Thesis (M.Sc.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Thesis (MPhil)--University of Melbourne, Centre for Neuroscience, The Howard Florey Institute, 2009. / Typescript. Includes bibliographical references (leaves 137-160)

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 145-165). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 September 20

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 75-84).

Les mouvements locomoteurs rythmiques nécessitent l’intervention de circuits neuronaux qui coordonnent l’activité motrice des deux parties du corps. Ces circuits sont formés majoritairement par les projections des interneurones commissuraux de la moelle épinière. Des facteurs de guidage comme la Nétrine, les Slits jouent un rôle fondamental dans la mise en place de ces projections. Une étude a également montré qu’une signalisation impliquant le récepteur Neuropiline2 (Nrp2) des signaux Sémaphorines de la classe 3 (Sema3), participe au guidage de ces projections et cela uniquement après la traversée de la ligne médiane (Zou et al. 2000). Ma thèse porte sur l’étude fonctionnelle d’un ligand de la Nrp2, la Sema3B dans le développement de ce système de projections. J’ai analysé une souris invalidée pour Sema3B et observé de nombreuses erreurs de trajectoires après la traversée de la ligne médiane. Je me suis ensuite intéressée aux mécanismes sous-jacents au gain de réponse : par une approche pharmacologique et biochimique j’ai pu montrer que le signal de la plaque du plancher inhibe une activité de dégradation dépendante de la calpaine1. L’inhibition de cette voie conduit à la stabilisation d’un co-récepteur de la Nrp2, la Plexine A1 dont l’expression est très faible dans les axones n’ayant pas encore traversé la ligne médiane. Cette régulation permet alors l’assemblage d’un complexe récepteur fonctionnel de Sema3B, comprenant cette Plexine associée à la Nrp2 au niveau des cônes de croissance. J’ai identifié la molécule d’adhérence NrCAM, et le facteur neurotrophique GDNF comme étant les facteurs de la plaque du plancher déclencheurs de la réponse / Rhythmic locomotor movements require neuronal circuits ensuring left-right coordination. Spinal commissural projections participate to left-right coordination of limb movements by mediating reciprocal inhibition in synchrony. Extensive research of the mechanisms governing the formation of commissural pathways focused on dorsally-located spinal commissural neurons, establishing a fundamental role for multiple guidance cues derived for the midline and surrounding tissues, including Netrins, Slits and various morphogens. Semaphorin (Sema2)/Neuropilin-2 (Nrp2) signaling has been proposed to contribute to the guidance of commissural projections in the spinal cord at the post- but not pre-crossing stage (Zou et al, 2000). My PhD project aimed at analyzing the role of a Nrp2 ligand, Sema3B, in the guidance of spinal commissural projections, whose expression is dynamic and restricted to some territories, including the floor plate in which axons cross the midline. Analysis of Sema3B null mice showed that the loss of Sema3B induces a range of guidance defects of post-crossing commissural pathways. I investigated the underlying mechanisms and found that the floor plate signal induces through blockade of a calpain 1-dependant pathway the stabilization of the Nrp2 co-receptor Plexin-A1, and enable the assembly of Nrp2/Plexin-A1 sub-units into functional complexes for Sema3B in post-crossing commissural growth cones. I identified the cell adhesion molecule NrCAM and the neurotrophic factor GDNF as being the floor-platederived signals triggering the gain of response

Axon pathfinding is an important process required for the establishment of proper neuronal connections during development. An increasing number of secreted and membrane-anchored molecules have been identified as axon guidance cues, which can act as positive or negative factors to increase or decrease the growth of axons and influence the direction of axonal growth. These axon guidance factors present in the extracellular environment interact with receptors present on the growth cone, a structure located at the tip of the axon which functions as the motor unit for the axon. Upon binding to their receptors on the growth cone, the guidance factors then elicit an intracellular signaling cascade within the axon that ultimately influences the direction of axon growth, often through a direct, non-transcriptional mechanism. In this dissertation, we show that Sonic hedgehog (Shh) acts as an axon guidance factor for chick retinal ganglion cell (RGC) axons in a concentration-dependent manner. At a low concentration, Shh functions as a positive factor that induces axon growth and attractive turning while, at a high concentration, Shh functions as a negative factor that induces axon retraction and repulsive axon turning. We further characterized the effects of Shh on macropinocytosis, a fluid-phase type of endocytosis, in the axons. A high concentration of Shh significantly increased macropinocytosis in the axons. Macropinocytosis resulted in the generation of large, dextran-positive, clathrinindependent vesicles in the axonal growth cones, prior to growth cone collapse, axon retraction and repulsive axon turning. These vesicles were found to require dynamic F-actin, nonmuscle myosin II and dynamin for their formation but were formed independently of PI3 kinase signaling. Interestingly, a low concentration of Shh had an opposite effect on macropinocytosis. A low concentration of Shh and soluble laminin decreased macropinocytosis and additionally increased the turnover of these vesicles within the axons, suggesting positive axon guidance factors can additionally regulate downstream processing or maturation of these vesicles. The effect of Shh on regulating the motility of macropinosomes within the axons was investigated. A low concentration of Shh appeared to increase the motility of these vesicles along axonal microtubules in a cAMPdependent manner. However, a high concentration of Shh did not appear to affect the motility of the macropinosomes, suggesting that it likely plays a more predominant role in the formation of these vesicles within the growth cone. When we began this work, a large body of research existed describing the effects of guidance factors on regulating the cytoskeleton during axon motility. However, the role of membrane trafficking events during axon growth and guidance were very poorly characterized. Since we began this project, an increasing number of reports have shown that endo- and exocytosis are important for axon growth and, here, we show that macropinocytosis induced by negative axon guidance factors plays a critical role in growth cone collapse, axon retraction and repulsive axon turning. Positive axon guidance factors also affect macropinocytosis within the axons and additionally regulate their maturation, suggesting that membrane trafficking events mediated by axon guidance factors are important for regulating axon growth and pathfinding.

Glia, whose name derives from the original Greek word, meaning “glue,” have long been understood to be cells that play an important functional role in the nutritive and structural support of the central nervous system, yet their full involvement has been historically undervalued. Despite the strong evidence that glial reactions to cellular debris govern the health of the nervous system, the specific properties of damaged axonal debris and the mechanisms by which glia sense them, morphologically adapt to their presence, and initiate phagocytosis for clearance, have remained poorly understood. The work presented in this thesis was aimed at addressing this fundamental gap in our understanding of the role for glia in neurodegenerative processes. I demonstrate that the cellular machinery responsible for the phagocytosis of apoptotic cell corpses is well conserved from worms to mammals. Draper is a key component of the glial response machinery and I am able to show here, for the first time, that it signals through Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70. Shark binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. I show that Shark activity is essential for Draper-mediated signaling events in vivo, including the recruitment of glial membranes to axons undergoing Wallerian degeneration. I further show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. Therefore I propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the subsequent downstream activation of the Draper pathway. I observed that these Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signaling events in myeloid and lymphoid cells. Thus, Draper appears to be an ancient immunoreceptor with an extracellular domain tuned to modified-self antigens and an intracellular domain that promotes phagocytosis through an ITAM domain-SFK-Syk-mediated signaling cascade. I have further identified the Drosophila guanine-nucleotide exchange factor (GEF) complex Crk/Mbc/dCed-12, and the small GTPase Rac1 as novel modulators of glial clearance of axonal debris. I am able to demonstrate that Crk/Mbc/dCed-12 and Rac1 function in a non-redundant fashion with the Draper pathway to promote a distinct step in the clearance of axonal debris. Whereas Draper signaling is required early during glial responses, promoting glial activation and extension of glial membranes to degenerating axons, the Crk/Mbc/dCed-12 complex functions at later stages of glial response, promoting the actual phagocytosis of axonal debris. Finally, many interesting mutants have been identified in primary screens for genes active in neurons that are required for axon fragmentation or clearance by glia, and genes potentially active in glia that orchestrate clearance of fragmented axons. The further characterization of these genes will likely unlock the mystery surrounding “eat me” and “find me” cues hypothesized to be released or exposed by neurons undergoing degeneration. Illuminating these important glial pathways could lead to a novel therapeutic approach to brain trauma or other neurodegenerative conditions by providing a druggable means of inducing early attenuation of the glial response to injury down to levels less damaging to the brain. Taken together, my combined work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris, and the termination of glial responses to injury are genetically separable events mediated by distinct signaling pathways.

The early postnatal period is marked by the rapid acquisition of sensorimotor processing capabilities. Initially responding to a limited set of environmental stimuli with a restricted repertoire of behaviors, mammals exhibit a remarkable proliferation of sensorimotor abilities in the early postnatal period. Central to action selection, reinforcement, and contingency learning are a subcortical set of evolutionarily conserved nuclei called the basal ganglia. The striatum, which is the primary input nucleus of the basal ganglia, receives afferent innervation from throughout the CNS. Its projection neurons (SPNs) integrate these diverse inputs, regulating movement and encoding salient cue-outcome contingencies. Here, using electrophysiological, electrochemical, imaging, and behavioral approaches in mice, I will explore the postnatal maturation of the striatal cholinergic interneuron (ChI), a critical modulator of dopamine signaling, afferent excitation, and SPN excitability. In Chapter 1, I will set the stage for this exploration by reviewing the current literature on striatal postnatal development, including cellular physiology, axonal elaboration and synapse formation, and plasticity expression. I will survey striatal deficits observed in clinical neurodevelopmental conditions such as autism, ADHD, tic disorders, and substance use disorders. I will additionally summarize evidence that the striatum is uniquely vulnerable to physiological and immunological insult, as well as early life adversity. In Chapter 2, I turn my focus specifically to the striatal ChI, uncovering fundamental cell-intrinsic changes that occur postnatally in this population. I will also elaborate on the postnatal maturation of dopamine release properties and regulation thereof by cholinergic signaling from the ChI. In Chapter 3, I investigate the circuit connectivity and circuit-driven firing dynamics of ChIs as they mature postnatally. I utilize a brain slice preparation retaining thalmostriatal afferents in order to assay the ChI pause, a synchronized transient quiescence in ChIs thought to facilitate cue learning and behavioral flexibility. I find that the ChI pause is refined postnatally, dependent on developmental changes in thalamic input strength and the cell- intrinsic expression of specific ionic conductances. Finally, in Chapter 4, I present preliminary evidence that ChI circuit maturation as defined in preceding chapters is delayed by chronic stress exposure postnatally. Following the maternal separation model of early life stress, ChI intrinsic characteristics mature normally, but they retain heightened thalamic innervation and thalamus-driven pause expression.

Activity plays diverse roles in shaping neuronal development and function. These roles range from aiding in synaptic refinement to triggering cell death during traumatic brain injury. Though the importance of activity-dependent mechanisms is widely recognized, the genetic underpinnings of these processes have not been fully described. In this thesis, I use the motor circuit of Caenorhabditis elegans as a model system to explore the functional and morphological consequences of modulating neuronal activity. First, I used a gain-of-function ionotropic receptor to hyperactivate motor neurons and asked how increased excitation affects neuronal function. Through this work, I identified a cell death pathway triggered by excess activation of motor neurons. I also showed that suppression of cell body death failed to block motor axon destabilization, providing evidence that death of the cell body and of motor axons can be genetically separated. Secondly, I removed excitatory drive from a simple neural circuit and asked how loss of excitatory activity alters circuit development and function. I identified excitatory motor neurons as master regulators of inhibitory synaptic connectivity. Additionally, I was able to identify previously undescribed activity-dependent mechanisms for regulating inhibitory synapses in both developing and mature neural circuits. Finally, I show data to implicate the highly conserved genes neurexin and neuroligin in determining inhibitory synapse connectivity. Collectively this work has lent insight into activity-dependent mechanisms in place to regulate neuronal development and function, a core function of neurobiology that is relevant to the study of a wide range of neurological disorders.