The cornea is the most densely innervated tissue in the body, and pain is the primary experience resulting from corneal stimulation. While physiological corneal pain (nociceptive pain) protects the eye from injury, inflammation and/or nerve damage can result in prolonged or chronic corneal pain. Corneal afferents represent a diverse population of neurons, with specialized properties related to maintaining ocular health. The full diversity of these neurons, and their responses to injury are unknown. The first set of experiments will determine the mRNA transcript signatures of mouse corneal neurons in the trigeminal ganglion (TG) and their transcriptional responses to corneal injury and compare with cell-type-specific transcriptional and epigenomic signatures of human TG neurons. Corneal afferents are known to project to two main regions in the spinal trigeminal nucleus (Vsp), each with distinct roles in nociception and maintaining corneal homeostasis. We have preliminary data demonstrating an additional projection to the lateral parabrachial nucleus (lPBN), a region critical in regulating complex motivational-affective responses to aversive stimuli. Its contribution to corneal pain is unknown. The second set of experiments will examine central processing of corneal input in the lPBN. We will determine the contribution of corneal->lPBN primary afferent projections to corneal nociceptive responses and the function of lPBN neurons in corneal nociceptive and chronic pain behaviors. Additional studies will perform single-nucleus transcriptome analysis to identify molecular profiles of corneal-activated brainstem neurons, followed by multiplex in situ hybridization to provide spatial resolution in regions that receive direct corneal afferent input. The cornea is also endowed with resident corneal leukocytes (RCLs) residing in close proximity to corneal nerves, suggesting the possibility of neuro-immune crosstalk in the cornea. However, current knowledge is limited on possible direct regulation of RCLs through corneal nerves, or the influence of RCLs on corneal nerve function. The third set of experiments will characterize the cell populations and molecular mechanisms involved in neuroimmune crosstalk resulting in peripheral nerve sensitization in the cornea. Corneal single cell mRNA transcript signatures associated in murine corneal pain models will be used to identify transcriptional changes that underly nociceptor sensitization. Crossing these immune cell transcripts with transcript profiles of corneal afferents will provide evidence for ligand-receptor pairs. In vitro studies will confirm the ability of the identified modulators to sensitize TG neurons, and the functional significance will be assessed using behavior and ex vivo electrophysiology. Employing a multidisciplinary approach, these experiments will provide a comprehensive analysis of cellular and molecular mechanisms of nociceptive and chronic corneal pain, leading to the identification nov...