Functional and molecular characterization of the central amygdala in the context of oxycodone withdrawal

ABSTRACT Prescription opioids, such as oxycodone, remain a common treatment for pain disorders. Although effective in the short-term, long-term use can result in tolerance, dependence and fatal overdose. While oxycodone is not the primary driver of fatal overdoses from opioids, the exposure to and subsequent withdrawal from oxycodone can lead susceptible individuals to relapse, potentially substituting intake fentanyl or other more potent synthetic opioids. Thus, understanding the mechanisms of oxycodone-induced withdrawal will provide a better foundation for the treatment of opioid use disorder. The central nucleus of the amygdala (CeA) is an integrative brain region that contributes to the generation of negative affective states. Prior work has demonstrated increased neuronal excitability within the CeA in rodent models of morphine withdrawal, and inhibition or lesion of the CeA resulted in a reduction of the aversive, and, to a lesser extent, somatic withdrawal behaviors from morphine. Most studies investigating the CeA’s role in opioid withdrawal have done so in a global sense, ignoring potential contributions of specific cell types to the manifestation of opioid withdrawal behaviors. The central hypothesis in the present application is that activation of molecularly distinct neuronal subpopulations within the CeA are necessary and sufficient for the expression of somatic opioid withdrawal and contribute to future drug seeking behaviors. Uncovering the specific subtypes responsible for the expression of opioid withdrawal has the potential to lead to a more effective and specialized approach for the development of treatments of opioid use disorder. To this end, I will employ the use of targeted recombination in active populations mice, which express the tamoxifen-dependent CreERT2 recombinase from the Fos promoter to gain genetic access to oxycodone withdrawal activated (OWA) neurons. Using this strategy, I propose to evaluate the activity of OWA during oxycodone withdrawal and evaluate their contributions to the expression of behaviors related to somatic withdrawal and negative affect. In Aim 2, I will seek to use both known probes and single-cell RNA sequencing to identify CeA neuronal populations preferentially activated during oxycodone withdrawal. In the independent (R00) phase I will evaluate different subpopulations of CeA neurons based on hits from Aim 2. Highly enriched projections and genes will be evaluated for their ability to influence behaviors pertaining to oxycodone withdrawal and seeking. These experiments will provide me with an outstanding technical skillset and an enriched data set to build the foundation of my independent research program studying the framework of the CeA during opioid dependence and withdrawal. The results of these experiments will provide a clearer understanding of the CeA’s role during opioid withdrawal, potentially to leading to more targetable treatments for opioid use disorder.