Date of Award

Spring 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Interdepartmental Neuroscience Program

First Advisor

Cafferty, William

Abstract

The corticospinal tract (CST) is the major descending motor tract responsible for voluntary movement in all mammals. Corticospinal neurons (CSNs) have cell bodies in layer 5 of the primary motor cortex, with axons that descend through the internal capsule, decussate at the medullary pyramids, and innervate every spinal segment along the entire spinal neuro-axis. Injury, disease, and neurodegeneration within this pathway result in chronic, irreversible functional deficits in motor and sensory function due to inhibitory extrinsic substrates, including central nervous system (CNS) myelin and chondroitin sulfate proteoglycans (CSPGs) in the extracellular matrix, and poor intrinsic growth capacity. Current strategies for repairing the CST after injury remain woefully incomplete due to the underexplored molecular diversity among neurons in this tract. To study the heterogeneity of uninjured adult CSNs, we developed a method for robust dissociation of cortical layer 5 pyramidal neurons, ensuring optimal cytoplasmic integrity. Using our protocol, we combined retrograde tracing from the cervical and lumbar spinal cord with single-cell RNA sequencing (scRNAseq) to build a transcriptional atlas of adult CSNs. Using publicly available datasets, we ascribed anatomical identity to molecularly distinct CSNs, showing that CSNs segregate based on supraspinal connectivity, in addition to spinal connectivity. By leveraging machine learning tools, we built a classifier that can reliably identify CSNs in M1 from layers 2, 3 and 5 pyramidal neurons. To explore CSN diversity in the context of injury, we performed bulk RNA sequencing (RNAseq) of CSNs from mice that had undergone spinal cord injury (SCI) and coupled it with two models of enhanced plasticity, a genetic knockout of Nogo Receptor 1 (Ngr1) or task-specific rehabilitation. Combining these bulk RNAseq studies with data from the single-cell CSN atlas enabled us to putatively assign each CSN with a plasticity index, revealing that intratelencephalic CSNs have an enhanced, innate plasticity potential. Lastly, by comparing CSNs during uniquely defined phases of postnatal patterning, we identified putative positive and negative regulators of long-distance axon growth. Together, these studies represent the first transcriptional characterization of the CST at the single-cell level. These data enable future studies that will explore the molecular mechanisms associated with CST plasticity and repair, and ultimately facilitate development of therapies that enhance functional recovery for individuals with SCI.

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