Mass Spectrometric Studies of Neuropeptides in Feeding

项目摘要/摘要 摄食行为是动物生存的关键，也是能量平衡的一个基本方面。这 这个过程是由高度复杂的神经化学途径调节的，涉及多种神经肽和 生物胺。尽管在单个神经化学系统方面做了几十年的工作，但总的组织 神经调节的基本原理仍然知之甚少。这主要是因为 到目前为止，对神经回路的调制研究主要是一次一个调制器，而不知道 关于网络的共调制。后一种信息将需要开发敏感的和 选择性分析工具来精确识别这些低丰度的内源信号分子和 在复杂的微环境中准确测量它们与行为相关的浓度。我们的建议 研究旨在通过开发新的生物分析来解决这一关键知识和技术差距 方法阐明神经肽在食物摄取中的复杂身份和功能作用 质谱学和生理学方法。我们用甲壳类动物的胃部神经 作为技术试验台的系统、心血管系统及其相关的神经内分泌器官 由于该模型系统的独特优势和生物学意义，进行了开发和验证。在……里面 同时，我们的目标是将我们的神经肽发现和分析技术开发转化为 哺乳动物的中枢神经系统。为此，我们建议将重点放在大鼠模型的关键大脑区域上 与喂食有关的信息处理逐渐变得更加复杂。具体目标包括：(1) 开发和应用基于质量缺陷的、与数据无关的胺反应化学标签 基于获取(DIA)质谱学(MS)的神经肽变化多路定量策略 在不同的饲养条件下；(2)建立了纳秒光化学反应(NsPCR)辅助 基于MALDI的MSI技术用于绘制个体的共定位模式 已鉴定神经元中扩展多肽家族和胺类神经递质的异构体与摄食 电路，具有增强的灵敏度和化学信息；以及(3)评估功能作用 体内整合多管齐下的神经肽在摄食和心脏调节中的作用 微透析和体外生理和行为测量。将对新型神经肽进行评估 在神经网络和系统水平上的摄食调节的功能作用。选举的结果是 拟议的研究将是一套新的分析工具，能够定量评估 具有高空间、化学和时间信息的神经肽和生物胺。平行线 这些新方法在甲壳类和哺乳动物的神经系统中的应用 加快开发治疗进食障碍的新疗法。

PROJECT SUMMARY/ABSTRACT Feeding behavior is critical for animal survival, and is also a fundamental aspect of energy homeostasis. This process is regulated by highly complex neurochemical pathways involving a multitude of neuropeptides and biogenic amines. Despite decades of work on individual neurochemical systems, the general organizational principles underlying neuromodulation are still poorly understood. This is mainly due to the fact that modulation of neural circuit has so far been studied primarily one modulator at a time without the knowledge about co-modulation of networks. The latter information would require the development of sensitive and selective analytical tools to precisely identify these low abundance endogenous signaling molecules and accurately measure their behaviorally-relevant concentrations in a complex microenvironment. Our proposed research aims to address this critical knowledge and technological gap by developing new bioanalytical methods to elucidate the complex identities and functional roles of neuropeptides in food intake via combined mass spectrometric and physiological approaches. We employ the crustacean stomatogastric nervous system, cardiovascular system, and its associated neuroendocrine organs as a test-bed for technology development and validation due to the unique advantages and biological significance of this model system. In parallel, we aim to translate our technology development for neuropeptide discovery and analysis to the mammalian central nervous system. To this end, we propose to focus on key brain regions in a rat model at progressively more complex levels of feeding-related information processing. The specific aims include: (1) Developing and applying mass defect-based, amine reactive chemical tags coupled with data-independent acquisition (DIA) mass spectrometry (MS)-based strategy for multiplexed quantitation of neuropeptide changes under different feeding conditions; (2) Developing a nanosecond photochemical reaction (nsPCR)-assisted MALDI-based mass spectral imaging (MSI) technique for mapping co-localization patterns of individual isoforms of extended peptide families and amine neurotransmitters in identified neurons and the feeding circuits, with enhanced sensitivity and chemical information; and (3) Assessing functional roles of neuropeptides in feeding and cardiac regulation using a multi-pronged approach integrating in vivo microdialysis and ex vivo physiological and behavioral measurements. Novel neuropeptides will be evaluated for functional roles in feeding regulation at the neuronal network and system levels. The outcome of the proposed research will be a suite of new analytical tools enabling quantitative assessment of the interplay of neuropeptides and biogenic amines with high spatial, chemical and temporal information. The parallel application of these new methods to both crustacean and mammalian nervous systems in feeding will accelerate our pace towards the development of new therapeutics for feeding disorders.