Chronic pain is a frequent, intractable complaint of patients with classic Ehlers-Danlos Syndrome (cEDS), a heritable connective tissue disorder, characterized by hyperextensible skin, hypermobile joints and generalized connective tissue fragility. cEDS is caused by genetic defects in type V collagen which leads to a disorganized extracellular matrix. Research into pain characteristics and mechanisms in EDS is highly limited. Fibroblasts are the principal source of abnormal type V collagen in cEDS, and molecules secreted by fibroblasts may contribute to peripheral sensitization of nociceptors, but studies addressing this are limited. We hypothesize that mutant cEDS fibroblasts in tissues, such as skin and/or dorsal root ganglia (DRG), may cause hyperexcitability and/or abnormal growth of nociceptors, resulting in a pain-promoting phenotype. We will perform in-depth studies on the cellular composition and cell-type specific gene expression of skin and DRG from Col5a1+/- mice, a model of cEDS with a proven pain phenotype. Next, the neuron-fibroblast crosstalk will be examined in microfluidic co-cultures of murine skin fibroblasts and DRG neurons, as well as by neurite outgrowth assays and Ca2+ imaging. Findings will be validated in skin biopsies from human cEDS patients. We anticipate that our study on neuron-fibroblast interactions will generate important novel information into mechanisms of pain in cEDS and more prevalent painful conditions.