Support by Centre National de la Recherche Scientifique (CNRS, UMR 6612 and 7339) is gratefully acknowledged.

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+phenotyping+of+rodent+ASD+models+enrichment+analysis+of+mouse+and+human+neurobiological+phenotypes+associated+with+high-risk+autism+genes+identifies+morphological,+electrophysiological,+neurological,+and+behavioral+features.">Optimizing the phenotyping of rodent ASD models enrichment analysis of mouse and human neurobiological phenotypes associated with high-risk autism genes identifies morphological, electrophysiological, neurological, and behavioral features.</a> Mol Autism. 3, 1 (2012).</li><li>Papaioannou, V., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mouse Phenotypes A Handbook of Mutation Analysis. , (2004).</li><li>Yu, F. 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T., Pietropaolo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Behavioral Genetics of the Mouse Genetics of Behavioral Phenotypes. Vol. 1, (2013).</li><li>Viola, A., Saywell, V., Villard, L., Cozzone, P. J., Lutz, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+fingerprints+of+altered+brain+growth,+osmoregulation+and+neurotransmission+in+a+Rett+syndrome+model.">Metabolic fingerprints of altered brain growth, osmoregulation and neurotransmission in a Rett syndrome model.</a> PLoS ONE. 2, e157 (2007).</li><li>Lutz, N. W., Sweedler, J. V., Wevers, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methodologies for Metabolomics. , (2013).</li><li>Rabinowitz, J. D., Purdy, J. 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J., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+biochemical+pathways+in+experimental+autoimmune+encephalomyelitis+and+adjuvant+arthritis+a+comparative+metabolomic+study.">Cerebral biochemical pathways in experimental autoimmune encephalomyelitis and adjuvant arthritis a comparative metabolomic study.</a> PLoS ONE. 8, e56101 (2013).</li><li>Lutz, N. W., Cozzone, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+multiparametric+optimization+for+phospholipidomics+by+31P+NMR+spectroscopy.">Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy.</a> Biophys Rev. 5, 295-304 (2013).</li><li>Pearson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Shimming an NMR magnet. , (1991).</li><li>Lane, A. N., Fan, T. W. M., Higashi, R. M., Correia, J. J., Detrich, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biophysical tools for biologists. Vol. 1, In vitro techniques, (2008).</li><li>Peeling, J., Wong, D., Sutherland, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+study+of+regional+metabolism+after+forebrain+ischemia+in+rats.">Nuclear magnetic resonance study of regional metabolism after forebrain ischemia in rats.</a> Stroke. 20, 633-640 (1989).</li></ol>

The authors have nothing to disclose.

NMR光谱法是用于测量溶液中的化学化合物的浓度在很重现的和准确的方式的一种有效方法。然而，为了获得高质量的数据，必须坚持有关样品制备和分析的某些规则。中代谢物浓度的NMR谱的测定，既不产生也不NMR信号的接收占主导地位的定量误差，除非观察到的信号的强度接近的检测阈值（特别是弱信号）。在所有其他情况下的生物变异性，样品制备技术（提取效率，化合物的物理和化学稳定性...

小鼠模型已被广泛利用在大脑研究1。基因型-表型的相关性进行了研究在小鼠和大鼠的大脑通过研究基因表达在一方面的RNA和/或蛋白水平，和形态，功能，电和/或行为的表现型上的其它2-6。然而，为了完全理解联的表型基因型的机制，就必须进行调查的分子事件的下游蛋白的表达， 即 。生化基板在其酶作用7的代谢。这要求领导，在过去10至15年，以代谢研究在生物学8,9的许多分支的复兴。虽然古典代谢的研究往往集中在特定途径的细节，新的代谢途径是面向正在考虑组织的全球性代谢谱的全方位调查。这个概念的一个后果是用于分析工具，最大限度地减少对特定的代谢途径和/或类型的化合物的偏压明显需要。然而，传统的生化测定是基于需要进行化验之前被指定的特定分析物的特定的化学反应。其中每一个与此相反，如核磁共振（NMR）光谱法和质谱（MS）（ⅰ）的光谱技术是基于生化化合物的特定分子（物理）性能产生了一个或若干个不同信号中的频谱在一个实验的过程中检测到的;以及（ii）检测大量每个实验的不同化合物。 因此，每一个光谱包含代谢物的整个范围的综合信息。出于这个原因，光谱方法可用于代谢组适当的工具，如不事先选择需要进行关于分析物的性质进行测定8 </suP&gt;。其结果是，这些技术自然地借给自己探索性的研究，因为它们极大地方便了意想不到的代谢变化的检测。 虽然NMR光谱法和质谱可以互换使用，用于许多代谢物的分析中，每个方法具有特定的优点和缺点，最近已审查10。简言之，核磁共振光谱，通常可以从粗提取物中进行，且不需要样品化合物的分析前的色谱分离。相比之下，MS可与气体或液体色谱（GC或LC）分离，除去最近特别发展，如质谱成像。在一些特殊情况下，如糖 ​​的分析，液相色谱分离可能成为一种必然的核磁共振光谱为好，因为不同的糖的共振线质子显著重叠（1H）NMR谱。然而，1 H NMR光谱没有字符omatographic分离仍然是最流行的，几乎普遍应用代谢组学核磁共振方法。通常，样品制备是更费时和复杂的MS比为核磁共振光谱。由于基体效应的严重问题是许多在NMR谱，其中它们可以导致相当衰减的信号不太常见的比在MS。代谢物的定量可以用任何一种方法来实现。然而，需要为MS多种标准化合物由于变化的代谢物之间的基体效应和离子化效率。与此相反，每个样品只有一个标准是需要的NMR波谱分析，因为在适当的测量条件下，后者的方法在本质上是定量由于通过所观察到的细胞核的严格线性的核磁共振响应。 NMR的主要缺点是其相对低的灵敏度。 MS，尤其是LC-MS，是由数个数量级比核磁共振更敏感;因为这个原因，MS要优于核磁共振对分析在非常低的浓度存在的化合物。另一方面，在NMR实验的无损性质是通过MS具有明显的优势;以这种方式，NMR可以反复对同一样品， 例如进行，不同的NMR活性核，例如1 H，磷-31（31 P），碳-13（13 C），氟-19（19 F）的等 ，如无材料以NMR消耗相对于MS的测量。 既NMR和MS可以在不同的模式下被使用，每一个是最适合于特定化学特性的化合物的检测。例如，31 P NMR往往更适合于1 H-NMR进行的适度浓缩的磷酸化化合物的分析，虽然几乎所有的磷酸化代谢物还含有质子。然而，它们的1 H NMR信号可以通过从其它的非磷酸化的化合物的1 H NMR信号被遮蔽，而后者则显然不会造成在31 P NMR谱的背景信号。在模拟的情况下，19 F NMR分析是优选的氟化合物， 如氟化的药物（从内源性代谢物没有背景信号），而13 C-NMR的特例是感兴趣的几乎全部，如果13 C-命运标记的外源性代谢前体需要遵循，由于13 C同位素（ 约 1％）的极低的天然丰度。许多质谱仪在任负离子模式或正离子模式下工作。因此，重要的是要知道前面的分析是否被观察的离子是带负电或带正电的。我们在此关注的协议对脑组织代谢的通过1 H和31 P NMR光谱法的分析，因为这种方法会产生在（ⅰ）需要的样品制备时间方面有大量的重要代谢物浓度以低成本ND所需的代谢物的定量（ⅱ）的努力。所有实验中可以使用一个标准的湿化学实验室的设备和高分辨率核磁共振光谱设施来进行。而且要求在下面的协议部分进行了描述。

<table><tbody><tr><td>Isoflurane</td><td>Virbac</td><td>Vetflurane</td><td>Anesthetic for animals</td></tr><tr><td>Isoflurane vaporizer</td><td>Ohmeda</td><td>Isotec 3</td><td>Newer model available: Isotec 4</td></tr><tr><td>Scalpel, scissors, forceps, clamps</td><td>Harvard Apparatus&lt;br/&gt; Fisher Scientific</td><td>various&lt;br/&gt; various</td><td>Surgical equipment for animals</td></tr><tr><td>Freeze-clamp tool</td><td>homebuilt</td><td>n/a</td><td>Tong with aluminium plates, to be inserted&lt;br/&gt; in liquid nitrogen for cooling</td></tr><tr><td>Dewar</td><td>Nalgene</td><td>4150-4000</td><td></td></tr><tr><td>Liquid nitrogen</td><td>Air Liquide</td><td>n/a</td><td></td></tr><tr><td>Nitrogen gas</td><td>Air Liquide</td><td>n/a</td><td></td></tr><tr><td>Nitrogen evaporator</td><td>Organomation Associates</td><td>N-EVAP 111</td><td>Can be replaced by homebuilt device</td></tr><tr><td>Mortar</td><td>Sigma-Aldrich</td><td>Z247472</td><td></td></tr><tr><td>Pestle</td><td>Sigma-Aldrich</td><td>Z247510</td><td></td></tr><tr><td>Tissue homogenizer</td><td>Kinematica</td><td>Polytron</td><td>With test tubes fitting homogenizer shaft</td></tr><tr><td>Electronic scale</td><td>Sartorius</td><td>n/a</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>M3641</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>366910</td><td></td></tr><tr><td>Glass centrifuge tubes</td><td>Kimble</td><td>45500-15, 45500-30</td><td>Kimax 15 ml, 30 ml tube</td></tr><tr><td>Microcentrifuge tubes</td><td>Kimble</td><td>45150-2</td><td>Kimax 2 ml tube; should replace &quot;Eppendorf&quot; tube if compatible with centrifuge rotor</td></tr><tr><td>Polystyrene pipettes</td><td>Costar Corning</td><td>Stripettes</td><td>5 and 10 ml volumes</td></tr><tr><td>Deuterochloroform</td><td>Sigma-Aldrich</td><td>431915</td><td>99.96% deuterated</td></tr><tr><td>Deuterium oxide</td><td>Sigma-Aldrich</td><td>423459</td><td>99.96% deuterated</td></tr><tr><td>Deuterium chloride</td><td>Alpha Aesar</td><td>42406</td><td>20% in deuterium oxide</td></tr><tr><td>Sodium deuteroxide</td><td>Sigma-Aldrich</td><td>164488</td><td>30% in deuterium oxide</td></tr><tr><td>Lyophilizer</td><td>Christ</td><td>Alpha 1-2</td><td></td></tr><tr><td>Cold centrifuge</td><td>Heraeus</td><td>Megafuge 16R</td><td></td></tr><tr><td>pH meter</td><td>Eutech Cybernetics</td><td>Cyberscan</td><td></td></tr><tr><td>CDTA</td><td>Sigma-Aldrich</td><td>D0922</td><td></td></tr><tr><td>Cesium hydroxide</td><td>Sigma-Aldrich</td><td>516988</td><td></td></tr><tr><td>NMR tubes</td><td>Wilmad</td><td>528-PP</td><td></td></tr><tr><td>NMR stem coaxial insert</td><td>Sigma-Aldrich</td><td>Z278513</td><td>By Wilmad</td></tr><tr><td>NMR pipettes</td><td>Sigma-Aldrich</td><td>Z255688</td><td></td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>Research</td><td>With tips for volumes from 0.5 to 1,000 &amp;mu;l</td></tr><tr><td>Pipet-Aid</td><td>Drummond</td><td>XP</td><td></td></tr><tr><td>NMR spectrometer</td><td>Bruker</td><td>AVANCE 400</td><td>including probe and other accessories</td></tr><tr><td>NMR software</td><td>Bruker</td><td>TopSpin 1.3</td><td>newer version available: Topspin 3.2</td></tr><tr><td>Water-soluble standard compounds</td><td>Sigma-Aldrich</td><td>various</td><td></td></tr><tr><td>Phospholipid standard compounds</td><td>Avanti Polar Lipids&lt;br/&gt; Doosan Serdary&lt;br/&gt; Sigma-Aldrich</td><td>various&lt;br/&gt; various&lt;br/&gt; various</td><td>Source for plasmalogens, but may be &amp;lt;70 - 80% purity</td></tr><tr><td>Methylenediphosphonate</td><td>Sigma-Aldrich</td><td>M9508</td><td></td></tr><tr><td>TSP-d4</td><td>Sigma-Aldrich</td><td>269913</td><td></td></tr></tbody></table>

metabolomic analysis of rat brain by high resolution nuclear magnetic resonance spectroscopy of tissue extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

为了获得最佳的分辨率在脑和其它组织提取物的代谢NMR谱，这早已是普遍的做法，以消除或掩蔽的金属离子（最重要的是:顺磁性离子）存在的提取液。这已经实现或者通过添加螯合剂，如EDTA或CDTA的提取物19，或通过将提取物通过离子交换树脂，如CHELEX-100 20。在图1中呈现的结果表明，该步骤不是必需的1 H NMR光谱分析，如果脑提取物是根据上述协议精心的准备...

Watch this Scientific Journal Video about Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts at JoVE.com

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

大鼠脑组织被提取物的高分辨核磁共振代谢组学分析

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

metabolomic-analysis-rat-brain-high-resolution-nuclear-magnetic

Research

JoVE Journal

Neuroscience

14.7K 次观看.  Aix-Marseille Université. The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

视频: Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

科研

医学

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

免疫与感染

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

在啮齿类动物的存活立体定向手术

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

生物学

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

神经科学

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not only provides quantitative data but also permits structural identification of the metabolites present in complex mixtures.
Metabolic profiling by 1H NMR has proven useful in the study of various types of plant scenarios, which include the evaluation of crop&#160;conditions, harvest and post- harvest treatments, metabolic phenotyping, metabolic pathways, gene regulation, identification of biomarkers, chemotaxonomy, quality control, denomination of origin, among others. However, signal overlapping of the large number of resonances with expanded J-coupling multiplicities complicates the spectra analysis and its interpretation, and represents a limitation for classical 1H NMR profiling.
In the last decade, novel NMR broadband homonuclear decoupling techniques through which multiplet signals collapse into single resonance lines - commonly called Pure Shift methods - have been developed to overcome the spectra resolution problem inherent to 1H NMR classical spectra.
Here a step-by-step protocol of the plant extract preparation and the procedure to record optimal Pure Shift PSYCHE and SAPPHIRE-PSYCHE spectra in three different plant matrices - Vanilla plant&#160;leaves, potato tubers (S. tuberosum), and Cape gooseberries (P. peruviana) - is presented. The effect of the gain in resolution in metabolic identification, correlation analysis and multivariate analyses, as compared against classical spectra, is discussed.

<meta charset="utf8"></head><body>Pure Shift 核磁共振:植物代谢组学的新工具

This paper presents the use of PSYCHE and SAPPHIRE-PSYCHE in the metabolic profiling of plants and includes detailed procedures for sample preparation and optimal Pure Shift NMR spectra recording. Examples through which the gain in resolution achieved by homonuclear decoupling allows a more comprehensive understanding of the system are discussed.

Pure Shift 核磁共振:植物代谢组学的新工具

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not ...

生物化学

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

代谢物的浓度从低密度浮游社区的环境代谢，利用核磁共振光谱

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

大鼠桶内皮层 7 t 的功能磁共振波谱在晶氏体活化过程中的应用

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

大鼠脑组织被提取物的高分辨核磁共振代谢组学分析

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此视频中的章节

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

在无菌无鼠标进定植评估肝代谢的变化¹ H核磁共振光谱

在啮齿类动物的存活立体定向手术

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Pure Shift 核磁共振:植物代谢组学的新工具

Pure Shift 核磁共振:植物代谢组学的新工具

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

代谢物的浓度从低密度浮游社区的环境代谢，利用核磁共振光谱

大鼠桶内皮层 7 t 的功能磁共振波谱在晶氏体活化过程中的应用

新生大鼠脑组织在神经端子突形圆锥花序分布超微结构形态分析中的制备