Lipidomics analysis of identified single neurons in the adult rodent brain

成年啮齿动物大脑中已识别的单个神经元的脂质组学分析

• 批准号: 9488668
• 负责人: Daniele Piomelli
• 金额: $ 0.85万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9488668
• 关键词: Adult; Alzheimer' s Disease; Anatomy; Behavior; Biocompatible Materials; Biological; Biological Neural Networks; Birth; Brain; Brain Diseases; Brain Pathology; Caliber; Cells; Ceramides; Cholesterol; Complex; Coupled; Cytoplasmic Granules; Data; Development; Diglycerides; Discipline; Disease; Drug Addiction; Electroencephalogram; Fiber; Frequencies; Genetic Fingerprintings; Glycerophospholipids; Goals; Golgi Apparatus; Health; Hippocampus (Brain); Human; Individual; Lateral; Libraries; Lipids; Liquid Chromatography; Mass Spectrum Analysis; Membrane; Messenger RNA; Methods; Modernization; Modification; Molecular; Morphology; Mus; Neurons; Neurosciences; Nucleic Acids; Pathologic; Pathology; Perforant Pathway; Physiological; Physiological Processes; Physiology; Play; Procedures; Process; Property; Protocols documentation; Research; Research Personnel; Resolution; Rest; Rodent; Role; Sampling; Schizophrenia; Scientist; Signal Transduction; Slice; Sphingolipids; Staining method; Sterols; Stimulus; Structure; Synaptic plasticity; Techniques; Testing; Time; Tissue Sample; Tissues; Triglycerides; Validation; Variant; Vesicle; Weight; Work; base; brain tissue; dentate gyrus; experimental study; flexibility; granule cell; hippocampal pyramidal neuron; instrument; nano; neuronal cell body; neuroregulation; public health relevance; relating to nervous system; stem; tandem mass spectrometry; tissue preparation; tool; young adult

 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.

 描述（由申请人提供）：脂质在大脑功能中发挥着关键作用，并对药物成瘾、精神分裂症和阿尔茨海默氏病等病理学产生重要影响。从成年小鼠的活体海马切片中收集齿状回 (DG) 颗粒细胞（大脑中发现的最小神经元）的单个体细胞，我们分析了每只小鼠的脂质提取物。通过纳流液相色谱 (nano-LC) 与高分辨率飞行时间质谱 (MS) 相结合，我们能够可靠地检测与膜结构、能量储存和细胞信号传导有关的许多重要脂质。我们发现，侧向穿通路径（一种向 DG 颗粒细胞提供主要兴奋性输入的纤维束）的生理刺激引起细胞脂质谱的快速而强烈的变化。本申请建议开发这些。我们将初步研究结果纳入优化和验证的方案中，该方案可广泛应用于整个大脑神经元的脂质组学分析：（1）我们的初始方案高度敏感，但存在三个源于消失的限制。单个神经元提供的生物材料量较少：(a) 仅覆盖脂质组的一小部分；(b) 仅允许对最丰富的脂质种类进行串联 MS 结构确认；(c) 提供相对而非绝对定量；检测到的我们将 (i) 通过系统地修改关键分析参数来提高我们的程序的灵敏度；(ii) 扩大程序的定量范围；以及 (iii) 建立脂质 MS 数据的参考库； (2)我们的初步工作使我们能够识别静息 DG 细胞中的大量脂质种类，并检测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) 分析脂质组。 海马 CA1 和 CA3 区域的锥体神经元在解剖学和功能上与颗粒细胞不同；(ii) 确定各种生理刺激对 DG 颗粒细胞和 CA1/CA3 锥体神经元脂质组的影响。为了定义单细胞和全组织制剂之间的相似性和差异，我们将在完全验证和刺激条件下比较单个颗粒细胞和 DG 组织微孔的脂质组。经过优化，本方法将提供一种灵活而强大的新工具来研究脂质分子在从活体脑组织中分离出的已识别神经元中的作用，为研究神经脂质以及神经元多样性在健康和疾病中的作用开辟令人兴奋的新途径。

项目成果

A Primary Cortical Input to Hippocampus Expresses a Pathway-Specific and Endocannabinoid-Dependent Form of Long-Term Potentiation.

海马体的初级皮质输入表达了一种途径特异性和内源性大麻素依赖性的长期增强形式。

• DOI: 10.1523/eneuro.0160-16.2016
• 发表时间: 2016
• 期刊: eNeuro
• 影响因子: 3.4
• 作者: Wang,Weisheng;Trieu,BrianH;Palmer,LindaC;Jia,Yousheng;Pham,DanielleT;Jung,Kwang-Mook;Karsten,CarleyA;Merrill,CollinB;Mackie,Ken;Gall,ChristineM;Piomelli,Daniele;Lynch,Gary
• 通讯作者: Lynch,Gary

Author Correction: Patch clamp-assisted single neuron lipidomics.

作者更正：膜片钳辅助单神经元脂质组学。

• DOI: 10.1038/s41598-018-24605-7
• 发表时间: 2018
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Merrill,CollinB;Basit,Abdul;Armirotti,Andrea;Jia,Yousheng;Gall,ChristineM;Lynch,Gary;Piomelli,Daniele
• 通讯作者: Piomelli,Daniele