1 ABSTRACT 2 Chronic systemic opioid use leads to misuse, addiction and fatal overdoses. Topical and peripheral application of 3 low doses of opioids are safer alternatives, as they provide efficient local analgesia with limited CNS penetration. 4 Unfortunately, tolerance decreases peripheral opioid analgesia and the mechanisms of this phenomenon are as yet, 5 unknown. We propose the innovative hypothesis that peripheral tolerance develops, at least in part, because of opioid 6 action on skin keratinocytes, which leads to changes in peripheral afferent signaling. Central tolerance involves 7 activation of the platelet-derived growth factor receptor beta (PDGFRβ) by the platelet-derived growth factor B (PDGF- 8 B) ligand. Centrally, PDGF-B is released in response to activation of the µ-opioid receptor (MOR). Accordingly, intrathecal 9 injections of imatinib, a PDGFRβ inhibitor, prevented opioid tolerance. Previous studies have hypothesized that central 10 and peripheral tolerance occur by different mechanisms. However, we found that imatinib can also prevent tolerance to 11 peripherally administered morphine, suggesting a shared mechanism that involves PDGFRβ signaling. In separate 12 studies, using mice that express channelrhodopsin (ChR2) in keratinocytes (Krt14-ChR2), we found that optogenetic 13 stimulation of keratinocytes activates a subpopulation of peripheral sensory neurons (PSNs), likely through release of 14 neuroactivators from activated keratinocytes. These findings led to the idea that keratinocytes are a component of 15 peripheral tolerance as PDGF-B is expressed in keratinocytes. In exciting preliminary studies we discovered that 16 repeated optogenetic stimulation of keratinocytes generated peripheral tolerance that could be blocked by imatinib, 17 providing evidence that peripheral tolerance involves keratinocyte and PDGFRβ signaling. Taken together, we now 18 postulate that peripheral tolerance is mediated by the release of PDGF-B from opioid-activated keratinocytes, which 19 activates PDGFRβ signaling in primary afferents. We will test this hypothesis in three specific aims. First, we will 20 determine if PDGF-B is released from keratinocytes and contributes to peripheral tolerance, using behavioral 21 pharmacology, optogenetics, Luminex protein quantification and mRNA/protein imaging methods. Second, we will 22 localize the cellular basis of MOR responsible for peripheral tolerance, by generating mice in which MOR will be 23 specifically deleted either from keratinocytes or PSNs. Third, we will test the effectiveness of FDA approved PDGFRβ 24 inhibitors in preventing peripheral tolerance. 25 This project explores new neuroanatomical and molecular substrates for peripheral tolerance that involve 26 epithelial-neural communication. Outcomes from this study could rapidly lead to new and highly effective therapeutic 27 strategies to treat chronic pain by repurposing PDGFRβ inhibitors to prevent peripheral opioid tolerance....