Motor Neurites Research Articles

Kinesin-5 inhibition improves neural regeneration in experimental autoimmune neuritis

BackgroundAutoimmune neuropathies can result in long-term disability and incomplete recovery, despite adequate first-line therapy. Kinesin-5 inhibition was shown to accelerate neurite outgrowth in different preclinical studies. Here, we evaluated the potential neuro-regenerative effects of the small molecule kinesin-5 inhibitor monastrol in a rodent model of acute autoimmune neuropathies, experimental autoimmune neuritis.MethodsExperimental autoimmune neuritis was induced in Lewis rats with the neurogenic P2-peptide. At the beginning of the recovery phase at day 18, the animals were treated with 1 mg/kg monastrol or sham and observed until day 30 post-immunisation. Electrophysiological and histological analysis for markers of inflammation and remyelination of the sciatic nerve were performed. Neuromuscular junctions of the tibialis anterior muscles were analysed for reinnervation. We further treated human induced pluripotent stem cells-derived secondary motor neurons with monastrol in different concentrations and performed a neurite outgrowth assay.ResultsTreatment with monastrol enhanced functional and histological recovery in experimental autoimmune neuritis. Motor nerve conduction velocity at day 30 in the treated animals was comparable to pre-neuritis values. Monastrol-treated animals showed partially reinnervated or intact neuromuscular junctions. A significant and dose-dependent accelerated neurite outgrowth was observed after kinesin-5 inhibition as a possible mode of action.ConclusionPharmacological kinesin-5 inhibition improves the functional outcome in experimental autoimmune neuritis through accelerated motor neurite outgrowth and histological recovery. This approach could be of interest to improve the outcome of autoimmune neuropathy patients.

The Effect of Botulinum Neurotoxin Serotype a Heavy Chain on the Growth Related Proteins and Neurite Outgrowth after Spinal Cord Injury in Rats

(1) Background: The botulinum toxin A (BoNT-A) heavy chain (HC) can stimulate the growth of primary motor neurites. (2) Methods: A recombinant BoNT/A HC was injected locally plus interval intrathecal catheter of BoNT/A HC to rats with ipsilateral semi-dissociated lumbar spinal cord injuries (SCIs). First, 2D gel with a silver nitrate stain was applied to detect the general pattern of protein expression. Growth associated protein 43 (GAP-43) and superior cervical ganglion 10 (SCG10) were chosen to represent the altered proteins, based on their molecular weight and pI, and were used to further detect their expression. Meanwhile, the neuronal processes were measured. The measurements of thermal hyperalgesia and grasp power at the ipsilateral hindlimb were used to evaluate spinal sensory and motor function, respectively. (3) Results: The local injection of BoNT/A HC followed by its intrathecal catheter intervally altered the spinal protein expression pattern after an SCI; protein expression was similar to normal levels or displayed a remarkable increase. The changes in the expression and distribution of phosphorylated growth associated protein 43(p-GAP 43) and superior cervical ganglion 10 (SCG 10) indicated that the administration of BoNT/A HC to the SCI significantly amplified the expression of p-GAP43 and SCG10 (p < 0.05). Meanwhile, the positive immunofluorescent staining for both p-GAP43 and SCG10 was mainly present near the rostral aspect of the injury, both in the cytoplasm and the neuronal processes. Moreover, the outgrowth of neurites was stimulated by the BoNT/A HC treatment; this was evident from the increase in neurite length, number of branches and the percentage of cells with neuronal processes. The results from the spinal function tests suggested that the BoNT/A HC did not affect sensation, but had a large role in improving the ipsilateral hindlimb grasp power (p < 0.05). (4) Conclusions: The local injection with the intermittent intrathecal administration of BoNT/A heavy chain to rats with SCI increased the local expression of GAP-43 and SCG 10, which might be affiliated with the regeneration of neuronal processes surrounding the injury, and might also be favorable to the relief of spinal motor dysfunction.

Immunohistochemical Expression of Synaptophysin in the Adult and Developing Cervical and Lumbar Enlargements of the Spinal Cord of Rabbit

Background: The principle findings of synaptophysin immunoreactivity (SynpIR) during the ontogeny of rabbit spinal cord are: At E14, SynpIR precedes in the entire marginal layer especially at the entrance zone of dorsal root and motor neurite outgrowth emerged from the basal plate. At E21, SynpIR is expressed in the motoneurons of ventral and lateral horns of mantle layer growing into the ventrolateral columns of marginal layer. Methods: We found intensely stained thick tracts and diffuse axons among proliferating neuroblasts of mantle layer. The peripheral parts of ventral horns were occupied with closely packed multipolar neurons from which long dendrites departed toward the surface of marginal layer. Results: At E28, pronounced SynpIR presented in the ventral grey horn while the white matter was faintly stained., meanwhile the dorsal horn was more cellular than ventral and lateral horns. Few intensively SynpIR fibers cross the dorsal and ventral commissures. In adult, profuse SynpIR appeared in the entire grey matter, and stained dendrites departed from neurons in the lateral laminae into the adjacent funiculi as finger-like projections. These projections did not reach the surface, so that the outer one-third to onefourth of the funiculi contained little or no SynpIR. In the periphery of ventral horns, we found large multipolar neurons with faintly stained cytoplasm. The white matter and the neuroepithelial cells surrounding the central canal were almost unstained. Conclusion: Synaptophysinis a reliable marker for fiber outgrowth and synapse formation in therabbit spinal cord, and its differential expression levels is specific and almost completed before birth.

Neuronal cell culture from transgenic zebrafish models of neurodegenerative disease.

ABSTRACTWe describe a protocol for culturing neurons from transgenic zebrafish embryos to investigate the subcellular distribution and protein aggregation status of neurodegenerative disease-causing proteins. The utility of the protocol was demonstrated on cell cultures from zebrafish that transgenically express disease-causing variants of human fused in sarcoma (FUS) and ataxin-3 proteins, in order to study amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type-3 (SCA3), respectively. A mixture of neuronal subtypes, including motor neurons, exhibited differentiation and neurite outgrowth in the cultures. As reported previously, mutant human FUS was found to be mislocalized from nuclei to the cytosol, mimicking the pathology seen in human ALS and the zebrafish FUS model. In contrast, neurons cultured from zebrafish expressing human ataxin-3 with disease-associated expanded polyQ repeats did not accumulate within nuclei in a manner often reported to occur in SCA3. Despite this, the subcellular localization of the human ataxin-3 protein seen in cell cultures was similar to that found in the SCA3 zebrafish themselves. The finding of similar protein localization and aggregation status in the neuronal cultures and corresponding transgenic zebrafish models confirms that this cell culture model is a useful tool for investigating the cell biology and proteinopathy signatures of mutant proteins for the study of neurodegenerative disease.

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors.

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation.

The role of semaphorin3A in myogenic regeneration and the formation of functional neuromuscular junctions on new fibres.

Current research on skeletal muscle injury and regeneration highlights the crucial role of nerve-muscle interaction in the restoration of innervation during that process. Activities of muscle satellite or stem cells, recognized as the 'currency' of myogenic repair, have a pivotal role in these events, as shown by ongoing research. More recent investigation of myogenic signalling events reveals intriguing roles for semaphorin3A (Sema3A), secreted by activated satellite cells, in the muscle environment during development and regeneration. For example, Sema3A makes important contributions to regulating the formation of blood vessels, balancing bone formation and bone remodelling, and inflammation, and was recently implicated in the establishment of fibre-type distribution through effects on myosin heavy chain gene expression. This review highlights the active or potential contributions of satellite-cell-derived Sema3A to regulation of the processes of motor neurite ingrowth into a regenerating muscle bed. Successful restoration of functional innervation during muscle repair is essential; this review emphasizes the integrative role of satellite-cell biology in the progressive coordination of adaptive cellular and tissue responses during the injury-repair process in voluntary muscle.

Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons.

Although peripheral axons can regenerate after nerve transection and repair, functional recovery is usually poor due to inaccurate reinnervation. Neurotrophic factors promote directional guidance to regenerating axons and their selective application may help to improve functional recovery. Hence, we have characterized in organotypic cultures of spinal cord and dorsal root ganglia the effect of GDNF, FGF-2, NGF, NT-3, and BDNF at different concentrations on motor and sensory neurite outgrowth. In vitro results show that GDNF and FGF-2 enhanced both motor and sensory neurite outgrowth, NGF and NT-3 were the most selective to enhance sensory neurite outgrowth, and high doses of BDNF selectively enhanced motor neurite outgrowth. Then, NGF, NT-3, and BDNF (as the most selective factors) were delivered in a collagen matrix within a silicone tube to repair the severed sciatic nerve of rats. Quantification of Fluorogold retrolabeled neurons showed that NGF and NT-3 did not show preferential effect on sensory regeneration whereas BDNF preferentially promoted motor axons regeneration. Therefore, the selective effects of NGF and NT-3 shown in vitro are lost when they are applied in vivo, but a high dose of BDNF is able to selectively enhance motor neuron regeneration both in vitro and in vivo.

Sensory neurons do not induce motor neuron loss in a human stem cell model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells

Background contextTransplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. PurposeTo characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). Study design/settingPreviously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. MethodsWe have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar–associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. ResultsAs has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. ConclusionsThese findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states.

In vitro study of axonal migration and myelination of motor neurons in a three‐dimensional tissue‐engineered model

Primary motor neurons are difficult to study in conventional culture systems because of their short-term survival without trophic support from glia. In addition, axonal migration on a two-dimensional Petri dish does not reflect the three-dimensional (3D) environment in vivo. A unique in vitro 3D model of motor nerve regeneration was developed to study motor neuron axonal migration and myelination. Mouse spinal cord motor neurons were seeded on a collagen sponge populated with Schwann cells and fibroblasts. This fibroblast-populated sponge was intended to mimic the connective tissue through which motor axons have to elongate in vivo. Addition of conventional neurotrophic supplements was not required for motor neuron survival but was necessary to promote deep neurite outgrowth, as assessed by immunostaining of neurofilament M. A vigorous neurite elongation was detected inside the sponge after only 14 days of neuron culture, reaching more than 850 microm. The model also allowed the maturation of motor fibers as one-third of them were positive for neurofilament H. Neurites growing in the sponge were subject to myelination when Schwann cells were present, as shown by myelin basic protein immunostaining and electron microscopy. We demonstrated in this model the spontaneous formation of numerous thick myelin sheaths surrounding motor fibers after long-term culture (28 days). Thus, this model might be a valuable tool to study the effect of various cells and/or attractive or repulsive molecules on motor neurite outgrowth in vitro and also for the study of myelination and pathogenesis of motor neuron diseases.

The Role of Rapid, Local, Postsynaptic Protein Synthesis in Learning-Related Synaptic Facilitation in Aplysia

The discovery that dendrites of neurons in the mammalian brain possess the capacity for protein synthesis stimulated interest in the potential role of local, postsynaptic protein synthesis in learning-related synaptic plasticity. But it remains unclear how local, postsynaptic protein synthesis actually mediates learning and memory in mammals. Accordingly, we examined whether learning in an invertebrate, the marine snail Aplysia, involves local, postsynaptic protein synthesis. Previously, we showed that the dishabituation and sensitization of the defensive withdrawal reflex in Aplysia require elevated postsynaptic Ca(2+), postsynaptic exocytosis, and functional upregulation of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Here, we tested whether the synaptic facilitation that underlies dishabituation and sensitization in Aplysia requires local, postsynaptic protein synthesis. We found that the facilitatory transmitter, serotonin (5-HT), enhanced the response of the motor neuron to glutamate, the sensory neuron transmitter, and this enhancement depended on rapid protein synthesis. By using individual motor neurites surgically isolated from their cell bodies, we showed that the 5-HT-dependent protein synthesis occurred locally. Finally, by blocking postsynaptic protein synthesis, we disrupted the facilitation of the sensorimotor synapse. By demonstrating its critical role in a synaptic change that underlies learning and memory in a major model invertebrate system, our study suggests that local, postsynaptic protein synthesis is of fundamental importance to the cell biology of learning.

Motor neurite outgrowth is selectively inhibited by cell surface MuSK and agrin

During neuromuscular development, “stop signals” present on the target myotube inhibit motor axon growth. Mice lacking either the neuronal form of agrin or the muscle-specific tyrosine kinase (MuSK) lose stop signal activity, suggesting that they are part of such signals or induce them in myotubes. To test whether MuSK complexes form stop signals in the absence of myotube signaling, we cultured ciliary ganglion (CG) neurons with nonmuscle cells expressing cell-surface MuSK. Expression of MuSK had no effect on neuronal adhesion. MuSK expression, however, inhibited neurite outgrowth from CG neurons, but not retinal ganglion cell neurons. The neurite-inhibitory effect could be completely reversed by an antibody to the MuSK extracellular domain, and partially reversed by an antibody to agrin, suggesting that inhibition is mediated by a complex of these proteins. Thus, an agrin/MuSK complex may form part of a motor neuron stop signal involved in “reverse signaling” to the motor neuron.

Neuromuscular synapses mediate motor axon branching and motoneuron survival during the embryonic period of programmed cell death

The embryonic period of motoneuron programmed cell death (PCD) is marked by transient motor axon branching, but the role of neuromuscular synapses in regulating motoneuron number and axonal branching is not known. Here, we test whether neuromuscular synapses are required for the quantitative association between reduced skeletal muscle contraction, increased motor neurite branching, and increased motoneuron survival. We achieved this by comparing agrin and rapsyn mutant mice that lack acetylcholine receptor (AChR) clusters. There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching, and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared with their wild-type littermates at embryonic day 18.5 (E18.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests that specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin-induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development.

Development of inhibition by ephrin-A5 on outgrowth of embryonic spinal motor neurites.

The spinal motor pool maps systematically onto the surface of muscles. This map is detectable in rat embryonic muscles, and is partially restored after reinnervation. Recent evidence shows that either overexpression or deletion of the ephrin-A5 gene significantly disrupts the map, suggesting that ephrin-A5 plays a critical role in the formation of this topography. Several studies have demonstrated that ephrin-A5 is a repulsive molecule in the nervous system, including the neuromuscular system. To examine the development of sensitivity of ventral spinal axons to this inhibitory ligand, slices of E11 to E15 embryonic rat spinal cords were cocultured with membranes derived from ephrin-A5-expressing cell lines. We detected a progressive expression of inhibition by ephrin-A5 between E11 and E15. By E15, rostral and caudal spinal neurites showed clear differences in responsiveness to the ephrin-A5 ligand. Further, we found that at this age caudal neurites are more sensitive to changes of ephrin-A5 concentration along a gradient. In addition, growth cones of caudal, more than rostral, neurites tended to assume a collapsed shape in the presence of the ligand. These results demonstrate a progressive development of sensitivity to ephrin-A5, and suggest a divergence in this sensitivity between rostral and caudal spinal cord neurites. These results provide further insight into how subtle rostrocaudal differences in the development of sensitivity to ephrin-A5 may explain, in part, neuromuscular topography.

F-spondin is a contact-repellent molecule for embryonic motor neurons.

The floor plate plays a key role in patterning axonal trajectory in the embryonic spinal cord by providing both long-range and local guidance cues that promote or inhibit axonal growth toward and across the ventral midline of the spinal cord, thus acting as an intermediate target for a number of crossing (commissural) and noncrossing (motor) axons. F-spondin, a secreted adhesion molecule expressed in the embryonic floor plate and the caudal somite of birds, plays a dual role in patterning the nervous system. It promotes adhesion and outgrowth of commissural axons and inhibits adhesion of neural crest cells. In the current study, we demonstrate that outgrowth of embryonic motor axons also is inhibited by F-spondin protein in a contact-repulsion fashion. Three independent lines of evidence support our hypothesis: substrate-attached F-spondin inhibits outgrowth of dissociated motor neurons in an outgrowth assay; F-spondin elicits acute growth cone collapse when applied to cultured motor neurons; and challenging ventral spinal cord explants with aggregates of HEK 293 cells expressing F-spondin, causes contact-repulsion of motor neurites. Structural-functional studies demonstrate that the processed carboxyl-half protein that contains the thrombospondin type 1 repeats is more prominent in inhibiting outgrowth, suggesting that the processing of F-spondin is important for enhancing its inhibitory activity.

Electrical Activity and Postsynapse Formation in Adult Muscle: γ-AChRs Are Not Required

Skeletal muscle fibers will not accept hyperinnervation by foreign motor axons unless they are paralyzed, suggesting that paralysis makes them receptive to innervation, e.g., by upregulating extrasynaptic expression of γ-AChRs and/or of the agrin receptor MuSK. To examine the involvement of these parameters in paralysis-mediated synapse induction, ectopic expression of agrin, a factor from motor neurons controlling neuromuscular synapse formation, was made dependent on the administration of doxycycline in innervated adult muscle fibers. In response to doxycycline-induced agrin secretion, adult fibers did form ectopic postsynaptic specializations, even when they were electrically active, lacked fetal AChRs, and expressed normal low levels of MuSK. These data demonstrate that paralysis and changes associated with it are not required for agrin-induced postsynapse formation. They suggest that paralyzed muscle induces synapse formation via the release of factors that make motor neurites contact muscle fibers and secrete agrin.

Axonal regeneration in severed peripheral facial nerve of the rabbit: relation of the number of axonal regenerates to behavioral and evoked muscle activity.

The minimum number of regenerating facial nerve myelinated motor axons that are required to innervate and activate the mimetic musculature is not known. We compare rabbit facial nerve regeneration following complete transectional injuries of the buccal division to the evoked and behavioral muscle activities of the quadratus labii superioris muscle of the rabbit in three experimental models: end-to-end direct anastomosis (N = 4), 8-mm autologous nerve grafts (N = 8), and 10-mm silicone chamber implants (N = 40). Data are presented as total numbers of regenerating myelinated axons that traverse the surgical repair and innervate the fascicles of the transected distal nerve stump, as well as the percentage of regenerating neurites, as compared to the preoperative normal controls. Five weeks after neural repair, direct end-to-end anastomosis regained more myelinated axons across the reconstructed defect (2,632 +/- 1,232; 67%) than silicone tube implants (2,006 +/- 445; 51%) or autologous cable graft repairs (1,660 +/- 1,169; 42%). However, only a small percentage of myelinated fibers innervated the intrafascicular region of the distal transected neural stump in direct anastomosis (948 +/- 168; 24%), silicone tube implants (670 +/- 275; 17%), or autologous nerve grafts (445 +/- 120; 12%) in rabbits that regained evoked and behavioral mimetic muscle activity. All rabbits with direct anastomosis and neural cable grafts regained motor activity, despite the fact that 66% of regenerating motor neurites in cable graft repairs and 54% in direct anastomosis were collateral sprouts that did not contribute to effective muscle activity. In 17 rabbits with neural regenerates within the silicone tube implants that did not regain mimetic activity, the mean number of regenerating myelinated motor axons across the defect was 504 +/- 419 (13%), and the mean number of axons that innervated the distal transected nerve stump fascicles was 277 +/- 128 (7%). Therefore, the minimal number of motor axons that is required to activate the quadratus labii superioris muscle is 12% of the original motor axon population of the normal buccal nerve division.

Expression Pattern of Collagen IX and Potential Role in the Segmentation of the Peripheral Nervous System

Segmentation of the peripheral nervous system of vertebrates requires guidance cues located in the adjacent somitic mesoderm. Recent experiments suggest that inhibitory molecules in the posterior somite may influence segmentation by restricting the outgrowth of axons and the migration of neural crest cells to the anterior somite. A potential candidate for an inhibitory molecule is collagen IX, a chondroitin sulfate proteoglycan made by sclerotome cells of the somite and by the notochord. Immunohistochemical localization of collagen IX demonstrated that its expression in the posterior sclerotome of the somite correlates with axon outgrowth and neural crest cell migration through the anterior sclerotome.In vitro,sensory neurites on fibronectin, and motor neurites on basal lamina extract, avoid regions which contain substrate-bound collagen IX. This effect can be abolished by chondroitinase treatment, suggesting that the glycosaminoglycan component of the molecule is responsible for this activity. Further, collagen IX elicits a similar avoidance behavior by neural crest cellsin vitro.These data suggest that collagen IX contributes to the segmentation of the peripheral nervous systemin vivo.

Cellular Interactions That Guide Sensory and Motor Neurites Identified in an Embryo Slice Preparation

We used cultured cross sections (“slices”) of avian embryos to identify interactions that guide neurites during their encounters with seven tissues that impose a stereotyped gross anatomical nerve pattern. We show that cultured slices retain tissue morphology, molecular distribution patterns, and guidance cues. They also allow us to directly visualize responses of labeled sensory and motor neurons deposited on the slice's surface. This assay has high predictive power. Contact-mediated avoidance or stimulation and long-range attraction or repulsion are each distinguishable because each predicts different neurite lengths and trajectories. The analysis shows that all but one of these mechanisms contributes to guidance. Three tissues similarly stimulated neurite elongation, suggesting common responses to a contact-mediated stimulation. Four tissues similarly elicited avoidance on contact, suggesting a common contact-mediated inhibition. Neurite orientations implicate a previously unsuspected long-distance attraction to one tissue, dorsal anterior sclerotome. Long-range repulsion plays no detectable role. Each tissue elicits the same response in two different neural populations, sensory and motor neurons. These results suggest that a small set of repeated mechanisms mediates responses to tissues that axons contact serially during pathfinding.

Grafts of fibroblasts genetically modified to secrete NGF, BDNF, NT-3, or basic fgf elicit differential responses in the adult spinal cord

Neuronal and axonal responses to neurotrophic factors in the developing spinal cord have been relatively well characterized, but little is known about adult spinal responses to neurotrophic factors. We genetically modified primary rat fibroblasts to produce either nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or basic fibroblast growth factor (bFGF), then grafted these neurotrophic factor-secreting cells into the central gray matter of the spinal cord in adult rats. Spinal cord lesions were not made prior to grafting. From 2 wk to 6 mo later, sensory neurites of dorsal root origin extensively penetrated NGF-, NT-3-, and bFGF-producing grafts, whereas BDNF-secreting grafts elicited no growth responses. Putative noradrenergic neurites also penetrated NGF-secreting cell grafts. Local motor and corticospinal motor axons did not penetrate any of the neurotrophic factor-secreting grafts. These results indicate that unlesioned or minimally lesioned adult spinal cord sensory and putative noradrenergic populations retain significant neurotrophic factor responsiveness, whereas motor neurites are comparatively resistant even to those neurotrophic factors to which they exhibit survival dependence during development. Grafts of genetically modified cells can be a useful tool for characterizing neurotrophic factor responsiveness in the adult spinal cord and designing strategies to promote axonal regeneration after injury.