We have employed the orthograde transport of wheat germ agglutinin conjugated to horseradish peroxidase to study the organization of rubrospinal connections in adult and pouch young opossums. Our results suggest that: (1) in the adult opossum rubrospinal axons are distributed more widely than suggested by previous studies; (2) rubrospinal projections are formed postnatally in the opossum, but much earlier than corticospinal connections; (3) rubrospinal axons do not grow synchronously, as a massive bundle following a few leading axons, but by addition of axons over a protracted period of time; and (4) the growth of rubral axons into the spinal gray matter follows a predictable rostral to caudal gradient as well as a proximal to distal one relative to the tract. Rubrospinal development is discussed in light of the growth of cerebellar and cortical axons into the red nucleus and the development of motor function.

We have employed axonal transport and degeneration techniques to study the development of selected rubral connections in the North American opossum. Opossums were chosen for study because they are born in an immature state, 12 days after conception, and have a lengthy postnatal development. The results of our studies suggest that: (1) the red nucleus innervates the spinal cord early in development, but not as early as some areas of the brainstem; (2) rubrospinal development occurs postnatally in the opossum; (3) rubrospinal axons do not grow synchronously into the spinal cord, but are added over time; (4) rubrospinal development follows rough rostral to caudal and lateral to medial gradients; (5) the red nucleus is innervated by the cerebellum well before it receives projections from the cerebral cortex; and (6) cortical axons do not grow into the red nucleus until after rubrospinal axons have reached most of their adult targets.

Much of our understanding of the fascinating complexity of neuronal circuits comes from anatomical tracing studies that use dyes or fluorescent markers to highlight pathways that run through the brain and spinal cord. Viral vectors have been utilized by many previous groups as tools to highlight pathways or deliver transgenes to neuronal populations to stimulate growth after injury. In a series of studies, we explore anterograde and retrograde tracing with viral vectors to trace spinal pathways and explore their contribution to behavior in a rodent model. In a separate study, we explore the effect of stimulating intrinsic growth programs on regrowth of corticospinal tract (CST) axons after contusive injury. In the first study, we use self-complimentary adeno associated viral (scAAV) vectors to trace long descending tracts in the spinal cord. We demonstrate clear and bright labeling of cortico-, rubro- and reticulospinal pathways without the need for IH, and show that scAAV vectors transduce more efficiently than single stranded AAV (ssAAV) in neurons of both injured and uninjured animals. This study demonstrates the usefulness of these tracers in highlighting pathways descending from the brain. Retrograde tracing is also a key facet of neuroanatomical studies involving long distance projection neurons. In the next study, we highlight a lentivirus that permits highly efficient retrograde transport (HiRet) from synaptic terminals within the cervical and lumbar enlargements of the spinal cord. By injecting HiRet, we can clearly identify supraspinal and propriospinal circuits innervating MN pools relating to forelimb and hindlimb function. We observed robust labeling of propriospinal neurons, including high fidelity details of dendritic arbors and axon terminals seldom seen with chemical tracers. In addition, we examine changes in interneuronal circuits occurring after a thoracic contusion, highlighting populations that potentially contribute to spontaneous behavioral recovery in this lesion model. In a related study, we use a modified version of HiRet as part of a multi-vector system that synaptically silences neurons to explore the contribution of the rubrospinal tract (RST) and CST to forelimb motor behavior in an intact rat. This system employs Tetanus toxin at the neuronal synapse to prevent release of neurotransmitter via cleavage of vesicle docking proteins, effectively preventing the propagation of action potentials in those neurons. We find that shutdown of the RST has no effect on gross forelimb motor function in the intact state, and that shutdown of a small population of CST neurons in the FMC has a modest effect on grip strength. These studies demonstrate that the HiRet lentivirus is a unique tool for examining neuronal circuitry and its contribution to function. In the final study, we explore stimulation of the Phosphoinositide 3-kinase/Rac-alpha serine/threonine Protein Kinase (PI3K/AKT) growth pathway by antagonizing phosphatase and tensin homolog (PTEN), a major inhibitor, to encourage growth of CST axons after a contusive injury. We use systemic infusions of four distinct PTEN antagonist peptides (PAPs) targeted at different sites of the PTEN protein. We find robust axonal growth and sprouting caudal to a contusion in a subset of animals infused with PAPs targeted to the PTEN enzymatic pocket, including typical morphology of growing axons. Serotonergic fiber growth was unaffected by peptide infusion and did not correlate with CST fiber density. Though some variability was seen in the amount of growth within our animal groups, we find these PTEN antagonist peptides a promising and clinically relevant tool to encourage CST sprouting, and a potentially useful addition to therapies using combinatory strategies to enhance growth. These studies demonstrate that viral tracing is a powerful tool for mapping spinal pathways and elucidating their ability to reform spinal circuits after injury. Viral vectors can be used in both anterograde and retrograde tracing studies to highlight intricacies of neuronal cell bodies, axons and dendritic arbors with a high degree of fidelity. In the injured state, these tools can help identify pathways that contribute to spontaneous recovery of function by highlighting those that reform circuits past an injury site. In the uninjured state, these vectors can contain neuronal silencing methods that help define the contribution of specific pathways to behavior. / Neuroscience

No description available.

The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration.

The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration.

The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration. / Medicine, Faculty of / Graduate

To investigate the roles of different dorsal spinal pathways in controlling movements in rats, I performed lesions of specific spinal pathways and measured the behaviour abilities of rats using different sensorimotor behavioural tests. The first experiment was designed to understand the contribution of sensory pathways traveling in the dorsal funiculus during locomotion and skilled movements using sensitive behavioural tests. I demonstrated that ascending sensory fibers play an important role during overground locomotion and contribute to skilled forelimb movements. The second experiment compared the differences in sensorimotor abilities caused by dorsal funicular lesions performed at two different levels of rat spinal cord. My results showed that the pathways present in the cervical and thoracic dorsal funiculus exert different functional effects over control of limb movement during locomotion. The third experiment investigated the compensatory potential of dorsal funicular pathways after dorsolateral funicular injuries in rats. My results showed that dorsal funicular pathways do not compensate for loss of dorsolateral pathways during the execution of locomotor tasks, though there is indirect evidence that rats with dorsolateral funicular lesions might rely more on ascending sensory pathways in the dorsolateral funiculus during skilled forelimb movements. Finally, the fourth experiment was designed to investigate the compensation from dorsolateral funicular pathways after injuries to pyramidal tract in rats. I demonstrated that pathways running in the spinal dorsolateral funiculus do provide compensatory input to spinal circuitry to maintain skilled reaching abilities after lesions of the pyramidal tract but these same pathways do not appear to compensate during either overground locomotion or skilled locomotion. Thus, this compensatory response is task-specific. These results highlight the fact that behavioural context determines the nature of compensation from spared pathways after spinal cord injuries.

To investigate the roles of different dorsal spinal pathways in controlling movements in rats, I performed lesions of specific spinal pathways and measured the behaviour abilities of rats using different sensorimotor behavioural tests. The first experiment was designed to understand the contribution of sensory pathways traveling in the dorsal funiculus during locomotion and skilled movements using sensitive behavioural tests. I demonstrated that ascending sensory fibers play an important role during overground locomotion and contribute to skilled forelimb movements. The second experiment compared the differences in sensorimotor abilities caused by dorsal funicular lesions performed at two different levels of rat spinal cord. My results showed that the pathways present in the cervical and thoracic dorsal funiculus exert different functional effects over control of limb movement during locomotion. The third experiment investigated the compensatory potential of dorsal funicular pathways after dorsolateral funicular injuries in rats. My results showed that dorsal funicular pathways do not compensate for loss of dorsolateral pathways during the execution of locomotor tasks, though there is indirect evidence that rats with dorsolateral funicular lesions might rely more on ascending sensory pathways in the dorsolateral funiculus during skilled forelimb movements. Finally, the fourth experiment was designed to investigate the compensation from dorsolateral funicular pathways after injuries to pyramidal tract in rats. I demonstrated that pathways running in the spinal dorsolateral funiculus do provide compensatory input to spinal circuitry to maintain skilled reaching abilities after lesions of the pyramidal tract but these same pathways do not appear to compensate during either overground locomotion or skilled locomotion. Thus, this compensatory response is task-specific. These results highlight the fact that behavioural context determines the nature of compensation from spared pathways after spinal cord injuries.