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

<ol><li>Manger, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+21st+century+neuroscience+too+focused+on+the+rat%2Fmouse+model+of+brain+function+and+dysfunction%5BTitle%5D)+AND+%22Front+Neuroanat%22%5BJournal%5D)">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/pubmed?term=((Optimizing+the+phenotyping+of+rodent+ASD+models+enrichment+analysis+of+mouse+and+human+neurobiological+phenotypes+associated+with+high-risk+autism+genes+identifies+morphological%2C+electrophysiological%2C+neurological%2C+and+behavioral+features%5BTitle%5D)+AND+%22Mol+Autism%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPapaioannou%2C+V+%22Mouse+Phenotypes+A+Handbook+of+Mutation+Analysis%22">Mouse Phenotypes A Handbook of Mutation Analysis</a>. , Cold Spring Harbor Laboratory Press. Woodbury. (2004).</li>
<li>Yu, F. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced+sodium+current+in+GABAergic+interneurons+in+a+mouse+model+of+severe+myoclonic+epilepsy+in+infancy%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">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/pubmed?term=((A+polymorphism+in+the+EAAT2+promoter+is+associated+with+higher+glutamate+concentrations+and+higher+frequency+of+progressing+stroke%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCrusio%2C+WE+%22Behavioral+Genetics+of+the+Mouse+Genetics+of+Behavioral+Phenotypes%22">Behavioral Genetics of the Mouse Genetics of Behavioral Phenotypes</a>. Vol. 1, Cambridge University Press. New York. (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/pubmed?term=((Metabolic+fingerprints+of+altered+brain+growth%2C+osmoregulation+and+neurotransmission+in+a+Rett+syndrome+model%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLutz%2C+NW+%22Methodologies+for+Metabolomics%22">Methodologies for Metabolomics</a>. , Cambridge University Press. New York. (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/pubmed?term=((Metabolomics+in+drug+target+discovery%5BTitle%5D)+AND+%22Cold+Spring+Harb+Symp+Quant+Biol%22%5BJournal%5D)">Metabolomics in drug target discovery.</a> Cold Spring Harb Symp Quant Biol. 76, 235-246 (2011).</li>
<li>Wishart, D. S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWishart%2C+DS+%22Methodologies+for+Metabolomics%22">Methodologies for Metabolomics</a>. Wevers, R. A., Lutz, N. W., Sweedler, J. V., et al. , Cambridge University Press. New York. (2013).</li>
<li>Ponten, U., Ratcheson, R. A., Salford, L. G., Siesjo, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+freezing+conditions+for+cerebral+metabolites+in+rats%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">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/pubmed?term=((Toward+dynamic+isotopomer+analysis+in+the+rat+brain+in+vivo+automatic+quantitation+of+13C+NMR+spectra+using+LCModel%5BTitle%5D)+AND+%22NMR+Biomed%22%5BJournal%5D)">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/pubmed?term=((Multiparametric+optimization+of+%2831%29P+NMR+spectroscopic+analysis+of+phospholipids+in+crude+tissue+extracts+2+Line+width+and+spectral+resolution%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">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/pubmed?term=((Multiparametric+optimization+of+%2831%29P+NMR+spectroscopic+analysis+of+phospholipids+in+crude+tissue+extracts.+1.+Chemical+shift+and+signal+separation%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">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. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLutz%2C+NW+%22Methodologies+for+Metabolomics%22">Methodologies for Metabolomics</a>. Lutz, N. W., Sweedler, J. V., Wevers, R. A. , Cambridge University Press. New York. (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/pubmed?term=((Cerebral+biochemical+pathways+in+experimental+autoimmune+encephalomyelitis+and+adjuvant+arthritis+a+comparative+metabolomic+study%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Principles+of+multiparametric+optimization+for+phospholipidomics+by+31P+NMR+spectroscopy%5BTitle%5D)+AND+%22Biophys+Rev%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPearson%2C+GA+%22Shimming+an+NMR+magnet%22">Shimming an NMR magnet</a>. , University of Iowa. Iowa City. (1991).</li>
<li>Lane, A. N., Fan, T. W. M., Higashi, R. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLane%2C+AN+%22Biophysical+tools+for+biologists%22">Biophysical tools for biologists</a>. Correia, J. J., Detrich, H. W. Vol. 1, In vitro techniques, Academic Press. Waltham. (2008).</li>
<li>Peeling, J., Wong, D., Sutherland, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+magnetic+resonance+study+of+regional+metabolism+after+forebrain+ischemia+in+rats%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Nuclear magnetic resonance study of regional metabolism after forebrain ischemia in rats.</a> Stroke. 20, 633-640 (1989).</li>
</ol>

The authors have nothing to disclose.

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

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

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

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 ...

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

Research

JoVE Journal

Neuroscience

14.8K Views.  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).

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

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

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

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

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

Medicine

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

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

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

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

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

Immunology and Infection

Survivable Stereotaxic Surgery in Rodents 

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

Survivable Stereotaxic Surgery in Rodents

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

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

Biology

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

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

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

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

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

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

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

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

Biochemistry

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Studying Metabolic Brain Connectivity Using 2-Deoxy-2-[18F]Fluoro-D-Glucose Dynamic Positron Emission Tomography at the Single-subject Level

To this day, metabolic brain connectivity is mostly studied on a group level through the acquisition of static positron emission tomography (PET) data of multiple subjects. Our research groups are currently studying changes in metabolic connectivity across multiple time points following an intracerebral hemorrhage on an intrasubject level in rats. To investigate intrasubject metabolic brain connectivity, temporal information of the tracer uptake in different brain regions is required, which can be achieved through dynamic PET. In this publication, we give a detailed description of our data acquisition and analysis protocol.
Dynamic PET data of the rat brain are acquired on a dedicated preclinical PET system using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) as tracer. The tracer is injected intravenously as a bolus at the start of the PET scan. During the 60 min acquisition, animals are sedated with medetomidine.
After acquisition, the PET data are reconstructed into thirty 2 min time frames using an iterative reconstruction algorithm (Maximum-Likelihood Expectation-Maximization). A parcellated atlas consisting of multiple volumes of interest (VOIs) is used to extract time-activity curves of each VOI, which are then used to calculate the Pearson correlation coefficient between each pair of VOIs.
This dynamic PET protocol enables the assessment of metabolic connectivity differences between two single scans, rather than between groups of scans. This approach allows for the study of changes in metabolic connectivity within a single subject across different time points, or for the comparison of an individual&#39;s metabolic connectivity to a normal database. Such comparisons could be useful for tracking disease progression or aiding in the diagnosis of neurological disorders characterized by disrupted communication between brain regions, such as epilepsy or dementia.

The acquisition of dynamic positron emission tomography (PET) data and reconstruction into time frames allows for metabolic brain connectivity analysis at the single-subject level. We describe a method to acquire [18F]FDG dynamic PET data of the rat brain and obtain a connectivity matrix through the extraction of time-activity curves of volumes of interest.

To this day, metabolic brain connectivity is mostly studied on a group level through the acquisition of static positron emission tomography (PET) data of ...

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

Summary

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

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

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

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

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

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

Studying Metabolic Brain Connectivity Using 2-Deoxy-2-[¹⁸F]Fluoro-D-Glucose Dynamic Positron Emission Tomography at the Single-subject Level