Project Summary The goals of this research are two fold: to better understand the role of dynorphin during fentanyl withdrawal and to develop innovative, novel approaches to allow in vivo detection of endogenously release opioids. The ability to better understand how dynorphin drives negative affect associated with drug withdrawal will be critical in reducing the incidence of relapse and overdose and allow us to consider alternative more affective long-term treatments for addiction. The US Department of Health states that 42,249 people died from overdosing on opioids in 2016, 2.1 million people had an opioid use disorder and the economic cost of this epidemic was $504 billion, but is still rising along with the number of people afflicted. The reasons for this are multifaceted, but include a deep lack of understanding of how opioids alter brain circuitry to cause analgesia- which is good, but also what happens to brain circuitry to drive addiction- which is bad. We also know that pain and addiction are co-morbid with other mental health diseases such as anxiety, depression and stress, which makes cohesive research challenging. The first aim of this proposal will directly measure in vivo changes in dynorphin levels in the nucleus accumbens during withdrawal from Fentanyl. Fentanyl is 80 times more potent than morphine in vivo; its rapid onset of action not only increases the risk of addiction but likely increases the severity of withdrawal symptoms. We will use liquid chromatography/mass spectrometry which will allow detection in sub-fmol range. The second aim focuses on the development of micro-immunoelectrodes to allow more spatiotemporally controlled detection of opioid peptide release in vivo. This technology will allow us to detect peptides changes on a second to minute timescale. The combination of these techniques will, for the first time, allow us to detect dynamic changes in opioid peptides in vivo. Importantly, this will facilitate our knowledge and ability to better treat addiction, but is also very much applicable to multiple disease states and neuropeptides biology in general.