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（由适用提供）：脂质在大脑功能中起关键作用，并以重要的方式为诸如药物成瘾，精神分裂症和阿尔茨海默氏病等病理做出贡献。在初步实验中，我们利用一种最初为单神经元mRNA分析而设计的移液捕获方法来收集齿状回（DG）颗粒细胞的单个somata，这是来自大脑中最小的神经元 - 来自成年小鼠的海马切片中最小的神经元。我们通过与高分辨率飞行时间质谱法（MS）结合的纳米液相色谱（Nano-LC）分析了每个颗粒细胞的脂质提取物。我们能够检测到许多参与膜结构，储能和细胞信号传导的重要脂质。重要的是，我们发现对横向穿孔路径的物理模拟，这是一种为DG颗粒细胞提供主要令人兴奋的输入的纤维道，引起了细胞脂质谱的快速变化。目前的应用程序提案将这些缩写调查结果开发为优化且经过验证的方案，该方案可以广泛应用于整个大脑神经元的脂肪组学分析。我们有两个具体的目标：（1）方法优化。我们的初始方案高度敏感，但具有三个限制，这是由于单个神经元提供的消失的生物材料量消失的限制：（a）它仅覆盖脂质组的一小部分； （b）仅对最丰富的脂质物种允许串联MS结构确认； （c）它提供了对检测到的脂质的相对而不是绝对定量。我们将通过系统修改关键分析参数来提高程序的敏感性； （ii）扩展程序的定量范围； （iii）构建脂质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止的DG颗粒细胞中鉴定大量脂质物种，并在生理刺激后检测细胞脂质谱的特定改变。为了测试协议的一般适用性，我们将（i）介绍 海马的CA1和CA3场中的锥体神经元，在解剖学和功能上与颗粒细胞不同。 （ii）确定各种物理刺激对DG颗粒细胞和CA1/CA3锥体神经元的脂多组的影响。最后，为了定义单细胞和整个组织制剂之间的相似性和差异，我们将比较在对照和刺激条件下，单个颗粒细胞和DG组织的微置脂质组。当完全验证和优化时，目前的方法将提供一种灵活且健壮的新工具，以研究脂质分子在从活脑组织中分离出的鉴定神经元中的作用，为神经元脂质的研究开辟了令人兴奋的新途径以及神经元多样性在健康和疾病中的作用。

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