PROJECT SUMMARY Over the past several years we have studied changes in critical blood-brain barrier (BBB) tight junction(TJ) proteins (i.e. claudin-5 occludin) in response to diseases or drugs. Our work has shown that claudin-5 andoccludin expression and trafficking is modulated by pain (i.e. TJ dysregulation) an effect that increases brainuptake of opioids such as codeine. Our preliminary data show that acetaminophen (APAP) can modulateexpression of claudin-5 at the BBB and increase paracellular permeability (i.e. leak). Leak has previously beenshown to occur with low dose acute cocaine administration as well as methamphetamine. Both APAP and paintarget transforming growth factor- (TGF-) signaling a pathway that controls TJ expression and BBB integrity. In this grant we hypothesize that APAP by itself and in the setting of pain can increase BBBleak and enhance CNS opioid delivery. These studies are highly significant because they will uncovermechanisms of altered opioid antinociception and adverse drug events that can occur in individualswho abuse or misuse opioids or APAP. Additionally APAP is taken with other centrally acting drugswhere APAP leak can lead to therapeutic toxicity. Aim 1: To investigate in vivo changes in TJ protein expression and trafficking at the BBBfollowing APAP administration. We will study how APAP alters CNS effects and adverse events of opioids.We will investigate the time course of changes in transmembrane TJ protein (i.e. claudin-5 occludin) expressionand trafficking and the dose-response relationship of APAP on changes in TJ protein complexes in male andfemale Sprague-Dawley rats (Aim 1A). We will then study the temporal relationship between CNS opioiddelivery opioid-associated antinociception respiratory depression and opioid-associated reward behavior (Aim1B). Since we have shown that transforming growth factor- (TGF-) signaling regulates BBB integrity we willstudy the role of this pathway on claudin-5 and occludin expression and trafficking (Aim 1C). Aim 2: To examine involvement of APAP on BBB integrity in pain. Using established in vivo painmodels (i.e. l-carrageenan-induced acute inflammatory pain chronic pain spinal nerve ligation) we will studythe time course of APAP effects on TJ protein expression/trafficking (i.e. claudin-5 occludin) as well as on CNSopioid uptake (Aim 2A). We will demonstrate effects of single versus multiple doses of APAP on TGF- signalingpathways in both pain models (Aim 2B). We will study how changes in CNS opioid delivery affect opioidantinociception respiratory depression and opioid-associated reward behavior (Aim 2C). Since we have shownthat APAP increases functional expression of the critical opioid transporter P-glycoprotein we will examine thecontribution of claudin-5/occludin modulation and P-gp changes to CNS opioid delivery (Aim 2D). Our group is uniquely positioned to provide a mechanistic explanation (i.e. signaling activitytrafficking) for adverse drug events in individuals who abuse/misuse prescription pain drugs.