PROJECT SUMMARY/ABSTRACT Opioids derived from opium are amid the oldest medications dating back to the 1800s, originally used for gastrointestinal distress and now are among the most effective treatments for pain management. It wasn’t until the mid-1800s that the abuse potential for opioids was evident, and this problem has since multiplied on an international scale. Within the last decade, the number of opioid overdose deaths per year in the United States alone has more than tripled. It is therefore urgent to not only find solutions to combat opioid overdoses, but to also develop new treatments for pain management. Opioid-induced respiratory depression (OIRD), the main cause of opioid overdose deaths, is primarily mediated through mu opioid receptors, particularly in two small brainstem regions, the preBötzinger complex (preBötC) and the parabrachial complex (PBN) that are sufficient and necessary to drive OIRD. Recent reports have also highlighted important cell types and downstream molecular mechanisms within these brain regions that mediate OIRD. Moreover, several laboratories within the last few decades have been exploring the function of the mu opioid receptor gene, Oprm1, which undergoes extensive alternative splicing to generate two classes of splice variants: Exon 1- (E1) and Exon 11- (E11) associated variants. The functional relevance of these splice variants has been demonstrated in mediating the actions of various mu opioids, including analgesia, tolerance, physical dependence and reward. However, it remains unknown how the two sets of Oprm1 splice variants influence OIRD. Preliminary data and unpublished data from our laboratory demonstrate differential actions of E1- and E11-associated variants in OIRD from varying doses of fentanyl and morphine in our rat models. Therefore, the overarching hypothesis is that the two classes of Oprm1 alternatively spliced variants (i.e., E1- and E11-associated variants) in the preBötC and PBN differentially influence OIRD. Aim 1 examines the role of E1- and E11-associated variants within two key brain regions modulating respiration, preBötC and PBN, on OIRD in rats, and will be completed in Year 1 of this fellowship training. Aim 2 identifies the expression of E1- and E11-associated Oprm1 variants within various cell types of the preBötC & PBN in rats (Year 2). To test these aims, we generated two gene targeted rat models, in which Oprm1 E1- or E11-associated splice variants can be conditionally deleted. Comprehension of the distinct roles of E1- and E11-dependent variants on OIRD in these two brain regions and in key cell types within these regions is expected at the end of this proposed project. Investigation of how these effects are altered by different opioids, doses, and sexes will also be assessed. The proposed study will advance our understanding of Oprm1 alternative splicing mechanisms and their role in OIRD, allowing us to develop therapeutic strategies to combat OIRD.