Monitoring Real-Time Neuropeptide Dynamics

摘要： 中脑边缘阿片肽在动机行为和奖赏相关决策中的作用 不清楚，尽管大量证据表明这些分子是享乐性的重要媒介 以及奖励处理的激励方面，以及对滥用药物的基本反应。这 这在很大程度上是由于对这些分子何时何地释放的理解存在严重差距， 因为缺乏监测细胞外阿片肽的检测方法 太空。我们已经确定了使用快速扫描循环伏安法(FSCV)和碳原子吸收光谱的可行性。 组织中的纤维微电极用于实时检测内源性脑啡肽(ENK)的波动。 这项建议的目标是优化和充分说明这一方法，以便提供 社区有一个已建立的工具，可以用来研究Enk在复杂生理过程中的作用 从基本的内分泌功能到动力的过程。第一个目标是充分描述 选择性。我们将评估将表征良好的Nafion复合膜纳入到 传感器设计作为增强选择性检测的物理手段。我们还将调查 分别测定20种天然氨基酸的电化学性质，并在多变量中使用这些数据 识别单个氨基酸对伏安信号有贡献的方法 结合成更长的氨基酸链。第二个目标是通过系统地优化敏感度 研究电化学参数(扫描速度、保持电位和采样频率)，例如 以及前景看好的纳米级电极材料。最后，第三个目标是评估实时ENK 在大鼠肾上腺和脑组织中的动态变化，以便深入了解正常浓度范围， Enk的细胞外寿命和扩散分布(影响范围)，以及生理和 能引起肽能信号变化的药理条件。我们也会直接比较 用FSCV研究小分子递质和ENK的刺激-反应关系。这个项目是 朝着我们的长期目标迈出的关键一步，即阐明释放和清除动力学是如何 几种神经肽和小分子构成了动机行为的不同方面。会的 澄清关于内源性阿片肽信号的基本性质的悬而未决的问题，以及 使FSCV能够被自信地使用，以揭示关键的机械细节，从而为基于证据的 治疗包括物质滥用障碍在内的各种疾病的药物疗法。

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.