Project Summary/Abstract The cochlea exploits an intricate tonotopic organization of afferent innervation to effectively process highly complex auditory stimuli. To create this precisely organized pattern, the neurites from spiral ganglion neurons (SGNs) navigate a complex milieu of cells, extracellular matrix, and biochemical gradients to reach their peripheral and central targets in the organ of Corti and cochlear nuclei, respectively. This process of a neurite growing through the environment is called pathfinding. In pathfinding, the tip of the neurite, the growth cone, senses, turns, and grows toward a target in response to biochemical and biophysical cues. This proposed research focuses on understanding how different substrate cues (topography, chemoattractive, and chemorepulsive) can promote an SGN neurite to turn as well as by which by signaling pathways a growth cone relies on to execute this basic biological process. Previous work has identified that engineered micropatterned substrates can be used to both (1) direct SGN growth and (2) study the signaling pathways activated by this phenomenon in vitro. We have demonstrated that neurons align their outgrowth to various engineered patterned substrates (topographical, chemoattractive, and chemorepulstive). Additionally, we have implicated RhoA-GTPase and calcium signaling in SGNs aligning their outgrowth to topographical cues. I propose to use this engineered micropatterned substrates to study (1) the hierarchy and interaction of topographical and biochemical cues in dictating neurite turning and (2) the biochemical pathways necessary for a growth cone to sense and turn in response to these cues. In particular, I will study how chemoattractive (laminin) and chemorepulsive (EphA4) interact with topographical growth cues when placed in complimentary (attractive in troughs) or antagonistic (repulsive in troughs) patterns. Additionally, I will research the role of Rho/ROCK and IP3 signaling in this basic biological process using pharmacology and imaging the activation of these pathways in real time when growing on various patterned substrates. Overall, the goal of this research is to better understand the key, basic biological process of how an SGN neurite senses and turns in response to substrate cues. I expect to contribute knowledge to this field by utilizing novel 3D combination micropatterned substrates, real time imaging approaches of the pathways of interest, and a machine learning image sorting model to use an unbiased approach in assessing neurite behavior in response to the micropatterned substrates. These novel insights will inform many aspects of SGN pathfinding through (1) determining if similar fundamental signaling pathways are used by SGNs when turning in response to both biophysical and biochemical cues, (2) clarifying the mechanisms of how the tonotopic organization of the cochlea develops, and (3) though this is not directly a translationally aimed proposal, the findings will...