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口胃神经系统 及其相关的神经内分泌器官作为技术开发和验证的试验台， 该模型系统的独特优势和生物学意义。同时，我们的目标是将我们的 用于哺乳动物中枢神经系统神经肽发现和分析的技术开发。 为此，我们建议将重点放在大鼠模型中的关键脑区，逐渐提高复杂水平， 喂食相关的信息处理。具体目标包括：（1）开发一套新的质量缺陷- 基于多重二甲基嘧啶基鸟氨酸（DiPyrO）标签，用于精确和高通量的基于MS 1的 不同饲养条件下神经肽在体内表达变化的相对定量;（2） 开发一种基于MALDI的亚环境电离质谱成像（MSI）技术， 神经肽和胺神经递质在确定的神经元和喂养电路，增强空间 （3）建立了多靶点亲和增强微透析体内进样技术 对于响应食物摄入的循环神经肽和生物胺的MS检测和定量， 通过使用同量异位二甲基化亮氨酸的新的混合绝对和相对定量（HARQ）策略， （DiLeu）和同位素DiLeu标签;和（4）确定神经肽同种型的功能后果 以及通过结合质谱，电生理学， 和行为研究。新的神经肽将被评估在摄食调节中的功能作用。的 拟议研究的成果将是一套新的分析工具， 具有高度空间、化学和时间信息的神经肽和生物胺的相互作用。的 将这些新方法同时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 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 a set of novel mass defect- based multiplex dimethyl pyrimidinyl ornithine (DiPyrO) tags for accurate and high throughput MS1-based relative quantification of in vivo expression changes of neuropeptides under different feeding conditions; (2) Developing a sub-ambient ionization MALDI-based mass spectral imaging (MSI) technique for mapping neuropeptides and amine neurotransmitters in identified neurons and feeding circuits, with enhanced spatial resolution and speed; (3) Developing a multi-target affinity-enhanced microdialysis in vivo sampling technique for MS detection and quantitation of circulating neuropeptides and biogenic amines in response to food intake, via a novel hybrid absolute and relative quantification (HARQ) strategy using isobaric dimethylated leucine (DiLeu) and isotopic DiLeu tags; and (4) Determining the functional consequences of neuropeptide isoforms and assaying functional activities of hormonal cocktails by combining mass spectrometric, electrophysiological, and behavioral studies. Novel neuropeptides will be evaluated for functional roles in feeding regulation. 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.