CORE--ANALYTIC NEUROCHEMISTRY AND SPECTROSCOPY CORE

在过去的二十年里，我们对大脑功能的理解有了巨大的增长，特别是那些 形成突触传递基础的事件。数以百计的实验室辛勤工作， 表征神经递质和突触的代谢，药理学，运输和发育 设备.这项事业的幸运结果是对信息的复杂性有了详细的了解 神经系统的交换。我们可以确定快速和慢速神经传递的分子基础。我们 认识到神经胶质细胞在维持神经元内离子稳态，终止神经元内细胞凋亡中的重要性。 在神经传递和提供给神经元的前体补充的神经递质池。 我们已经开始认识到，突触-就像大脑本身-不是一个成品，而是一个结构， 一种永恒的进化状态，其发展按照精心制定的时间表进行， 促进获取新信息和适应不断变化的外部环境。 对突触生物化学的科学兴趣既反映了这一学科的根本重要性， 认识到神经递质失衡是许多人类疾病的核心，其中包括大脑缺氧 损伤、先天性代谢缺陷、癫痫、药物滥用、抑郁症等。 发育障碍通常由突触生物化学和/或突触发生的紊乱引起。 有效治疗这些疾病的前提是了解相关的病理生物学。 我们构建了分析神经化学和光谱学核心，为我们的用户提供一系列 服务，适应全面审查的突触-从体外分析的个人 从代谢物到离子流的原位表征到神经递质的体内测量， 神经递质受体和转运蛋白。我们已经能够提供先进的技术- 质谱，正电子发射断层扫描，光学成像-这超过了分析能力的一个 个人实验室此外，我们通过让每个用户熟悉我们的分析工具， 并帮助每个用户解释实验结果。 在MRDDRC最初提交时，该核心赞助了三项服务：液相色谱（HPLC） 神经递质及其代谢物的分析、GC-MS技术和神经元细胞培养设施。在 1995年，目前的细胞神经科学核心（核心A）被“戏弄”出来，作为一个单独的核心，以改善我们的神经系统。 细胞和病理学服务。当前的分析神经化学和光谱学核心 (Core D）然后假设其当前配置。我们仍然提供HPLC资源，用于定量氨基 酸和生物胺具有高灵敏度，我们提供质谱服务的测定 用于体内和体外动力学研究的稳定同位素富集。2000年，我们增加了基于细胞的检测， 测量由细胞死亡和/或凋亡激活的分子（包括Ca 2+、反应性 氧类、Ca 2+激活的蛋白酶和半胱天冬酶-3。我们还测量了DNA片段化（一种晚期标记， 细胞凋亡）、乳酸脱氢酶（细胞死亡的细胞质标志物）和甲瓒3-（4，5-二甲基噻唑- 2-基）-2，5-二苯基四唑溴化铵（MTT），活细胞的线粒体标志物。我们加了一个正电子 发射断层扫描（PET）设备，用于阐明活体中的分子和细胞突触过程 有机体一个新的倡议是目前提出的蛋白质组学服务，将使蛋白质鉴定， 复杂的混合物以及翻译后修饰位点的表征。 该核心的重要性反映在核心服务的许多出版物和赠款申请中。 成功启用。由于丰富的分析基础设施， 核心所能提供的一个例子是罕见疾病研究联盟，一个研究尿素的企业。 这是其他几个MRDDRC之间合作的一个显著例子1。我们 随着我们为核心增加新的服务，包括蛋白质组学设施，预计这一趋势将继续下去。 该核心包含几种不同的分析组件：HPLC、质谱、基于细胞的测定、PET和 蛋白质组学我们承认这个指令集是广泛的，但我们希望通过创建一个 一个便于生物化学研究的单一单元，沿着从单个分析物（HPLC，质谱）延伸的连续体 光谱分析和蛋白质组学）到完整细胞（基于细胞的测定）到整个生物体（PET）。这不仅 这样的安排既能使组织统一，又能使节约得到加强，这是我们必须加以权衡的一个因素 在MRDDRC的总体预算受到严格限制的情况下，创建更多核心将 使这种节约变得不可能。相反，目前的结构允许我们利用机构 支持，其中包括迄今为止为我们的用户提供这些状态的实际成本的更大比例- 艺术服务。

The past two decades have seen enormous growth in our understanding of brain function, especially of those events that form the basis of synaptic transmission. Hundreds of laboratories have labored diligently to characterize the metabolism, pharmacology, transport and development of the neurotransmitters and the synaptic apparatus. The fortunate outcome of this enterprise is a detailed understanding of the intricacies of information exchange in the nervous system. We can identify the molecular basis for fast and slow neural transmission. We appreciate the importance of glial cells in maintaining ionic homesostasis in the neuropil, in terminating neurotransmission and in furnishing to neurons the precursors for replenishment of the neurotransmitter pool. We have begun to appreciate that the synapse - like the brain itself- is not a finished product, but a structure in a state of perpetual evolution, the development of which proceeds according to a carefully wrought schedule that facilitates the acquisition of new information and the adaptation to an ever changing external environment. Scientific interest in synaptic biochemistry reflects both the fundamental importance of the subject and the recognition that neurotransmitter imbalance is at the heart of many human diseases, among them hypoxic brain injury, inborn errors of metabolism, epilepsy, substance abuse, depression, etc. Mental retardation and other developmental disabilities commonly result from derangements of synaptic biochemistry and/or synaptogenesis. Effective treatments for these disorders presuppose an understanding of the relevant pathobiology. We structured the Analytical Neurochemistry and Spectroscopy Core to provide our users with an array of services that accommodates comprehensive scrutiny of the synapse - from the in vitro analysis of individual metabolites to in situ characterization of ionic flux to in vivo measurement of neurotransmitters and neurotransmitter receptors and transporters. We have been able to make available sophisticated technologies - mass spectrometry, positron emitting tomography, optical imaging - that exceed the analytic capability of an individual laboratory. Furthermore, we support our users by familiarizing each of them with our analytical potential and by helping each user to interpret experimental results. At the initial submission of the MRDDRC, this core sponsored three services: liquid chromatographic (HPLC) analysis of neurotransmitters and their metabolites, GC-MS technology, and a neuronal cell culture facility. In 1995 the current Cellular Neuroscience Core (Core A) was "teased" out as a separate core to improve our repertoire of cellular and pathology services. The current Analytical Neurochemistry and Spectroscopy Core (Core D) then assumed its present configuration. We still provide HPLC resources for the quantitation of amino acids and biogenic amines with high sensitivity and we furnish mass spectrometry services for the determination of stable isotope enrichment for kinetic studies, both in vivo and in vitro. In 2000 we added a Cell Based Assay Facility that measures molecules that are activated by cell death and/or apoptosis, including Ca2+, reactive oxygen species, Ca2+-activated proteases, and caspase-3. We also measure DNA fragmentation (a late marker of apoptosis), lactate dehydrogenase (a cytoplasmic marker of cell death) and the formazan 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazoliumbromide (MTT), a mitochondrial marker for living cells. We added a Positron Emission Tomography (PET) Facility to elucidate molecular and cellular synaptic processes in the living organism. A novel initiative is the current proposed Proteomics service that will enable protein identification in complex mixtures as well as the characterization of sites of post-translational modification. The significance of this Core is reflected in the many publications and grant applications that the core services successfully enabled. Several new scientific initiatives were feasible because of the rich analytic infrastructure that the Core affords. One example is the Rare Disease Research Consortium, a venture that studies the urea cycle defects and that represents a notable example of collaboration among several other MRDDRCs1. We anticipate this trend to continue as we add novel services to the Core, including the Proteomics Facility. This Core contains several different analytic components: HPLC, mass spectrometry, cell-based assays, PET and proteomics. We acknowledge that this repertoire is broad, but we wanted to accommodate our users by creating a single unit that facilitates biochemical study along a continuum extending from individual analytes (HPLC, mass spectrometry and proteomics) to intact cells (cell-based assays) to the whole organism (PET). Not only does this arrangement confer organizational unity, it also allows increased economy, a consideration that we must weigh carefully at a time of strict limits on the overall budget of the MRDDRC. The creation of additional cores would have made impossible such economization. Instead, the current structure allows us to leverage institutional support, which comprises by far the greater proportion of the actual costs of providing our users,with these stateof- the-art services.