DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine perchlorate (DiI)¹. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching² (Fig. 1).The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)³. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself⁴ (Fig. 2), however, other options have been discussed as well⁵.This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.     

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.     

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.     

Stimulating regeneration in the damaged spinal cord.

Great progress has been made in recent years in experimental strategies for spinal cord repair. In this review we describe two of these strategies, namely the use of neurotrophic factors to promote functional regeneration across the dorsal root entry zone (DREZ), and the use of synthetic fibronectin conduits to support directed axonal growth. The junction between the peripheral nervous system (PNS) and central nervous system (CNS) is marked by a specialized region, the DREZ, where sensory axons enter the spinal cord from the dorsal roots. After injury to dorsal roots, axons will regenerate as far as the DREZ but no further. However, recent studies have shown that this barrier can be overcome and function restored. In animals treated with neurotrophic factors, regenerating axons cross the DREZ and establish functional connections with dorsal horn cells. For example, intrathecal delivery of neurotrophin 3 (NT3) supports ingrowth of A fibres into the dorsal horn. This ingrowth is revealed using a transganglionic anatomical tracer (cholera toxin subunit B) and analysis at light and electron microscopic level. In addition to promoting axonal growth, spinal cord repair is likely to require strategies for supporting long-distance regeneration. Synthetic fibronectin conduits may be useful for this purpose. Experimental studies indicate that fibronectin mats implanted into the spinal cord will integrate with the host tissue and support extensive and directional axonal growth. Growth of both PNS and CNS axons is supported by the fibronectin, and axons become myelinated by Schwann cells. Ongoing studies are aimed at developing composite conduits and promoting axonal growth from the fibronectin back into the spinal cord.     

Live imaging of dorsal root axons after rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo(1). These mice have been used extensively for in vivo imaging of muscle(2-4) and brain(5-7), and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy(8,9). Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished(10). Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord(11). In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.     

Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and "waiting periods"

We are studying how axons branch in vivo. Individual cortical neurons send axons to both the spinal cord and the basilar pons. Here we show that the corticopontine projection develops by an interstitial budding of collaterals from parent axons rather than a reported mechanism of axon branching, growth cone bifurcation. This mechanism is used regardless of whether the parent axon's postpontine segment, which forms the corticospinal projection, is permanent (motor cortex) or transient (visual cortex). Budding occurs days after the parent axons grow spinally past the pons, accounting for the "waiting period" reported in this system in contrast to an alternative explanation that the growth cones pause outside of their target. Timing and location of pontine collateral budding vary with cortical origin of the parent axon and are correlated with the temporal ordering of axon arrival.     

Netrin-1 signaling for sensory axons: Involvement in sensory axonal development and regeneration

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.Key words: netrin-1, dorsal root ganglion, axon guidance, chemorepellent, Unc5, spinal cord, axon regenerationDeveloping axons navigate to their targets by responding to attractive and repulsive guidance cues working in a contact-dependent or diffusible fashion in their environment (reviewed in ref. ¹). During early development of the primary sensory system, centrally projecting sensory axons from dorsal root ganglion (DRG) neurons extend toward the dorsolateral region of the spinal cord (Fig. 1A and C), where they enter the spinal cord exclusively through the dorsal root entry zone (DREZ), and never orient themselves toward the notochord or the ventral spinal cord (Fig. 1A; reviewed in ref. ²). We previously showed that the notochord but not the ventral spinal cord secretes semaphorin 3A (Sema3A), which is known to be a chemorepellent for DRG axons at early developmental stages (Fig. 1A).³ This is the reason why DRG axons never project toward the notochord. Along the same line, it is highly possible that the ventral spinal cord may secrete some chemorepulsive cue other than Sema3A for DRG axons.Open in a separate windowFigure 1Netrin-1 plays a critical role in sensory axonal guidance as an axon chemorepellent. (A) A schematic diagram of a thoracic transverse section of an E10 mouse embryo, summarizing the possible mechanism of netrin-1 action in early DRG axonal guidance. When DRG axons project toward the DREZ in the dorsal spinal cord (dSC), ventrally derived netrin-1 chemorepels DRG axons to prevent them from orienting aberrantly toward the ventral spinal cord (vSC) (upper). NC; notochord. In netrin-1-deficient embryos, some DRG axons misorient themselves toward the ventral spinal cord, because of the absence of netrin-1 proteins in the ventral spinal cord (lower). (B) At E12.5 when DRG axons grow to the marginal zone of the spinal cord longitudinally (arrows) to form the dorsal funiculus (DF), netrin-1 proteins are transiently expressed in a subpopulation of dorsal spinal cord cells adjacent to the dorsal funiculus (upper). In netrin-1-deficient embryos, the dorsal funiculus is disorganized because DRG axons are no longer waiting for invading the dorsal mantle layer (lower). (C) Gain-of-function experiments by electroporation confirm the repulsive activity of netrin-1 toward DRG axons. When netrin-1 is misexpressed in the dorsal spinal cord, the number of DRG axons that enter the DREZ is significantly reduced compared with the control, because some DRG axons fail to project toward the DREZ and turn in the wrong direction.After entering the spinal cord, DRG axons grow to the marginal zone of the spinal cord longitudinally to form the dorsal funiculus without projecting to the dorsal mantle layer for a few days (this delay of the axonal projection to the mantle layer is referred to as the ‘waiting period;’ Fig. 1B). A few days later, proprioceptive afferents of DRGs begin to send collaterals into the dorsal layers, and cutaneous afferents project ventrally through the dorsal layers.⁴ This evidence raises the possibility that some repulsive cues transiently prevent the collaterals of DRGs from penetrating the dorsal spinal cord during this waiting period.Netrins are a family of secreted proteins that play a key role in axonal guidance, cell migration, morphogenesis and angiogenesis.⁵ Netrin-1 is a bifunctional axonal guidance cue, attracting some axons including commissural axons via the Deleted in Colorectal Cancer (DCC) receptor and repelling others via Unc5 receptors (reviewed in ref. ⁶). However, it has not been clear whether netrin-1 plays a role in sensory axonal guidance during development.Several observations strongly suggest a role for netrin-1 in DRG axonal guidance as a repulsive guidance cue during development.^7,8 First, in the mouse embryo at embryonic day (E) 10–11.⁵ when many DRG axons orient themselves to reach the DREZ, netrin-1 is strongly expressed in the floor plate of the ventral spinal cord but not in the dorsal spinal cord (Fig. 1A). Second, at E12.5 when DRG neurons extend their axons longitudinally along the dorsolateral margin of the spinal cord, netrin-1 is expressed in the dorsolateral region adjacent to the DREZ (Fig. 1B), but its expression is down-regulated in the dorsal spinal cord at E13.5 when many collaterals have entered the mantle layer. Third, repulsive netrin-1 receptor Unc5c is expressed in the DRG neurons during development.These observations motivated us to explore whether netrin-1/Unc5c signaling contributes to DRG axonal guidance. We used cell and tissue cultures combined with tissues from netrin-1-deficient mice. We clearly showed that netrin-1 exerts a chemorepulsive activity toward developing DRG axons and that the ventral spinal cord-derived repulsive activity depends on netrin-1 in vitro.⁸ Additional evidence for a chemorepulsive role of netrin-1 came from the observation of DRG axonal trajectories in netrin-1-deficient mice.^7,8 In netrin-1-deficient embryos at E10, we showed that some DRG axons became misoriented toward the ventral spinal cord, probably because of the absence of netrin-1 proteins in the ventral spinal cord (Fig. 1A). In addition, at E12.5 when DRG axons grow to the marginal zone of the spinal cord longitudinally to form the dorsal funiculus, the dorsal funiculus is disorganized in netrin-1-deficient embryos, because in the absence of netrin-1 DRG axons are not waiting for invading the dorsal mantle layer adjacent to the dorsal funiculus (Fig. 1B). Gain-of-function experiments further confirmed the repulsive activity of netrin-1 toward DRG axons (Fig. 1C). These lines of evidence lead us to the conclusion that dorsally derived netrin-1 plays an important role in providing the ‘waiting period’ for extension of collaterals from sensory afferents and that ventrally derived netrin-1 prevents sensory axons from misorienting themselves toward the ventral spinal cord.At later developmental stages (E13.5), DRG axons still possess a weak responsiveness to the chemorepulsive activity of netrin-1 in vitro.⁸ In addition, both postnatal and adult DRG neurons respond to netrin-1-induced axon inhibition.⁹ Consistent with these results, DRG neurons at not only later developmental stages (E13.5) but also postnatal stages express the repulsion-mediating netrin-1 receptor Unc5c.^8,9Generally, lesioning of the dorsal column projection of sensory axons results in a complete lack of regeneration. The possible explanation for the complete lack of regeneration is that the environment, the lesion site itself and/or oligodendrocytes adjacent to the lesion, may be non-permissive for regenerating axons.¹⁰ Sema3A and chondroitin sulfate proteoglycans (CSPGs) are candidates as major inhibitors of sensory axonal regeneration in the spinal cord, because they are expressed in the lesion site and can inhibit DRG axonal growth in vitro.^3,11–14 Recently, Kaneko et al. showed that a selective inhibitor of Sema3A also enhances axonal regeneration and functional recovery in a subpopulation of sensory neurons after lesioning of the dorsal column.¹² More recently, McMahon''s group clearly demonstrated that enzymatic degradation of CSPGs on the dorsal column lesion of the spinal cord promotes sensory axonal regeneration and functional recovery.^13,14 Although these treatments greatly improved functional recovery, complete sensory axonal growth and functional recovery have not been yet achieved after the spinal cord injury. To promote further recovery of sensory axonal regeneration in the CNS, we should focus on other candidate inhibitors of CNS injury sites.Following spinal cord injury, the expression of the attraction- mediating netrin-1 receptor DCC decreases, while the expression level of the repulsive receptor Unc5c returns to normal.¹⁵ Levels of netrin-1 expression also stay unchanged in neurons and oligodendrocytes adjacent to the lesion site. Together with the in vitro evidence described above, these data strongly suggest a possible role for netrin-1 as a novel inhibitor of CNS myelin for regenerating DRG axons in the dorsal column-lesioned spinal cord. Further studies will be required to show directly the functional recovery of sensory axons in the spinal cord by perturbation of netrin-1 in and around the lesion site after spinal cord injury.     

Stage-dependent restoration of sensory dorsal columns following spinal cord transection in anuran tadpoles

In frogs sensory axons from the lumbar dorsal roots ascend in the dorsal column of the spinal cord to terminate in the medulla and cerebellum. The response of these axons to complete transection of the thoracic spinal cord has been analysed in Rana temporaria tadpoles at different stages of development. The presence and position of dorsal column axons were assessed by using the anterograde transport of horseradish peroxidase or by electrophysiological methods. Before developmental stage VIII, dorsal column axons can grow across the transection and reach their normal areas of termination in the brainstem. Axons that do cross the transection follow their normal pathways. From stage VIII onwards this capacity for growth is largely lost. These results are discussed in terms of the relation between neurogenesis, axon growth and axonal regeneration.     

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.     

Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant

Developing axons are guided to their targets by molecular cues in their local environment. Some cues are short-range, deriving from cells along axonal pathways. There is also increasing evidence for longer-range guidance cues, in the form of gradients of diffusible chemoattractant molecules, which originate from restricted populations of target cells. The guidance of developing commissural axons within the spinal cord depends on one of their intermediate cellular targets, the floor plate. We have shown previously that floor plate cells secrete a diffusible factor(s) that can alter the direction of commissural axon growth in vitro. Here we show that the factor is an effective chemoattractant for commissural axons. It can diffuse considerable distances through a collagen gel matrix and through dorsal and ventral neural epithelium in vitro to reorient the growth of virtually all commissural axons. The orientation of axons occurs in the absence of detectable effects on the survival of commissural neurons or on the rate of commissural axon extension. The regionally restricted expression of the factor suggests that it is present in the embryonic spinal cord in a gradient with its high point at the floor plate. These observations support the idea that the guidance of commissural axons to the ventral midline of the spinal cord results in part from the secretion of a chemoattractant by the floor plate.     

Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons

Axon bifurcation results in the formation of sister branches, and divergent segregation of the sister branches is essential for efficient innervation of multiple targets. From a genetic mosaic screen, we find that a lethal mutation in the Drosophila Down syndrome cell adhesion molecule (Dscam) specifically perturbs segregation of axonal branches in the mushroom bodies. Single axon analysis further reveals that Dscam mutant axons generate additional branches, which randomly segregate among the available targets. Moreover, when only one target remains, branching is suppressed in wild-type axons while Dscam mutant axons still form multiple branches at the original bifurcation point. Taken together, we conclude that Dscam controls axon branching and guidance such that a neuron can innervate multiple targets with minimal branching.     

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.     

Septin-driven coordination of actin and microtubule remodeling regulates the collateral branching of axons

Axon branching is fundamental to the development of the peripheral and central nervous system. Branches that sprout from the axon shaft are termed collateral or interstitial branches. Collateral branching of axons requires the formation of filopodia from actin microfilaments (F-actin) and their engorgement with microtubules (MTs) that splay from the axon shaft. The mechanisms that drive and coordinate the remodeling of actin and MTs during branch morphogenesis are poorly understood. Septins comprise a family of GTP-binding proteins that oligomerize into higher-order structures, which associate with membranes and the actin and microtubule cytoskeleton. Here, we show that collateral branching of axons requires SEPT6 and SEPT7, two interacting septins. In the axons of sensory neurons, both SEPT6 and SEPT7 accumulate at incipient sites of filopodia formation. We show that SEPT6 localizes to axonal patches of F-actin and increases the recruitment of cortactin, a regulator of Arp2/3-mediated actin polymerization, triggering the emergence of filopodia. Conversely, SEPT7 promotes the entry of axonal MTs into filopodia, enabling the formation of collateral branches. Surprisingly, septins provide a novel mechanism for the collateral branching of axons by coordinating the remodeling of the actin and microtubule cytoskeleton.     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

PlexinA1 signaling directs the segregation of proprioceptive sensory axons in the developing spinal cord

As different classes of sensory neurons project into the CNS, their axons segregate and establish distinct trajectories and target zones. One striking instance of axonal segregation is the projection of sensory neurons into the spinal cord, where proprioceptive axons avoid the superficial dorsal horn-the target zone of many cutaneous afferent fibers. PlexinA1 is a proprioceptive sensory axon-specific receptor for sema6C and sema6D, which are expressed in a dynamic pattern in the dorsal horn. The loss of plexinA1 signaling causes the shafts of proprioceptive axons to invade the superficial dorsal horn, disrupting the organization of cutaneous afferents. This disruptive influence appears to involve the intermediary action of oligodendrocytes, which accompany displaced proprioceptive axon shafts into the dorsal horn. Our findings reveal a dedicated program of axonal shaft positioning in the mammalian CNS and establish a role for plexinA1-mediated axonal exclusion in organizing the projection pattern of spinal sensory afferents.     

Molecular and cellular characterization of the glial roof plate of the spinal cord and optic tectum: a possible role for a proteoglycan in the development of an axon barrier.

Certain types of glial structures, located at strategic positions along axon pathways, may provide the mechanical and/or chemical elements for the construction of barriers which can grossly direct the elongation of axons during development. The roof plate, a putative axon barrier, is located along the dorsal midline of the developing spinal cord and may be important for the guidance of the commissural and dorsal column axons. We examined the roof plate to determine the developmental morphology of the region and to determine which molecules were correlated with the barrier function when axons were growing nearby. Light and electron microscopic observations of the roof plate revealed that this glial domain undergoes a dramatic change in shape from a "wedge" with large extracellular spaces between the cell apices at E12.5 to a thin, dense septum with reduced extracellular space at E15.5. Immunocytochemical techniques demonstrated that highly sialylated neural cell adhesion molecule (N-CAM), the carbohydrate recognized by L2 monoclonal antibody, cholinesterase, stage-specific embryonic antigen 1, and a ligand that binds tetragonolobus purpureas agglutinin are expressed by the roof plate. These molecules, however, were also found in other regions of the spinal cord which are permissive or attractive to axon growth. A molecule which is unique to the roof plate when axons grow close to, but do not cross, the dorsal midline is a glycosaminoglycan (GAG), keratan sulfate. Keratan sulfate is also present in the tectal midline and in other noninnervated regions such as the outer epidermis and developing cartilage. Our data suggest that keratan sulfate, alone or in combination with other molecules expressed by the roof plate, may be responsible, in part, for the inhibition of axon elongation through the roof plate in the embryonic spinal cord.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons

The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.