PROJECT ABSTRACT A rich network of sensory afferents in the ocular surface (OS), supplied by the ophthalmic branch of the trigeminal nerve, performs a multitude of physiological functions, including sensation, regulation of various reflexes, and secretion of trophic and growth factors. Of the OS structures, the cornea is the most highly innervated tissue in the body. As such, dysfunction of the corneal/OS nerves has been shown to underlie a wide range of diseases, including trauma, infections, metabolic imbalances, and therapeutic interventions such as refractive surgeries. Corneal diseases continue to be a major health problem in the US, and corneal nerve dysfunction contributes to many of these disorders, such as neurotrophic keratitis, corneal neuropathic pain, and dry eye disease (DED). DED alone has been shown to impact up to 75% of a given population, causing significant reduction in quality of life and increased risk for visually debilitating opacities. Therefore, a comprehensive characterization of corneal sensory nerves is critical for understanding the pathophysiology leading to OS diseases. To meet this challenge and in response to the FOA RFA-EY-21-004 on OS innervation, we have assembled an interdisciplinary team with a strong collaborative track record and complementary expertise, including eye disease models and wound healing (Lee); pain and the mammalian somatosensory system (Luo); intravital corneal nerve fiber imaging and regeneration (Rompolas); single-cell transcriptomics/epigenomics and related computational analyses (Wu); and neurophysiology and data analysis/machine learning (Ding). We propose to combine advanced imaging approaches, novel single-cell multi-omics, and cutting-edge mouse genetic models to perform three levels of analysis: 1) Morphology - using single neuron genetic labeling, we will resolve the fine morphology, spatial organization, and connectivity of corneal sensory neurons by visualizing axonal arborization in the cornea and brainstem/spinal cord. We will also examine interactions of individual sensory afferents and corneal epithelial cells. 2) Molecular - we will perform integrated single-cell transcriptomics and epigenomics to interrogate the subtype-specific marker gene expression and epigenetic landscape of the corneal sensory neurons. 3) Functional - we will perform real-time physiologic analyses of corneal sensory afferents using in vivo two-photon calcium imaging. To further elucidate the role of sensory nerves in OS pathophysiology, we will determine how their morphology, molecular profile, and responsiveness are altered in a surgically induced DED model. Moreover, we will implement a novel high-speed imaging and machine learning platform to quantify evoked responses to corneal sensory stimuli and spontaneous behavior in the DED model. Taken together, our study will generate a comprehensive data set poised for integration that will fill in critical knowledge gaps in the field, create a robust found...