Mass Spectrometric Studies of Neuropeptides in Feeding

项目总结/摘要 摄食行为对动物的生存至关重要，也是能量稳态的一个基本方面。这 这一过程受到高度复杂的神经化学途径的调节，涉及多种神经肽， 生物胺尽管几十年来对个体神经化学系统的研究， 神经调节的基本原理仍然知之甚少。这主要是因为， 迄今为止，神经回路的调制主要是一次一个调制器地研究， 关于网络的共同调制。后一种信息将需要制定敏感和 选择性分析工具，以精确鉴定这些低丰度内源性信号分子， 准确测量其在复杂微环境中的行为相关浓度。我们提出的 研究旨在通过开发新的生物分析技术来解决这一关键的知识和技术差距。 方法来阐明复杂的身份和功能作用的神经肽在食物摄入，通过联合 质谱和生理学方法。我们利用甲壳类动物的胃神经 系统，心血管系统及其相关的神经内分泌器官作为技术的试验台 由于该模型系统的独特优势和生物学意义，因此具有开发和验证的价值。在 同时，我们的目标是将我们的神经肽发现和分析技术发展转化为 哺乳动物中枢神经系统为此，我们建议将重点放在大鼠模型的关键脑区， 与进食相关的信息处理水平逐渐变得更加复杂。具体目标包括：（1） 开发和应用基于质量缺陷的胺反应性化学标签， 用于神经肽变化的多重定量的基于采集（DIA）质谱（MS）的策略 （2）发展了纳秒光化学反应（nsPCR）辅助的 基于MALDI的质谱成像（MSI）技术用于绘制个体的共定位模式 已鉴定神经元中扩展肽家族和胺神经递质的亚型和摄食 电路，具有增强的灵敏度和化学信息;和（3）评估的功能作用， 神经肽在摄食和心脏调节中的作用 微透析和离体生理和行为测量。新的神经肽将被评估 在神经元网络和系统水平上的摄食调节中的功能作用。的结果 拟议的研究将是一套新的分析工具，能够定量评估 神经肽和生物胺具有高的空间，化学和时间信息。并行 将这些新方法应用于甲壳类动物和哺乳动物的神经系统， 加快我们对喂养障碍的新疗法的发展步伐。

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.