Project Summary Currently our understanding of how the nervous system maintains ocular surface homeostasis is extremely limited. New technologies, methods and models are needed to advance our scientific understanding and address knowledge gaps. The ocular surface and tear film-secreting glands (including the lacrimal and meibomian glands, as well as the goblet cells) are carefully controlled to provide an optically smooth, low-scattering surface with appropriate immune and injury responses. Sensory feedback to maintain the structural and functional integrity of the ocular surface is provided by the corneal nerves, which send feedback from stimuli (chemical, thermal, mechanical) to ganglia (e.g., trigeminal) and brain regions (e.g., ventral posteromedial thalamus) to drive production of tear film components as well as the blink reflex. This delicate balance of neural control is disrupted by damage, peripheral neuropathies, inflammation and further complicated by a wide array of immune responses to various diseases. Dysfunction of this feedback loop can lead to a downward spiral of further dysregulation. Aberrant neural control of the ocular surface can lead to abnormal sensation and pain, which in the worst cases can be disabling. To find remedies, it is first essential to understand the underlying neural control system and how it adapts to its environment. In this proposal, we aim to bring new tools and models to study molecular, cellular, and functional interactions across systems responsible for neural control of the ocular surface and examine how they change under different inflammatory and pain conditions. We have assembled an excellent team with expertise across multiple fields including advanced 3D microscopy, neuroscience, electrophysiology, pain, ocular immunology, ocular lipid metabolism, ocular surface disorders, spatial statistics, and machine/deep learning. Here, we will utilize cutting edge techniques and technologies including optical clearing, tract tracing, ethologically-valid behavior analysis, machine/deep learning, spatial statistics, genetically encoded calcium imaging, light-sheet microscopy, multiplexed 3D fluorescence in situ hybridization (FISH) imaging, and multi-array electrodes implanted in the brain. These tools will help us assess molecular, cellular, and functional interactions across organs and begin to understand ocular surface control at the organism level. We will also employ several relevant animal models to assess ocular surface control under different inflammatory and pain conditions. Models include AWAT2 deficient mice that mimic evaporative dry eye disease (DED), diabetic mice, an epithelial debridement model with Pseudomonas aeruginosa that mimics bacterial keratitis, and human donor eyes. The mouse models all have gCaMP6f expressed in corneal nerves allowing functional imaging of calcium transients. With these models we will study both innate and adaptive immunity as well as nociceptive and neuropathic p...