Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.     

Regional specificity of developing reticulospinal, vestibulospinal, and vestibulo-ocular projections in the chicken embryo

The regional mapping of reticulospinal, vestibulospinal, and vestibulo-ocular neuron groups onto specific axonal pathways was determined in the chicken embryo by retrograde axonal tracing. Experiments were performed on in vitro preparations of the brain stem to allow for precisely localized tracer injections combined with selective lesions of axon tracts. Brain-stem neuron groups were labelled from 3 days of embryonic development, when the first reticulospinal axons reached the cervical spinal cord, to 9 days of embryonic development, when each of the three systems studied had acquired a relatively mature organization. A striking feature at all stages was the spatial segregation of many neuron groups that projected along different trajectories. Examples of such segregation were found for neuron groups projecting in the same tract on different sides of the brain stem, in different tracts on the same side of the brain stem, and rostrally versus caudally. The occurrence of this segregation from early stages suggests that the choice of projection pathway by many brain-stem neurons is in some way linked to cell position. In some regions of the brain stem, neuron groups projecting along different pathways are intermingled. At least some of this intermingling, however, appears to occur subsequent to the initial establishment of axon projection patterns. Comparison of the mapping patterns at progressively older stages, and with previous mapping in the 11-day-old embryo (Glover and Petursdottir, 1988; Petursdottir, 1990) suggests that these projections are established with little error. The one obvious example of remodelling involved the pontine reticulospinal projection, in which an early contralateral axon population appeared to retract from spinal to medullary levels over the course of a few days. A similar phenomenon may be involved in the elimination of part of the contralateral reticulospinal projection from the midmedulla.     

Regional specificity of developing reticulospinal,vestibulospinal, and vestibulo–ocular projections in the chicken embryo

The regional mapping of reticulospinal, vestibulospinal, and vestibulo–ocular neuron groups onto specific axonal pathways was determined in the chicken embryo by retrograde axonal tracing. Experiments were performed on in vitro preparations of the brain stem to allow for precisely localized tracer injections combined with selective lesions of axon tracts. Brain-stem neuron groups were labelled from 3 days of embryonic development, when the first reticulospinal axons reached the cervical spinal cord, to 9 days of embryonic development, when each of the three systems studied had acquired a relatively mature organization. A striking feature at all stages was the spatial segregation of many neuron groups that projected along different trajectories. Examples of such segregation were found for neuron groups projecting in the same tract on different sides of the brain stem, in different tracts on the same side of the brain stem, and rostrally versus caudally. The occurrence of this segregation from early stages suggests that the choice of projection pathway by many brain-stem neurons is in some way linked to cell position. In some regions of the brain stem, neuron groups projecting along different pathways are intermingled. At least some of this intermingling, however, appars to occur subsequent to the initial establishment of axon projection patterns. Comparison of the mapping patterns at progressively older stages, and with previous mapping in the 11-day-old embryo (Glover and Petursdottir, 1988; Petursdottir, 1990) suggests that these projections are established with little error. The one obvious example of remodelling involved the pontine reticulospinal projection, in which an early contralateral axon population appeared to retract from spinal to medullary levels over the course of a few days. A similar phenomenon may be involved in the elimination of part of the contralateral reticulospinal projection from the midmedulla.     

A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.     

Netrin-1 signaling for sensory axons

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a ‘waiting period’ of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.     

DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension

We have identified a 95 kd cell surface protein, DM-GRASP, that is expressed on a restricted population of axons. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons. We have cloned and sequenced the cDNA corresponding to this protein and find that it is a new member of the immunoglobulin superfamily of adhesion molecules. Consequently we have named this protein DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein that is expressed in the dorsal funiculus and ventral midline of the chick spinal cord.     

Soma position is correlated with time of development in three types of identified reticulospinal neurons

The relationship among neuronal type, position, and time course of development of identified neurons was examined in the zebrafish (Brachydanio rerio). The cells studied, the reticulospinal neurons Mauthner, MiM1, and MiV1, are located within the same small region in the hindbrain, differ stereotypically in their positions within this region and also in their axonal projections. All of the cell types were generated and had initiated axonal outgrowth by the second day after fertilization. The time that these events occurred was specific for each cell type, with axonal outgrowth occurring about 10 hr after the neuronal birthday. Furthermore, the time of the events varied systematically according to the dorsoventral location of the neuron within the set.     

Developmental expression of the axonal glycoprotein TAG-1: differential regulation by central and peripheral neurons in vitro.

TAG-1 is a 135,000 Mr axonal glycoprotein of the immunoglobulin superfamily that promotes axon extension in vitro. One distinguishing feature of TAG-1 is its transient expression on subsets of axons in the developing nervous system. To examine the mechanisms that regulate TAG-1, we have monitored the expression of this protein by developing central and peripheral neurons in vitro. TAG-1 was detected on the surface of a subset of E11 to E13 spinal cord neurons in vitro and was also released by these neurons. Expressions of TAG-1 on the cell surface was transient but it was possible to detect a released form of TAG-1 at all times in vitro. Spinal cord neurons isolated from older embryos did not express surface TAG-1 when they regenerated axons in vitro. Changes in the environment of spinal cord neurons did not alter the time course of TAG-1 expression, suggesting that regulation of the protein is cell autonomous. In contrast to these results with spinal cord neurons, surface expression of TAG-1 by DRG neurons persisted in vitro and adult DRG neurons re-expressed TAG-1 when grown in vitro. The cell surface and released forms of TAG-1 therefore appear to be regulated differently by central and peripheral neurons.     

Integration and Long Distance Axonal Regeneration in the Central Nervous System from Transplanted Primitive Neural Stem Cells

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.     

WNT-3, expressed by motoneurons,regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons

Sensory axons from dorsal root ganglia neurons are guided to spinal targets by molecules differentially expressed along the dorso-ventral axis of the neural tube. NT-3-responsive muscle afferents project ventrally, cease extending, and branch upon contact with motoneurons (MNs), their synaptic partners. We have identified WNT-3 as a candidate molecule that regulates this process. Wnt-3 is expressed by MNs of the lateral motor column at the time when MNs form synapses with sensory neurons. WNT-3 increases branching and growth cone size while inhibiting axonal extension in NT-3- but not NGF-responsive axons. Ventral spinal cord secretes factors with axonal remodeling activity for NT-3-responsive neurons. This activity is present at limb levels and is blocked by a WNT antagonist. We propose that WNT-3, expressed by MNs, acts as a retrograde signal that controls terminal arborization of muscle afferents.     

Interaction between hindbrain and spinal networks during the development of locomotion in zebrafish

Little is known about the role of the hindbrain during development of spinal network activity. We set out to identify the activity patterns of reticulospinal (RS) neurons of the hindbrain in fictively swimming (paralyzed) zebrafish larvae. Simultaneous recordings of RS neurons and spinal motoneurons revealed that these were coactive during spontaneous fictive swim episodes. We characterized four types of RS activity patterns during fictive swimming: (i) a spontaneous pattern of discharges resembling evoked high-frequency spiking during startle responses to touch stimuli, (ii) a rhythmic pattern of excitatory postsynaptic potentials (EPSPs) whose frequency was similar to the motoneuron EPSP frequency during swim episodes, (iii) an arrhythmic pattern consisting of tonic firing throughout swim episodes, and (iv) RS cell activity uncorrelated with motoneuron activity. Despite lesions to the rostral spinal cord that prevented ascending spinal axons from entering the hindbrain (normally starting at approximately 20 h), RS neurons continued to display the aforementioned activity patterns at day 3. However, removal of the caudal portion of the hindbrain prior to the descent of RS axons left the spinal cord network unable to generate the rhythmic oscillations normally elicited by application of N-methyl-d-aspartate (NMDA), but in approximately 40% of cases chronic incubation in NMDA maintained rhythmic activity. We conclude that there is an autonomous embryonic hindbrain network that is necessary for proper development of the spinal central pattern generator, and that the hindbrain network can partially develop independently of ascending input.     

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.     

Noggin is required for correct guidance of dorsal root ganglion axons

Members of the bone morphogenetic protein family of secreted protein signals have been implicated as axon guidance cues for specific neurons in Caenorhabditis elegans and in mammals. We have examined axonal pathfinding in mice lacking the secreted bone morphogenetic protein antagonist Noggin. We have found defects in projection of several groups of neurons, including the initial ascending projections from the dorsal root ganglia, motor axons innervating the distal forelimb, and cranial nerve VII. The case of the dorsal root ganglion defect is especially interesting: initial projections from the dorsal root ganglion enter the dorsal root entry zone, as normal, but then project directly into the gray matter of the spinal cord, rather than turning rostrally and caudally. Explant experiments suggest that the defect lies within the spinal cord and not the dorsal root ganglion itself. However, exogenous bone morphogenetic proteins are unable to attract or repel these axons, and the spinal cord shows only very subtle alterations in dorsal-ventral pattern in Noggin mutants. We suggest that the defect in projection into the spinal cord is likely the result of bone morphogenetic proteins disrupting the transduction of some unidentified repulsive signal from the spinal cord gray matter.     

Retrograde labeling of regenerated electromotor neurons with HRP in a teleost fish,Sternarchus albifrons: Relation to cell death

Summary Back-labeling of regenerated electromotor neurons in the teleost Sternarchus albifrons was performed to test the hypothesis that, in regenerated spinal cord, incorrectly located electromotor neurons are eliminated because their axons do not reach the correct target area (electric organ). In each cross section examined, all of the regenerated electromotor neurons ipsilateral to the implantation site were labeled with horseradish peroxidase, including those ectopic cells located at the edge of the cord, which are later eliminated by selective cell death. Retrograde labeling of these ectopic neurons demonstrates that their axons do extend into the correct target area (the regenerated electric organ). Thus total misdirection of the axons cannot be the cause of their subsequent cell death. We conclude that selective neuronal death in this system does not reflect the absence of axonal projection to the correct target area.A preliminary report on this work has been presented in Soc. Neurosci. Abstracts 10:48 (1984)     

The Adhesive Role of Acetylcholinesterase (AChE): Detection of AChE Binding Proteins in Developing Rat Spinal Cord

Acetylcholinesterase (AChE) is expressed by dorsal root ganglion (DRG) neurons during developmental periods when their central axons are growing into and through the spinal cord. Importantly, our previous studies have shown that AChE induces DRG axonal outgrowth by an adhesive mechanism and thus, have now employed a blot overlay technique to screen for potential AChE binding proteins in the developing spinal cord. Our results show that: (1) AChE binds to proteins with apparent molecular weights of 200, 110, 35, and 33k Da; (2) these proteins are developmentally expressed during periods of axonal outgrowth from DRG neurons; (3) all four proteins are synthesized by astrocytes; and (4) AChE binding to these proteins is highly dependent on ionic strength supporting an electrostatic mechanism of adhesion. Taken together, these data provide further documentation for the participation of AChE in adhesive interactions during morphogenesis of the central nervous system and suggest a role for astrocytes in regulating AChE-mediated axonal growth.Special issue dedicated to Lawrence. F. Eng.     

The neuroanatomy of an amphibian embryo spinal cord

Horseradish peroxidase has been used to stain spinal cord neurons in late embryos of the clawed toad (Xenopus laevis). It has shown clearly the soma, dendrites and axonal projections of spinal sensory, motor and interneurons. On the basis of light microscopy we describe nine differentiated spinal cord neuron classes. These include the Rohon-Beard cells and extramedullary cells which are both primary sensory neurons, one class of motoneurons that innervate the segmental myotomes, two classes of interneurons with decussating axons, three classes of interneurons with ipsilateral axons and a previously undescribed class of ciliated ependymal cells with axons projecting ipsilaterally to the brain. We believe that all differentiated neuron classes are described and that this anatomical account is the most complete for any vertebrate spinal cord.