Growth Of Corticospinal Tract Axons Research Articles

Optogenetic Interrogation of Functional Synapse Formation by Corticospinal Tract Axons in the Injured Spinal Cord.

To restore function after injury to the CNS, axons must be stimulated to extend into denervated territory and, critically, must form functional synapses with appropriate targets. We showed previously that forced overexpression of the transcription factor Sox11 increases axon growth by corticospinal tract (CST) neurons after spinal injury. However, behavioral outcomes were not improved, raising the question of whether the newly sprouted axons are able to form functional synapses. Here we developed an optogenetic strategy, paired with single-unit extracellular recordings, to assess the ability of Sox11-stimulated CST axons to functionally integrate in the circuitry of the cervical spinal cord. Initial time course experiments established the expression and function of virally expressed Channelrhodopsin (ChR2) in CST cell bodies and in axon terminals in cervical spinal cord. Pyramidotomies were performed in adult mice to deprive the left side of the spinal cord of CST input, and the right CST was treated with adeno-associated virus (AAV)-Sox11 or AAV-EBFP control, along with AAV-ChR2. As expected, Sox11 treatment caused robust midline crossing of CST axons into previously denervated left spinal cord. Clear postsynaptic responses resulted from optogenetic activation of CST terminals, demonstrating the ability of Sox11-stimulated axons to form functional synapses. Mapping of the distribution of CST-evoked spinal activity revealed overall similarity between intact and newly innervated spinal tissue. These data demonstrate the formation of functional synapses by Sox11-stimulated CST axons without significant behavioral benefit, suggesting that new synapses may be mistargeted or otherwise impaired in the ability to coordinate functional output. As continued progress is made in promoting the regeneration of CNS axons, questions of synaptic integration are increasingly prominent. Demonstrating direct synaptic integration by regenerated axons and distinguishing its function from indirect relay circuits and target field plasticity have presented technical challenges. Here we force the overexpression of Sox11 to stimulate the growth of corticospinal tract axons in the cervical spinal cord and then use specific optogenetic activation to assess their ability to directly drive postsynaptic activity in spinal cord neurons. By confirming successful synaptic integration, these data illustrate a novel optogenetic-based strategy to monitor and optimize functional reconnection by newly sprouted axons in the injured CNS.

Short Hairpin RNA against PTEN Enhances Regenerative Growth of Corticospinal Tract Axons after Spinal Cord Injury

Developing approaches to promote the regeneration of descending supraspinal axons represents an ideal strategy for rebuilding neuronal circuits to improve functional recovery after spinal cord injury (SCI). Our previous studies demonstrated that genetic deletion of phosphatase and tensin homolog (PTEN) in mouse corticospinal neurons reactivates their regenerative capacity, resulting in significant regeneration of corticospinal tract (CST) axons after SCI. However, it is unknown whether nongenetic methods of suppressing PTEN have similar effects and how regenerating axons interact with the extrinsic environment. Herein, we show that suppressing PTEN expression with short-hairpin RNA (shRNA) promotes the regeneration of injured CST axons, and these axons form anatomical synapses in appropriate areas of the cord caudal to the lesion. Importantly, this model of increased CST regrowth enables the analysis of extrinsic regulators of CST regeneration in vivo. We find that regenerating axons avoid dense clusters of fibroblasts and macrophages in the lesion, suggesting that these cell types might be key inhibitors of axon regeneration. Furthermore, most regenerating axons cross the lesion in association with astrocytes, indicating that these cells might be important for providing a permissive bridge for axon regeneration. Lineage analysis reveals that these bridge-forming astrocytes are not derived from ependymal stem cells within the spinal cord, suggesting that they are more likely derived from a subset of mature astrocytes. Overall, this study reveals insights into the critical extrinsic and intrinsic regulators of axon regeneration and establishes shRNA as a viable means to manipulate these regulators and translate findings into other mammalian models.

A re-assessment of the effects of intracortical delivery of inosine on transmidline growth of corticospinal tract axons after unilateral lesions of the medullary pyramid

This study was undertaken as part of the NIH “Facilities of Research Excellence-Spinal Cord Injury”, which supports independent replication of published studies. Here, we repeat an experiment reporting that intracortical delivery of inosine promoted trans-midline growth of corticospinal tract (CST) axons in the spinal cord after unilateral injury to the medullary pyramid. Rats received unilateral transections of the medullary pyramid and 1day later, a cannula assembly was implanted into the sensorimotor cortex contralateral to the pyramidotomy to deliver either inosine or vehicle. The cannula assembly was attached to an osmotic minipump that was implanted sub-cutaneously. Seventeen or 18days post-injury, the CST was traced by making multiple injections of miniruby-BDA into the sensorimotor cortex. Rats were killed for tract tracing 14days after the BDA injections. Sections through the cervical spinal cord were stained for BDA and immunostained for GAP43 and GFAP. Our results revealed no evidence for enhanced growth of CST axons across the midline of the dorsal column in rats that received intracortical infusion of inosine. Possible reasons for the failure to replicate are discussed.

Regenerative Growth of Corticospinal Tract Axons via the Ventral Column after Spinal Cord Injury in Mice

Studies that have assessed regeneration of corticospinal tract (CST) axons in mice after genetic modifications or other treatments have tacitly assumed that there is little if any regeneration of CST axons in normal mice in the absence of some intervention. Here, we document a previously unrecognized capability for regenerative growth of CST axons in normal mice that involves growth past the lesion via the ventral column. Mice received dorsal hemisection injuries at thoracic level 6-7, which completely transect descending CST axons in the dorsal and dorsolateral column. Corticospinal projections were traced by injecting biotinylated dextran amine (BDA) into the sensorimotor cortex of one hemisphere either at the time of the injury or 4 weeks after injury, and mice were killed at 20-23 or 46 d after injury. At 20-23 d after injury, BDA-labeled CST axons did not extend past the lesion except in one animal. By 46 d after injury, however, a novel population of BDA-labeled CST axons could be seen extending from the gray matter rostral to the injury into the ventral column, past the lesion, and then back into the gray matter caudal to the injury in which they formed elaborate terminal arbors. The number of axons with this highly unusual trajectory was small ( approximately 1% of the total number of labeled CST axons rostral to the injury). The BDA-labeled axons in the ventral column were on the same side as the main tract and thus are not spared ventral CST axons (which would be contralateral to the main tract). These results indicate that normal mice have a capacity for CST regeneration that has not been appreciated previously, which has important implications in studying the effect of genetic or pharmacological manipulations on CST regeneration in mice.

Development of the rat corticospinal tract through an altered glial environment

The major corticospinal tract (CST) in the rat is located at the base of the dorsal funiculus. It is a late-developing tract, and the growth of its axons into the lumbosacral region of the spinal cord does not occur until postnatal days 5 and 6. This delay is taken advantage of in this study in order to evaluate the effects of a markedly reduced glial population on ingrowth of the CST axons into the lumbosacral spinal cord. A reduction of the glial population is achieved by exposure of this region of spinal cord to X-radiation at 3 days of age. Growth of CST axons into and through the lumbosacral spinal cord in rats in which this region has undergone a radiation-induced depletion of glial cells is compared with that in their non-irradiated littermate controls by axonal tracing techniques using horseradish peroxidase (HRP). The HRP was applied directly to the motor cortices of normal and irradiated rats, and at all ages studied, there was anterograde filling of CST axons and their growth cones. At 3 days postnatally, the age when the lumbosacral spinal cord was irradiated in the experimental animals, CST axons were present in the more rostral thoracic levels. CST axons were observed in the lumbar region of non-irradiated rats on day 5, and by day 7 they were present at sacral levels. This same pattern was also noted in the animals with irradiated lumbosacral spinal cords: thus, despite the marked, radiation-induced reduction of glia in the region to be occupied and/or traversed by the ingrowing CST axons, the development of the tract appeared to be normal with respect to location and to time of arrival of the axons in the lumbosacral region.

Growth of corticospinal axons on prosthetic substrates introduced into the spinal cord of neonatal rats

Nitrocellulose implants treated with biological materials known to support neurite growth in vitro were introduced at thoracolumbar levels of the neonatal rat spinal cord before the arrival of growing corticospinal tract (CST) axons. Implant placement was designed to interrupt the normal CST growth path and provide a potential, alternative growth path. Subsequent growth of CST axons within the spinal cord in the vicinity of the implants was evaluated by labeling the axons with the anterograde transport of horseradish peroxidase. Untreated implants either blocked further CST axon growth or deflected CST axons to abnormal positions. Implants bearing living cells from spinal cord primary cultures were able to support the adhesion and growth of CST axons. Similarly, acellular implants coated with laminin, but not with poly- l-lysine supported the adhesion and growth of CST axons, suggesting that laminin or some other adhesive factor produced by immature neuroglial cells may be normally involved in CST axon growth and guidance.