ABSTRACT: The role of mesolimbic opioid peptides in motivated behavior and reward-related decision making is unclear, despite extensive evidence indicating that these molecules are important mediators of hedonic and motivational aspects of reward processing, and the fundamental response to drugs of abuse. This is largely due to a critical gap in understanding when and where these molecules are released, because there is a paucity of detection methods for monitoring opioid peptides in the extracellular space. We have established the feasibility of using fast-scan cyclic voltammetry (FSCV) and carbon- fiber microelectrodes in tissue for the detection of endogenous enkephalin (ENK) fluctuations in real time. The objective of this proposal is to optimize and fully characterize this methodology, so as to provide the community with an established tool that can be used to study the role of the ENKs in complex physiological processes ranging from basic endocrine function to motivation. The first goal is to fully characterize selectivity. We will assess incorporation of well-characterized Nafion composite membranes into the sensor design as a physical means to enhance selective detection. We will also investigate the electrochemistry of each of 20 natural amino acids individually, and use these data in a multivariate approach to identify individual amino acid contributors to the voltammetric signal when these residues are incorporated into longer amino acid chains. The second goal is to optimize sensitivity by systematically investigating electrochemical parameters (scan rates, holding potentials, and sampling frequencies), as well as promising nanoscale electrode materials. Finally, the third goal is to evaluate real-time ENK dynamics in rat adrenal and brain tissue, so as to provide insight into the normal concentration range, extracellular lifetime, and diffusion profile (sphere of influence) of the ENKs, as well as physiological and pharmacological conditions that can induce changes in peptidergic signaling. We will also directly compare the stimulation-response relationship for small molecule transmitters and ENK, using FSCV. This project is a critical step toward our long-term goal of elucidating how the release and clearance dynamics of several neuropeptides and small molecules underlie discrete aspects of motivated behavior. It will clarify outstanding questions regarding the fundamental nature of endogenous opioid peptide signaling, and enable FSCV to be used confidently to reveal critical mechanistic details that will inform evidence-based pharmacotherapies for treating a wide range of disorders, including substance abuse disorders.