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. 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. 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The authors have nothing to disclose.

Espectroscopia de RMN é um método eficiente para a medição de concentrações de compostos químicos em solução de um modo muito preciso e reprodutível. No entanto, a obtenção de dados de alta qualidade, é necessário obedecer a certas regras respeitantes à preparação e análise de amostras. Na determinação das concentrações do metabolito por espectroscopia de RMN, nem a geração ou a recepção do sinal de RMN domina o erro de quantificação, a menos que a intensidade de um sinal observado se aproxim...

Modelos murinos têm sido utilizados extensivamente em pesquisas sobre o cérebro 1. Correlações genótipo-fenótipo foram investigados em ratinhos e ratos cérebros por estudar a expressão do gene no RNA e / ou os níveis de proteína em, por um lado, e os fenótipos morfológicos, funcionais, electrofisiológicos e / ou comportamentais no outro 2-6. No entanto, para compreender completamente os mecanismos que ligam fenótipo com o genótipo, é imperativo para investigar os eventos moleculares jusante da expressão da proteína, ie. o metabolismo dos substratos bioquímicos sobre o qual as enzimas actuam 7. Esta exigência levou, ao longo dos últimos 10 a 15 anos, a um renascimento da pesquisa metabólica em muitos ramos da biologia 8,9. Enquanto estudos metabólicos clássicos têm sido muitas vezes focado em detalhes de caminhos específicos, a nova abordagem metabolômica é voltada para uma investigação abrangente do perfil metabólico global do tecido em questão.Uma conseqüência desse conceito é uma necessidade óbvia de ferramentas analíticas que minimizem viés para as vias e / ou classes de compostos metabólicos específicos. No entanto, um ensaio bioquímico clássica baseia-se numa reacção química específica de um analito específico que precisa de ser especificado antes de o ensaio é realizado. Por outro lado, as técnicas espectroscópicas de ressonância magnética (RMN) nuclear e espectrometria de massa (MS) (i) são determinadas com base em propriedades moleculares (físicas) de compostos bioquímicos, cada um dos quais dá origem a um ou vários sinais distintos num espectro detectado no decurso de uma experiência; e (ii) detectar um grande número de compostos diferentes por experiência. Assim, cada espectro contém a informação combinada de toda uma gama de metabolitos. Por esta razão, métodos espectroscópicos são ferramentas adequadas para metabolômica, já que nenhuma seleção prévia precisa ser feita em relação à natureza da substância a medir 8 </sup&gt;. Como consequência, estas técnicas adequam-se naturalmente para estudos exploratórios porque facilitam grandemente a detecção de alterações metabólicas inesperados. Embora espectroscopia de RMN e MS pode ser utilizada indiferentemente para a análise de vários metabolitos, cada método possui vantagens e desvantagens que foram recentemente revisto 10 específicas. Resumidamente, a espectroscopia de RMN pode geralmente ser realizada a partir de extractos em bruto e não necessita de separação cromatográfica de compostos de amostra antes da análise. Por outro lado, MS trabalha com gás ou cromatografia líquida (GC ou LC) separação, exceto para os recentes desenvolvimentos específicos, tais como imagens de espectrometria de massa. Em alguns casos especiais, tais como a análise de açúcares, LC separação pode tornar-se uma necessidade para a espectroscopia de RMN, como bem, porque as linhas de ressonância de diferentes açúcares sobrepõem-se significativamente em protão (1H) Os espectros de RMN. No entanto, uma espectroscopia de RMN sem chrseparação omatographic continua a ser o método de RMN mais popular, quase universalmente aplicada metabolômica. Geralmente, a preparação da amostra é mais demorada e complexa para MS do que para espectroscopia de RMN. Sérios problemas devido a efeitos de matriz são muito menos comuns em espectroscopia de RMN que em MS, onde eles podem levar a sinais consideravelmente atenuados. Metabolito quantificação pode ser conseguida com um ou outro método. No entanto, vários compostos padrão são necessários para a MS, devido às variações nos efeitos de matriz e as eficiências de ionização entre os metabolitos. Pelo contrário, apenas um padrão por exemplo é necessário para uma análise de espectroscopia de RMN, porque sob as condições de medição adequadas, o último método é intrinsecamente graças quantitativos para a resposta RMN estritamente linear pelos núcleos observados. Uma grande desvantagem de RMN é a sua sensibilidade relativamente baixa. MS, em particular, LC-MS, é mais sensível do que a RMN de várias ordens de grandeza; por esta razão, MS é para ser preferido sobre RMN para oanálise de compostos que ocorrem em concentrações muito baixas. Por outro lado, a natureza não destrutiva da experiência de RMN é uma clara vantagem sobre a esclerose múltipla; desta forma, pode ser realizada RMN repetidamente com a mesma amostra, por exemplo, para diferentes núcleos de RMN-activo, tal como um H, fósforo-31 (31 P), carbono-13 (13 C), flúor-19 (19 M) etc., como nenhum material é consumido por RMN em oposição às medições de MS. Tanto a RMN e MS pode ser utilizada em modos diferentes, cada um sendo óptima para a detecção de compostos com características químicas específicas. Por exemplo, 31 P RMN é muitas vezes mais adequados do que 1 H RMN para a análise de compostos fosforilados moderadamente concentrado, embora quase todos os metabólitos fosforilados também contêm protões. No entanto, os sinais de 1 H NMR pode ser obscurecido por sinais de 1 H RMN de outros compostos, não fosforilada, enquanto o segundoobviamente não causam sinais de fundo em 31 P espectros de RMN. Em uma situação análoga, 19 F NMR análise é para ser preferido para os compostos fluorados, por exemplo, drogas fluorados (não há sinais de fundo de metabolitos endógenos), enquanto que o caso especial, de 13 C RMN de interesse é, quase exclusivamente, se o destino do 13C precursores metabólicos marcados exógenos deve ser seguido, devido à extremamente baixa abundância natural dos isótopos de 13 C (cerca de 1%). Muitos espectrómetros de massa de funcionar no modo de iões negativos ou modo de ião positivo. Portanto, é importante saber em frente da análise se os iões a serem observados são negativamente ou positivamente carregada. Nós nos focamos em um protocolo para a análise do tecido cerebral metabolome por 1 H e 31 P espectroscopia de RMN, porque este método produz um grande número de concentrações metabólicas importantes, a baixo custo, em termos de (i) tempo necessário para a preparação de amostras dend (ii) esforço necessário para o metabolito quantificação. Todos os ensaios podem ser realizados utilizando o equipamento de laboratório padrão de química húmida e uma maior facilidade de resolução espectroscopia de RMN. Outros requisitos estão descritos na seção de protocolo a seguir.

<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.

Para obter melhor resolução em espectros de RMN metabólica do cérebro e outros extratos de tecidos, tem sido prática comum para remover ou íons máscara de metal (o mais importante: íons paramagnéticos) presentes em soluções de extractos. Isto foi conseguido através da adição de um agente quelante tal como o EDTA ou o extracto de CDTA 19, ou fazendo passar o extracto através de uma resina de troca iónica, tais como Chelex-100 20. Os resultados apresentados na Figura 1</strong...

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).

Metabolômica Análise do cérebro de um rato de alta resolução Espectroscopia de Ressonância Magnética Nuclear de Extratos de Tecidos

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

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Neuroscience

14.6K visualizações.  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).

Vídeo: 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.

Espectroscopia de Ressonância Magnética de viver Drosophila melanogaster Usando Angle Spinning Magia

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

Neurociência

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.

De alta resolução funcionais Métodos ressonância magnética para Midbrain Humanos

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.

Micron escala Resolução óptica Tomografia de todo cérebro do rato com confocal Luz Folha de Microscopia

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.

O Uso de Espectroscopia de Ressonância Magnética como ferramenta para a Medição do Bi-hemisféricas transcranianas Estimulação Elétrica Efeitos sobre Motor Primário Cortex Metabolismo

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.

Alta resolução<em&gt; In Vivo</em&gt; Manual Protocolo Segmentação para hipocampo humano subcampos Utilizando Ressonância Magnética 3T

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.

De alta resolução de ressonância magnética estrutural do Subcórtex Humano<I&gt; In Vivo</I&gt; E 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 ...

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.

Funcionais de ressonância magnética espectroscopia em 7 T no córtex de barril de rato durante a ativação dos whiskers

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.

Imagem latente 3D de alta resolução da infecção do vírus da raiva no tecido solvente-cancelado do cérebro

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.

Preparação do tecido cerebral de rato recém-nascido para Análise morfométrica ultraestrutural da distribuição de vesículas sinápticas em terminais nervosos

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.

Um método de limpeza de tecido hidrofóbico para tecido cerebral de rato

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