Project Summary – Abstract Current neuroimaging methods to assess brain function are limited by poor spatial and temporal resolution as well as confounding vascular effects. This is particularly concerning for vasoactive drugs of abuse including fentanyl (FTY) that causes vasodilation. Also, current tools only indirectly measure neuronal activity likely missing patterns of neurons (i.e. neuronal ensembles) that are responsible for specific behaviors. FTY use significantly contributes to opioid overdoses in the United States and it is being abused by a large number of Americans. Yet, researchers know relatively little about this synthetic opioid agonist and assume it acts the same as other opioids and drugs of abuse on brain function. Research in animals and humans has shown that dysfunction of the prefrontal cortex (PFC) is a hallmark of drugs of abuse including opioids, and this dysfunction in the PFC is thought to contribute to compulsive drug taking behaviors. The technical limitations of current neuroimaging techniques and misassumption that FTY acts similar to other opioids and drugs of abuse on brain function present major research gaps that this grant proposal aims to address. Using photoacoustic imaging (PAI), a novel optical neuroimaging technique, combined with a Fos-LacZ transgenic rodent model, we propose a highly innovative project to study Fos-based neuroactivity, hemodynamics, and vascularization in female and male rats engaging in FTY self-administration (FTY-sa; i.e. FTY taking behavior). Our overall hypothesis is that PAI with multi-wavelength analysis in female and male Fos-LacZ transgenic rats provides a model system to study neuronal activity, vascularization and hemodynamics concurrently and in vivo to disentangle the neuronal and vascular components of FTY taking behavior. The studies proposed herein will use PAI to quantify neuronal ensembles and Fos-based neuroactivity, while simultaneously measuring hemodynamics and vascularization. Using transgenic Fos-LacZ rats we will study changes in PFC based on its known role in drug taking behavior and we will study females and males because of their unique patterns of drug taking. The proposed studies of this project address significant gaps, are highly innovative, and are forward looking. In short, the results of these studies will advance understanding of FTY and better integrate PAI in studies on the neurobiology of addiction.