Lipidomics analysis of identified single neurons in the adult rodent brain

 描述(由申请者提供)：脂质在大脑功能中发挥关键作用，并在药物成瘾、精神分裂症和阿尔茨海默病等病理过程中起重要作用。在初步实验中，我们利用最初为单神经元mRNA分析设计的吸管捕获方法，从成年小鼠的活海马片中收集齿状回(DG)颗粒细胞的单个体细胞-大脑中发现的最小神经元-的个体体细胞。我们用纳米液色谱结合高分辨飞行时间质谱仪分析了每个颗粒细胞的脂类提取物。我们能够可靠地检测到许多与膜结构、能量储存和细胞信号有关的重要脂类。重要的是，我们发现，对侧穿支路径的生理刺激，导致细胞的脂质谱快速而强劲的变化。侧穿支路径是为DG颗粒细胞提供主要兴奋性输入的纤维束。本申请建议将这些初始发现发展成一种优化和验证的方案，可以广泛应用于对整个大脑中神经元的脂质组学分析。我们有两个具体的目标：(1)方法优化。我们最初的方案是高度敏感的，但有三个限制，这源于单个神经元提供的生物材料数量极少：(A)它只覆盖脂体的一小部分；(B)它只允许对最丰富的脂类进行串联MS结构确认；以及(C)它提供了检测到的脂类的相对定量而不是绝对定量。我们将(I)通过系统地修改关键分析参数来提高程序的灵敏度；(Ii)扩大程序的定量范围；以及(Iii)建立脂类MS数据的参考库 单个神经元，使用单独捕获的颗粒DG细胞池。(2)方法验证。我们的初步工作使我们能够在静息的DG颗粒细胞中鉴定出大量的脂质物种，并检测到生理刺激后细胞脂质谱的特定变化。为了测试该协议的普遍适用性，我们将(I)描述 (Ii)确定不同生理刺激对DG颗粒细胞和CA1/CA3锥体细胞脂体的影响。最后，为了明确单个细胞和整个组织制剂的异同，我们将比较对照和刺激条件下单个颗粒细胞和DG组织微穿孔的脂质体。当充分验证和优化后，本方法将提供一个灵活和强大的新工具来研究从活脑组织中分离出来的已识别神经元中脂分子的作用，为研究神经脂质和神经元多样性在健康和疾病中的作用开辟了令人兴奋的新途径。

 DESCRIPTION (provided by applicant): Lipids play key roles in brain function and contribute in important ways to pathologies such as drug addiction, schizophrenia and Alzheimer's disease. In preliminary experiments, we utilized a pipette capture method originally devised for single-neuron mRNA analysis to collect individual somata of dentate gyrus (DG) granule cells - the smallest neurons found in the brain - from living hippocampal slices of adult mice. We analyzed lipid extracts of each granule cell by nanoflow liquid chromatography (nano-LC) coupled to high-resolution time-of-flight mass spectrometry (MS). We were able reliably to detect many important lipids involved in membrane structure, energy storage, and cellular signaling. Importantly, we found that physiological stimulation of the lateral perforant path, a fiber tract that provides major excitatory input to DG granule cells, caused rapid and robust changes in the cells' lipid profile. The present application proposes to develop these initials findings into an optimized and validated protocol that can be widely applied to lipidomics analyses of neurons throughout the brain. We have two specific aims: (1) Method optimization. Our initial protocol is highly sensitive, but has three limits that stem from the vanishingly low amount of biomaterial afforded by a single neuron: (a) it covers only a fraction of the lipidome; (b) it allows tandem MS structure confirmation only for the most abundant lipid species; and (c) it provides relative rather than absolute quantification of detected lipids. We will (i) increase te sensitivity of our procedure through systematic modifications of key analytical parameters; (ii) extend the procedure's quantitative reach; and (iii) build reference libraries of lipid MS data for individual neurons, using pools of individually captured granule DG cells. (2) Method validation. Our preliminary work allowed us to identify a substantial number of lipid species in resting DG granule cells, and to detect specific alterations in the cells' lipid profile following physiologicl stimulation. To test the general applicability of the protocol, we will (i) profile the lipidome of pyramidal neurons in the CA1 and CA3 fields of the hippocampus, which are anatomically and functionally different from granule cells; and (ii) determine the impact of various physiological stimuli on the lipidome of DG granule cells and CA1/CA3 pyramidal neurons. Lastly, to define similarities and differences between single-cell and whole tissue preparations, we will compare the lipidomes of individual granule cells and micropunches of DG tissue under control and stimulated conditions. When fully validated and optimized, the present method will provide a flexible and robust new tool to investigate the roles of lipid molecules in identified neurons isolated from live brain tissue, opening exciting new avenues for research on neural lipids and the role of neuronal diversity in health and disease.