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

<ol>
	<li>Manger, P. R., 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=Is+21st+century+neuroscience+too+focused+on+the+rat/mouse+model+of+brain+function+and+dysfunction.">Is 21st century neuroscience too focused on the rat/mouse model of brain function and dysfunction.</a> Front Neuroanat. 2, 5 (2008).</li><li>Buxbaum, J. 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. H., 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=Reduced+sodium+current+in+GABAergic+interneurons+in+a+mouse+model+of+severe+myoclonic+epilepsy+in+infancy.">Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy.</a> Nat Neurosci. 9, 1142-1149 (2006).</li><li>Mallolas, J., 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=A+polymorphism+in+the+EAAT2+promoter+is+associated+with+higher+glutamate+concentrations+and+higher+frequency+of+progressing+stroke.">A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke.</a> The Journal of Experimental Medicine. 203, 711-717 (2006).</li><li>Crusio, W. E., Sluyter, F., Gerlai, R. 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. G., Vastag, L., Shenk, T., Koyuncu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+in+drug+target+discovery.">Metabolomics in drug target discovery.</a> Cold Spring Harb Symp Quant Biol. 76, 235-246 (2011).</li><li>Wishart, D. S., Wevers, R. A., Lutz, N. W., Sweedler, J. V., 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=.">.</a> Methodologies for Metabolomics. , (2013).</li><li>Ponten, U., Ratcheson, R. A., Salford, L. G., Siesjo, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+freezing+conditions+for+cerebral+metabolites+in+rats.">Optimal freezing conditions for cerebral metabolites in rats.</a> Journal of Neurochemistry. 21, 1127-1138 (1973).</li><li>Henry, P. G., Oz, G., Provencher, S., Gruetter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+dynamic+isotopomer+analysis+in+the+rat+brain+in+vivo+automatic+quantitation+of+13C+NMR+spectra+using+LCModel.">Toward dynamic isotopomer analysis in the rat brain in vivo automatic quantitation of 13C NMR spectra using LCModel.</a> NMR Biomed. 16, 400-412 (2003).</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=Multiparametric+optimization+of+(31)P+NMR+spectroscopic+analysis+of+phospholipids+in+crude+tissue+extracts+2+Line+width+and+spectral+resolution.">Multiparametric optimization of (31)P NMR spectroscopic analysis of phospholipids in crude tissue extracts 2 Line width and spectral resolution.</a> Anal Chem. 82, 5441-5446 (2010).</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=Multiparametric+optimization+of+(31)P+NMR+spectroscopic+analysis+of+phospholipids+in+crude+tissue+extracts.+1.+Chemical+shift+and+signal+separation.">Multiparametric optimization of (31)P NMR spectroscopic analysis of phospholipids in crude tissue extracts. 1. Chemical shift and signal separation.</a> Anal Chem. 82, 5433-5440 (2010).</li><li>Lutz, N. W., Cozzone, P. J., 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>Lutz, N. W., Fernandez, C., Pellissier, J. F., Cozzone, P. 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 border="0" cellpadding="0" cellspacing="0" width="921">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Isoflurane</td>
			<td style="width:144px;">Virbac</td>
			<td style="width:180px;">Vetflurane</td>
			<td style="width:316px;">Anesthetic for animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane vaporizer</td>
			<td>Ohmeda</td>
			<td>Isotec 3</td>
			<td>Newer model available: Isotec 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel, scissors, forceps, clamps</td>
			<td>Harvard Apparatus 
			Fisher Scientific</td>
			<td>various 
			various</td>
			<td>Surgical equipment for animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-clamp tool</td>
			<td>homebuilt</td>
			<td>n/a</td>
			<td>Tong with aluminium plates, to be inserted 
			in liquid nitrogen for cooling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dewar</td>
			<td>Nalgene</td>
			<td>4150-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid nitrogen</td>
			<td>Air Liquide</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen gas</td>
			<td>Air Liquide</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen evaporator</td>
			<td>Organomation Associates</td>
			<td>N-EVAP 111</td>
			<td>Can be replaced by homebuilt device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mortar</td>
			<td>Sigma-Aldrich</td>
			<td>Z247472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pestle</td>
			<td>Sigma-Aldrich</td>
			<td>Z247510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue homogenizer</td>
			<td>Kinematica</td>
			<td>Polytron</td>
			<td>With test tubes fitting homogenizer shaft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electronic scale</td>
			<td>Sartorius</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>366910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass centrifuge tubes</td>
			<td>Kimble</td>
			<td>45500-15, 45500-30</td>
			<td>Kimax 15-mL, 30-mL tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td>Kimble</td>
			<td>45150-2</td>
			<td>Kimax 2-mL tube; should replace &#34;Eppen-dorf&#34; tube if compatible with centrifuge rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polystyrene pipettes</td>
			<td>Costar Corning</td>
			<td>Stripettes</td>
			<td>5 and 10-mL volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterochloroform</td>
			<td>Sigma-Aldrich</td>
			<td>431915</td>
			<td>99.96 % deuterated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterium oxide</td>
			<td>Sigma-Aldrich</td>
			<td>423459</td>
			<td>99.96 % deuterated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterium chloride</td>
			<td>Alpha Aesar</td>
			<td>42406</td>
			<td>20 % in deuterium oxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium deuteroxide</td>
			<td>Sigma-Aldrich</td>
			<td>164488</td>
			<td>30 % in deuterium oxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>Christ</td>
			<td>Alpha 1-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cold centrifuge</td>
			<td>Heraeus</td>
			<td>Megafuge 16R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>Eutech Cybernetics</td>
			<td>Cyberscan</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CDTA</td>
			<td>Sigma-Aldrich</td>
			<td>D0922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cesium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>516988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR tubes</td>
			<td>Wilmad</td>
			<td>528-PP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR stem coaxial insert</td>
			<td>Sigma-Aldrich</td>
			<td>Z278513</td>
			<td>By Wilmad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>Z255688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td>Research</td>
			<td>With tips for volumes from 0.5 to 1000 &#956;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipet-Aid</td>
			<td>Drummond</td>
			<td>XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR spectrometer</td>
			<td>Bruker</td>
			<td>AVANCE 400</td>
			<td>including probe and other accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR software</td>
			<td>Bruker</td>
			<td>TopSpin 1.3</td>
			<td>newer version available: Topspin 3.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water-soluble standard compounds</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phospholipid standard compounds</td>
			<td>Avanti Polar Lipids 
			Doosan Serdary 
			Sigma-Aldrich</td>
			<td>various 
			various 
			various</td>
			<td>&#160; 
			Source for plasmalogens, but may be 
			&#60; 70 - 80 % purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylenediphosphonate</td>
			<td>Sigma-Aldrich</td>
			<td>M9508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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?...

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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

14.6K 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

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS 1H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster. Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS 1H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS 1H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

生活磁共振波谱果蝇</em用魔角旋转

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

科研

神经科学

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

磁共振波谱的用途，用于对初级运动皮层新陈代谢双半球经颅电刺激效果的测量工具一

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

高分辨率结构磁共振人类皮质下的成像<I&gt;在体内</I&gt;与死亡

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

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 ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

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.

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

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...