Name: metabolomic analysis of rat brain by high resolution nuclear magnetic resonance spectroscopy of tissue extracts
Uploaded: 2014-09-21T14:00:00
Description: Watch this Scientific Journal Video about Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts at JoVE.com

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

NOTE: ANIMAL ETHICS STATEMENT 
Animal studies on rats followed the guidelines valid in France, and were approved by the local Ethics Committee (#40.04, University of Aix-Marseille Medical School, Marseille, France).
1. Harvesting and Freezing Rat Brain
<ol>
	<li>Prepare items required: liquid nitrogen (N2liq.) in Dewar that is large enough to keep a freezing clamp (at least 2-3&#160;L volume); anesthetic (e.g., isoflurane, or ketamine/xylazine); anesthesia chamber; s...

<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>

NMR spectroscopy is an efficient method for measuring concentrations of chemical compounds in solution in a very reproducible and accurate manner. However, to obtain high-quality data it is necessary to adhere to certain rules concerning sample preparation and analysis. In the determination of metabolite concentrations by NMR spectroscopy, neither the generation nor the reception of the NMR signal dominates the quantitation error, unless the intensity of an observed signal approaches the detection threshold (particularly...

Murine models have been utilized extensively in brain research1. Genotype-phenotype correlations have been investigated in mouse and rat brains by studying gene expression at the RNA and/or protein levels on the one hand, and morphological, functional, electrophysiological and/or behavioral phenotypes on the other2-6. However, to completely understand the mechanisms linking phenotype to genotype, it is imperative to investigate the molecular events downstream of protein expression, i.e. the metabolism of the biochemical substrates upon which enzymes act7. This requirement led, over the past 10 to 15 years, to a renaissance of metabolic research in many branches of biology8,9. While classical metabolic studies have often been focused on details of specific pathways, the new metabolomic approach is geared towards an all-encompassing investigation of the global metabolic profile of the tissue under consideration. One consequence of this concept is an obvious need for analytical tools that minimize bias towards specific metabolic pathways and/or classes of compounds. However, a classical biochemical assay is based on a particular chemical reaction of a specific analyte that needs to be specified before the assay is performed. By contrast, spectroscopic techniques such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) (i) are based on particular molecular (physical) properties of biochemical compounds, each of which gives rise to one or several distinct signals in a spectrum detected in the course of one experiment; and (ii) detect a large number of different compounds per experiment.
Thus, each spectrum contains the combined information of a whole range of metabolites. For this reason, spectroscopic methods are adequate tools for metabolomics, as no prior selection needs to be made regarding the nature of the analyte to be measured8. As a consequence, these techniques naturally lend themselves to exploratory studies because they greatly facilitate the detection of unexpected metabolic changes.
Although NMR spectroscopy and MS can be used interchangeably for the analysis of many metabolites, each method possesses specific advantages and disadvantages that have recently been reviewed10. Briefly, NMR spectroscopy can usually be performed from crude extracts and does not require chromatographic separation of sample compounds before analysis. By contrast, MS works with gas or liquid chromatography (GC or LC) separation, except for particular recent developments such as mass spectrometry imaging. In a few special cases such as the analysis of sugars, LC separation may become a necessity for NMR spectroscopy as well, because the resonance lines of different sugars overlap significantly in proton (1H) NMR spectra. Nevertheless, 1H NMR spectroscopy without chromatographic separation remains the most popular, almost universally applied metabolomic NMR method. Generally, sample preparation is more time-consuming and complex for MS than it is for NMR spectroscopy. Serious problems due to matrix effects are much less common in NMR spectroscopy than in MS where they may lead to considerably attenuated signals. Metabolite quantitation can be achieved with either method. However, multiple standard compounds are needed for MS due to variations in matrix effects and ionization efficiencies between metabolites. By contrast, only one standard per sample is needed for an NMR spectroscopic analysis because under appropriate measuring conditions, the latter method is intrinsically quantitative thanks to the strictly linear NMR response by the observed nuclei. A major drawback of NMR is its relatively low sensitivity. MS, in particular LC-MS, is more sensitive than NMR by several orders of magnitude; for this reason, MS is to be preferred over NMR for the analysis of compounds occurring at very low concentrations. On the other hand, the nondestructive nature of the NMR experiment is a clear advantage over MS; in this way, NMR can be performed repeatedly on the same sample, e.g., for different NMR-active nuclei such as 1H, phosphorus-31 (31P), carbon-13 (13C), fluorine-19 (19F) etc., as no material is consumed by NMR as opposed to MS measurements.
Both NMR and MS can be employed in different modes, each one being optimal for the detection of compounds with particular chemical characteristics. For instance, 31P NMR is often better suited than 1H NMR for the analysis of moderately concentrated phosphorylated compounds, although almost all phosphorylated metabolites also contain protons. However, their 1H NMR signals may be obscured by 1H NMR signals from other, non-phosphorylated compounds, while the latter obviously do not cause background signals in 31P NMR spectra. In an analog situation, 19F NMR analysis is to be preferred for fluorinated compounds, e.g., fluorinated drugs (no background signals from endogenous metabolites), while the special case of 13C NMR is of interest almost exclusively if the fate of 13C-labeled exogenous metabolic precursors needs to be followed, due to the extremely low natural abundance of the 13C isotope (ca. 1%). Many mass spectrometers work in either negative ion mode or positive ion mode. Therefore, it is important to know ahead of the analysis whether the ions to be observed are negatively or positively charged. We focus here on a protocol for the analysis of the brain tissue metabolome by 1H and 31P NMR spectroscopy because this method yields a large number of important metabolite concentrations at low cost in terms of (i) time needed for sample preparation and (ii) effort required for metabolite quantitation. All experiments can be performed using the equipment of a standard wet-chemistry laboratory and a high-resolution NMR spectroscopy facility. Further requirements are described in the Protocol section below.

<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.

To obtain best resolution in metabolic NMR spectra of brain and other tissue extracts, it has long been common practice to remove or mask metal ions (most importantly: paramagnetic ions) present in extract solutions. This has been achieved either by adding a chelating agent such as EDTA or CDTA to the extract19, or by passing the extract through an ion exchange resin such as Chelex-10020. The results presented in Figure 1 demonstrate that this step is not necessary for 1H...

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).

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

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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