TECHNOLOGIES FOR CELLULAR NEUROMETABOLOMICS

描述（由申请人提供）：了解健康和患病大脑的脑功能需要了解构成大脑的神经元和支持细胞内发生的生物化学。众所周知，大脑中的单个细胞具有不同的信号分子补体和蛋白质补体，但对细胞代谢组的影响却知之甚少。哺乳动物中枢神经系统中细胞代谢组的细胞间变异是什么？对于大多数代谢物，答案是未知的。使用无脊椎动物模型，已经测量了相邻神经元的代谢组的巨大差异，例如氮能神经元中的硝酸盐的毫摩尔水平，而在非NO产生神经元中没有可检测的硝酸盐。同样，氨基酸补体的巨大变化取决于神经元使用的信号分子。不幸的是，技术限制目前不允许在单个哺乳动物神经元内测量主要代谢物。提出了一套技术开发和连接的方法来创建仪器和协议，以测量神经元簇，神经元组，甚至单个神经元中的代谢物。这些开发工作包括独特的采样协议，基于微流体的样品调理单元与集成的电泳分离，其次是天然荧光和质谱检测，并在必要时，捕获适当的代谢物到纳升体积的毛细管纳升体积NMR光谱表征。这套独特的技术有望开辟一种新的体积机制，以分析细胞间代谢物的变化。该技术将针对已知最异质的样本-哺乳动物大脑进行定制和验证。

DESCRIPTION (provided by applicant): Understanding brain function in healthy and diseased brains requires an understanding of the biochemistry occurring within the neurons and supporting cells making up the brain. It is well known that individual cells in the brain have distinct signaling molecule complements and protein complement, but the effects on the cellular metabolome are much less well understood. What is the cell to cell variation of the cellular metabolome in the mammalian CNS? For most metabolites, the answer is unknown. Using invertebrate models, large differences in the metabolome of adjacent neurons have been measured such as millimolar levels of nitrate in nitrergic neurons with no detectable nitrate in non-NO producing neurons. Similarly, large changes in the amino acid complement occur depending on the signaling molecules used by the neuron. Unfortunately, technology limitations do not currently allow the major metabolites to be measured within individual mammalian neurons. A suite of technology development and hyphenated approaches are proposed to create instruments and protocols to measure the metabolites in neuronal clusters, groups of neurons and even individual neurons. These development efforts include unique sampling protocols, microfluidically-based sample conditioning unit with integrated electrophoretic separations, followed by native fluorescence and mass spectrometric detection, and where necessary, capture of the appropriate metabolites into nanolitervolume capillaries for nanoliter volume NMR spectroscopic characterization. This unique set of technology promises to open up a new volume regime to profile the cell to cell variations in the metabolites. The technology will be tailored to and validated on the most heterogeneous samples known - the mammalian brain.