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

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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.