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.



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