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）：脂质在脑功能中起关键作用，并以重要方式促成药物成瘾、精神分裂症和阿尔茨海默病等病理。在初步的实验中，我们利用移液管捕获方法最初设计的单神经元mRNA分析收集单个胞体的齿状回（DG）颗粒细胞-在大脑中发现的最小的神经元-从活的成年小鼠海马切片。我们分析了每个颗粒细胞的脂质提取物的纳流液相色谱（nano-LC）耦合到高分辨率飞行时间质谱（MS）。我们能够可靠地检测到许多参与膜结构，能量储存和细胞信号传导的重要脂质。重要的是，我们发现，生理刺激的横向穿孔路径，纤维束，提供主要的兴奋性输入DG颗粒细胞，引起细胞的脂质谱的快速和强大的变化。本申请提出将这些初始发现发展成优化和验证的方案，其可以广泛应用于整个大脑的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。我们最初的协议是高度敏感的，但有三个限制，从一个单一的神经元所提供的生物材料的消失低量的根源：（a）它只涵盖了一小部分的脂质体;（B）它允许串联MS结构确认仅为最丰富的脂质种类;和（c）它提供了相对的，而不是绝对的定量检测脂质。我们将（i）通过系统地修改关键分析参数来提高我们程序的灵敏度;（ii）扩展程序的定量范围;（iii）建立脂质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确定大量的脂质种类在休息DG颗粒细胞，并检测特定的改变，在细胞的脂质配置文件后，生理刺激。为了测试该方案的普遍适用性，我们将（i）分析 海马CA 1和CA 3区的锥体神经元，其在解剖学和功能上不同于颗粒细胞;和（ii）确定各种生理刺激对DG颗粒细胞和CA 1/CA 3锥体神经元的脂质体的影响。最后，为了确定单细胞和整个组织制剂之间的相似性和差异，我们将比较控制和刺激条件下的单个颗粒细胞和DG组织的微穿孔的脂质体。当充分验证和优化时，本方法将提供一种灵活和强大的新工具来研究脂质分子在从活脑组织分离的已鉴定神经元中的作用，为神经脂质的研究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