age from embryonic day 9 to posthatching. Within the retina, labeled retinofugal axons travel in a compact bundle but do not maintain strict neighbor relations, as they course to the optic fissure. The axons enter the contralateral tectum at its rostral edge and grow caudally. Many extend well past their appropriate terminal zone within rostral tectum; a proportion of these later reverse their direction of growth. Many axons grow onto the tectum at incorrect positions along the medial-lateral tectal axis. Some correct this error in a directed manner by altering their trajectory or extending collateral branches at right angles. About 80% of the positional changes of this type are made in the direction appropriate to correct axon position, and thus are likely a response to tectal positional cues. After maturation of retinotopic order, about half of the axons that project to a mature terminal zone have made abrupt course corrections along one or both tectal axes, indicating that initially mistargeted axons can establish appropriately positioned arbors and survive. The development of temporal axons within the tectum is characterized by 3 phases: elongation, branch and arbor formation, and remodeling. After considerable rostrocaudal elongation, an axon typically develops numerous side branches and arbors, many at inappropriate locations. Most arbors are formed by side branches that develop as interstitial collaterals; few axons grow directly to their appropriate terminal zone and arborize. Aberrant arbors, and axons and axon segments that fail to form arbors in the appropriate terminal zone, are rapidly eliminated over about a 2 d period. Axon degeneration appears to play a role in this remodeling process.

Ultrastructural alterations of primary afferent axons in the nucleus gracilis after peripheral nerve axotomy.

Alterations of primary afferent axons in the nucleus gracilis were studied at three weeks and four, 12, and 24 months after peripheral nerve axotomy by hindlimb amputation at the hip joint in 19 female cats. The contralateral side and two cats not subjected to amputation were controls. We observed two major types of fiber alterations. One, fibers showed changes of axonal atrophy, myelin remodelling, and degeneration. Adaxonal invagination occurred more frequently (p less than 0.005) at three weeks postamputation, in territories known to contain centrally directed axons of primary afferent neurons of the lower limb, than in controls. Perhaps adaxonal sequestration contributed to axonal attenuation. Two, there were filamentous, granular, central core, and other types of axonal swellings found in territories containing central axons of primary afferent neuron terminals of the lower limb. These changes occurred most frequently at 12 and 24 months and were significantly more frequent than in control tissue. These reactive/dystrophic axons therefore were associated with permanent axotomy, but we have not established that they occur in central axons of primary afferent neurons, or whether they are degenerative or abortive regenerative changes.

Peripheral axotomy induces neurofilament decrease, atrophy, demyelination and degeneration of root and fasciculus gracilis fibers

We have recently shown that peripheral axotomy by hindlimb amputation in adult cats sequentially results in neurofilament and microtubule decrease and axonal atrophy, myelin wrinkling, myelin remodeling (de- and remyelination), more atrophy and axonal degeneration in proximal sciatic and L7 segmental nerve fibers. The neurophatologic, morphometric and teased fiber alterations in the myelinated fibers (MF) of roots and sampled levels of fasciculus gracilis in groups of adult cats 24 months after hindlimb amputation have now been studied. We found: (1) a severe decrease of neurofilaments, axonal atrophy, myelin wrinkling, de- and remyelination and axonal loss in posterior root axons; (2) that these morphologic abnormalities extended up the fasciculus gracilis in the appropriate territories established from degenerative studies; (3) that the retrograde effect was less severe in ventral root fibers, although atrophy and sprouting were demonstrated here, and (4) that the cellular sequence of retrograde atrophic degeneration of ascending axons was similar to that observed in proximal stump axons. These findings confirm that primary afferent neurons are more vulnerable to axotomy than lower motot neurons and may provide an additional explanation for the poorer functional restoration of sensory than of motor deficit after root compression and in delayed nerve reconnection. Our observations also have important implications for interpretation of neuropathologic alterations in roots and fasciculus gracilis, since the observed features may be secondary to axotomy of peripheral nerve fibers induced by disease and not evidence of a primary derangement.

Axon membrane remodeling in the lead-induced demyelinating neuropathy of the rat

Single-teased fibers stained with the ferric ion-ferrocyanide method 14 allowed us to study axonal remodeling in the lead-induced demyelinating neuropathy of the rat. Our findings, in agreement with recent physiological data, pointed to a transitory reorganization of the demyelinated axons to maintain impulse conduction until remyelination and formation of new cytochemically normal nodes had restored a secure saltatory conduction.

Development of the optic nerve in Xenopus laevis IL Gliogenesis, myelination and metamorphic remodelling

ABSTRACT We studied the time of origin, development and location of glial elements in the developing optic nerve of Xenopus with light and electron microscopy. The first cells acting as a primitive glia are ependymal cells lying dorsal to the chiasmatic optic nerve (CON) at Nieuwkoop & Faber (1956) NF stage 39. Later (stage 47/48), immature astrocyte cell bodies migrate from the periphery of the middle optic nerve (MON) into the central fibre mass along cytoplasmic processes extending from the outer glia limitans. Shortly thereafter, oligodendrocyte cell bodies appear in the centre of the fibre mass and myelination begins, first in the middle of the MON, spreading from the centre distally towards the chiasm and proximally to the retina. In late tadpoles myelinated fibres appear first in the CON then in the retinal optic nerve (RON) increasing markedly in juveniles and adults. Segment-specific patterns of glia and myelination appear during optic nerve development. During metamorphic climax, the optic nerve shortens (Cullen & Webster, 1979), a process involving myelin and axon remodelling primarily in the MON. Neither the profound changes during metamorphosis, nor the processes of gliogenesis and myelination significantly alter the underlying chronotopic ordering in the tadpole nerve. In juvenile and adult optic nerves, however, as myelination and gliogenesis increase, and as more axons mature and grow in diameter, the dorsoventral chronotopic arrangement of axons becomes less apparent.

Remodelling of optic nerve myelin sheaths and axons during metamorphosis in Xenopus laevis.

Whole mounts and transverse sections of Xenopus optic nerves were examined with the light and electron microscopes before, during, and after metamorphosis. In stage 52--58 tadpoles, almost all myelin sheaths were circular in transverse sections. Early in metamorphosis (stages 60--61) large redundant myelin loops surrounded many large axons in central regions of the nerve. The loops subsequently were broken down into ovoids and lamellar segments that remained mostly within oligodendrocytes. These myelin changes were not observed in the chiasm or next to the eye. They were not associated with significant axonal degeneration and were no longer apparent in optic nerves of young frogs. Xenopus optic nerves also became shorter during metamorphosis. We therefore suggest that myelin sheaths with redundant loops which degenerate and disappear are being remodelled as the nerve decreases in length.