Ce travail a été soutenu en partie par un National Institutes of Health (NIH) aux subventions AI063433 Laurence G. Rahme, un des Instituts Institut national de la santé (NIH) Centre Grant (P50GM021700) à Ronald G. Tompkins (A. Aria Tzika, directeur de le cœur RMN), et l&#39;Hôpital des Shriners pour enfants de subvention de recherche (# 8893) à A. Aria Tzika. Nous remercions Dionyssios Mintzopoulos Ph.D. pour l&#39;assistance dans les phases initiales du développement de ce protocole et Ovidiu C. Andronesi Ph.D. pour l&#39;assistance à la séquence d&#39;impulsions TOBSY.

Partie 1: Préparation de la drosophile pour les mesures de HRMAS <ol><li> Utilisation de la norme flylab procédures 1, collecter nouvellement eclosing mouches pendant 3 jours et de les transférer à voler la nourriture volée flacons contenant frais. Incuber les mouches recueillies pendant 5 jours afin que les mouches deviennent 5-8 jours anciens, juste avant l&#39;expérience. Un seul sexe (généralement des hommes) est utilisé pour des expériences. </li><li> Utilisez saine, intacte mouches de type sauvage à comparer avec traitées par exemple traumatisés ou mouches génétiquement modifiées 1. </li><li> Placez mouches simples dans des tubes 2ml contenant un morceau (~ 0,2 ml) de voler la nourriture 24 heures avant l&#39;expérience. Ces tubes portent un trou dans la coupe tube en utilisant une aiguille d&#39;insuline flamme chauffé. </li><li> Peser les tubes immédiatement avant et après l&#39;insertion volée en utilisant une balance de haute précision et à définir le poids de chaque volée s en soustrayant la première valeur de ce dernier. Une seule mouche mâle pèse généralement de 0,7 à 1 mg. </li></ol> Partie 2: Préparation du rotor HRMAS. <ol><li> Placez un seul tube sur la glace pendant moins d&#39;une minute pour anesthésier l&#39;intérieur voler. </li><li> Lay à la volée sur une couche de papier d&#39;aluminium placé sur la glace et pousser à la volée en douceur dans l&#39;espace semi-sphérique creux de l&#39;insert du rotor RMN en utilisant une brosse douce pour assurer une insertion complète volée tout en évitant les blessures voler. </li><li> Placer l&#39;insert dans l&#39;oxyde de zirconium (ZrO2) tube de rotor (4 mm de diamètre, 50 pi) pour localiser les mouches entre l&#39;insert rotor et le fond du rotor (voir figure 1). </li><li> Fermer l&#39;insert avec une vis et le couvrir avec du parafilm (voir figure 1) pour empêcher le contact entre la mouche et la solution de TSP standard (TSP: triméthylsilyl-propionique-2 ,2,3,3-d4 acide, Mw = 172, δ = 0,00 ppm, 50 mM dans l&#39;eau deutérée-D 2 O, qui fonctionnent comme une référence tant pour le déplacement chimique de résonance et de quantification). </li><li> Ajouter 8 pl de TSP solution standard sur le dessus de l&#39;insert rotor. (Voir figure 1). </li><li> Sécurisé et serrer le tout mis en place dans les rotors avec un plafond haut (voir figure 1). </li></ol> Partie 3: Acquisition de données HRMAS <ol><li> Introduire le rotor dans la sonde HRMAS et le positionner à l&#39;angle magique 54,7 ° (voir figure 1). </li><li> Réglez la température à 4 ° C par une unité de BTO-2000 en combinaison avec l&#39;unité pneumatique MAS. Les mouches sont conservés à 4 ° C alors que dans le spectromètre pour maintenir l&#39;anesthésie. </li><li> Réglez le HRMAS 1 H MRS vitesse de rotation à 2 kHz MAS. </li><li> Stabiliser la fréquence de rotation MAS à 2,0 ± 0,001 kHz par un régulateur de vitesse MAS (fréquence de rotation). </li><li> Pour la préparation de l&#39;aimant: hauteur et faire correspondre la bobine pour des performances optimales et la cale de l&#39;aimant pour une qualité optimale des spectres. </li><li> Acquérir une spectres unidimensionnels H en utilisant du rotor synchronisée Carr-Purcell-Meiboom-Gill (CPMG) en écho de spin séquence d&#39;impulsions 2, [90 ° - (τ-180 ° - τ) n-acquisition], qui fonctionne comme un filtre pour T 2 retirez l&#39;élargissement spectral RMN 1H seule volée spectres acquis une dimension de données sur tous les échantillons. Synchroniser le retard entre les impulsions (τ = 500μs) à la fréquence de rotation MAS (2 kHz). Réglez le nombre de transitoires à 256 avec 32 768 (32k) points de données. Le temps d&#39;acquisition 9 min. </li><li> Acquérir en deux dimensions (2D) 1 H-1 H RMN HRMAS seule volée spectres sur tous les échantillons en utilisant une séquence d&#39;impulsions adiabatiques avec TOBSY 3. Les paramètres d&#39;acquisition sont les suivants: la dimension 2k points de données directes (11 ppm largeur spectrale), 1 s d&#39;eau pré-saturation pendant le délai de relaxation, 8 balayages par incrément de 128 incréments, le temps de 2 s répétition total, 45 ms temps de mélange, et un total d&#39;acquisition temps de 29 min. </li></ol> Partie 4: Traitement des données / Analyse <ol><li> Analyser les MRspectra de spécimens à l&#39;aide du logiciel MestReC (Mestrelab recherche, www.mestrec.com) </li><li> Utilisez une ligne d&#39;élargissement de la fonction d&#39;apodisation de 0,5 Hz et s&#39;appliquent à tous une HRMAS FID H avant la transformation de Fourier. </li><li> Référence des spectres MR à l&#39;égard de TSP à δ = 0,0 ppm (standard externe). </li><li> Phase spectres manuellement et appliquer un estimateur de base Whittaker pour soustraire les composantes de l&#39;éventail de base des calculs de superficie avant de pointe. </li><li> Estimation des domaines de pointe utilisant des logiciels MestReC. Échelle hauteurs de pics par rapport au TSP pour chaque spectre acquis (TSP pic de hauteur = 1). Utilisez les tests t (bilatéral, p &lt;0,05) pour comparer l&#39;intérieur des groupes d&#39;entre-groupes de ratios. </li><li> Paramètres 2D processus TOBSY sont: Qsine = fonction fenêtre 2 dans les deux dimensions, avec des points de FT 2k dans la dimension directe et zéro de remplissage pour 1k dans la deuxième dimension, la correction de phase dans les deux dimensions et la correction de base dans la deuxième dimension. </li><li> Produire les spectres 2D à l&#39;aide du programme Sparky (TD Goddard et DG Kneller, Sparky 3, USCF, <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/</a>Accueil / sparky /) </li></ol> Partie 5: spectres représentatifs de Live mouches Drosophila melanogaster Les procédures décrites ci-dessus permettent de collecter des spectres reproductibles à partir de mouches vivent Drosophila melanogaster. Les figures 2 et 3 montrent représentant de M. spectres acquis au type sauvage (WT) Oregon-R mouches. La figure 2 présente 1D 1 H HRMAS CPMG spectres. Principaux composants lipidiques [C 3 H (0,89 ppm), (C H 2) n (ppm 1,33), C H 2 C-CO (1.58ppm), C H 2 C = C (2,02 ppm), C H 2 C = O (2,24 ppm), C = C H H (5,33 ppm)], du glycérol (1,3-C H 4,10 ppm et 4,30 ppm; 2-CH 2 5,24 ppm), et des métabolites de petite taille: β-alanine (β-Ala , 2,57 ppm), acétate (Ac, 1,97 ppm), phosphocholine (PC, 3,22 ppm) et phophoetanolamine (PE, 3.23ppm) ont été détectées et attribuées conformément aux rapports antérieurs 4, 5. Signaux à 2,02 ppm ont été assignés à protons du méthylène du C H2-CH = CH fraction de mono-acides gras insaturés (ie palmitoléique). Les acides insaturés ont été identifiés par un signal à 5,33 ppm produites par les protons de l&#39;-CH = CH-fraction. Métabolites petits qui ne pouvaient pas être cédés ou n&#39;étaient pas visibles en utilisant le spectre 1D ont été détectés en utilisant 2D 1 H-1 H TOBSY HRMAS (voir figure 3). <img src="/files/ftp_upload/1710/1710fig1.jpg" alt="Figure 1" /> Figure 1. Expérimental mis en place in vivo HRMAS 1H SRM pour l&#39;enquête sur la drosophile vivons à 14,1 T. externe standard de triméthylsilyl-2-propionique ,2,3,3-d4 acide dans l&#39;eau deutérée (TSP / D 2 O). Dans le carré: la position du rotor à l&#39;angle magique en HRMAS sonde. <img src="/files/ftp_upload/1710/1710fig2.jpg" alt="Figure 2" /> Figure 2. En 1D HRMAS spectres 1 H CPMG vivo d&#39;une mouche Drosophila melanogaster vivre en poids. Composants lipidiques: C H 3 (0,89 ppm), (C H 2) n (ppm 1,33), C H 2 C-CO (1.58ppm), acétate (Ac), C H 2 C = C (2,02 ppm), C H 2 C = O (2,24 ppm), β-alanine (β-Ala), phosphocholine (PC) et phophoetanolamine (PE), Glycérol (1,3-C H 4,10, 4,30 ppm; 2-C H 2 5,22 ppm) , C = C H H (5,33 ppm). <img src="/files/ftp_upload/1710/1710fig3.jpg" alt="Figure 3" /> Figure 3. In vivo 2D 1H-1H TOBSY HRMAS spectre d&#39;une mouche Drosophila melanogaster direct au poids métabolites T. 14.1 Petites et composants lipidiques ont été identifiés. Métabolites: alanine (Ala), β-alanine (β-Ala), l&#39;arginine (Arg), de la glutamine (Gin), le glutamate (Glu), phosphocholine PC (PC), phophoetanolamine (PE), taurine (Tau), α-glucose (α-Glc) et du glycérol. Composants Lipides: C 3 H (0,89 ppm), (C H 2) n (ppm 1,33), C H 2 C-CO (1,58 ppm), C H 2 C = C (2,02 ppm), C H 2 C = O (2,24 ppm), C = C H H (5,33 ppm).

<ol>
	<li>Apidianakis, Y., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+host+for+studying+Pseudomonas+aeruginosa+infection.">Drosophila melanogaster as a model host for studying Pseudomonas aeruginosa infection.</a> Nat Protoc. 4, 1285-1294 (2009).</li><li>Meiboom, S., Gill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+spin-echo+method+for+measuring+nuclear+relaxation+time.">Modified spin-echo method for measuring nuclear relaxation time.</a> Rev Sci Instrum. 29, 688-691 (1958).</li><li>Andronesi, O. C., Mintzopoulos, D., Struppe, J., Black, P. M., Tzika, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-state+NMR+adiabatic+TOBSY+sequences+provide+enhanced+sensitivity+for+multidimensional+high-resolution+magic-angle-spinning+1H+MR+spectroscopy.">Solid-state NMR adiabatic TOBSY sequences provide enhanced sensitivity for multidimensional high-resolution magic-angle-spinning 1H MR spectroscopy.</a> J Magn Reson. 193, 251-258 (2008).</li><li>Astrakas, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton+NMR+spectroscopy+shows+lipids+accumulate+in+skeletal+muscle+in+response+to+burn+trauma-induced+apoptosis.">Proton NMR spectroscopy shows lipids accumulate in skeletal muscle in response to burn trauma-induced apoptosis.</a> Faseb J. 19, 1431-1440 (2005).</li><li>Fan, T. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+profiling+by+one-+and+two+dimensional+NMR+analysis+of+complex+mixtures.">Metabolite profiling by one- and two dimensional NMR analysis of complex mixtures.</a> Prog Nuc Magn Reson Spec. 28, 161-219 (1996).</li><li>Null, B., Liu, C. W., Hedehus, M., Conolly, S., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution,+in+vivo+magnetic+resonance+imaging+of+Drosophila+at+18.8+Tesla.">High-resolution, in vivo magnetic resonance imaging of Drosophila at 18.8 Tesla.</a> PLoS One. 3, e2817-e2817 (2008).</li><li>Zektzer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+signal+to+noise+in+high-resolution+magic+angle+spinning+total+correlation+spectroscopy+studies+of+prostate+tissues+using+rotor-synchronized+adiabatic+pulses.">Improved signal to noise in high-resolution magic angle spinning total correlation spectroscopy studies of prostate tissues using rotor-synchronized adiabatic pulses.</a> Magn Reson Med. 53, 41-48 (2005).</li></ol>

Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements stipulés par le Massachusetts General Hospital Animal Research Comité Institutional Review Board.

À l&#39;exception du récent rapport de la faisabilité de l&#39;IRM in vivo dans les mouches des fruits 6, études in vivo chez la drosophile MRS n&#39;ont pas encore été signalés. Dans le présent protocole, nous décrivons la mise en œuvre d&#39;un roman in vivo HRMAS 1 H-MRS approche pour la détection de molécules biologiquement importantes. Plus précisément, nous avons détecté des lipides et des métabolites de petites mouches drosoph...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Deuterium oxide</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>7789-20-0</td>
<td>&nbsp;</td>
<tr>
<td>3-(trimethylsilyl)propionic-2,2,3,3-d4 acid</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>24493-21-8</td>
<td>&nbsp;</td>
<tr>
<td>agar, sucrose, yeast, cornmeal</td>
<td>Food</td>
<td>Genesee Scientific Corporation</td>
<td>&nbsp;</td>
<td><a href="http://www.flystuff.com/">http://www.flystuff.com/</a></td>
</tr>
<tr>
<td>Oregon RS or Canton-S flies</td>
<td>Adult fly lines</td>
<td>Bloomington Stock Center</td>
<td>&nbsp;</td>
<td><a href="http://flystocks.bio.indiana.edu/">http://flystocks.bio.indiana.edu/</a></td>
</tr>
<tr>
<td>Paintbrush</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>(size 0)</td>
</tr>
<tr>
<td>2ml tubes</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>K749521-1590</td>
<td>&nbsp;</td>
<tr>
<td>Fly incubators</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>high humidity capacity (60-75%), adjustable temperature, and a 12 h:12 h light: dark cycle.</td>
</tr>
<tr>
<td>Bruker Bio-Spin Avance NMR spectrometer (600.13 MHz) 4mm triple resonance (1H, 13C, 2H) HRMAS probe </td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BTO-2000 unit in combination with a MAS pneumatic unit</td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>4mm zirconium oxide rotor (capacity 50 ul)</td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>B3829 (Bruker store)</td>
<td>&nbsp;</td>
<tr>
<td>MestReC (Mestrelab Research) </td>
<td>Software</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1D NMR spectra analysis <a href="http://mestrelab.com/">http://mestrelab.com/</a></td>
</tr>
<tr>
<td>SPARKY 3, USCF</td>
<td>Software</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2D NMR spectra
analysis
 <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/home/sparky/</a></td>
</tr>		</tbody>
		</table>
		</div>

magnetic resonance spectroscopy of live drosophila melanogaster using magic angle spinning

High-Resolution Magic Angle Spinning (HRMAS) protons spectroscopie par résonance magnétique (<sup&gt; 1</sup&gt; H-MRS) est un roman technique non-destructive qui améliore spectrale ligne largeurs et permet à haute résolution des spectres d&#39;être obtenu à partir d&#39;extraits, des cellules intactes, des cultures de cellules, tissus et plus important encore intactes pour étudier les relations entre les métabolites et les processus cellulaires.<em&gt; In vivo</em&gt; HRMAS<sup&gt; 1</sup&gt; H-MRS études doivent encore être rapportés dans la mouche à fruit direct<em&gt; Drosophila melanogaster.</em&gt;<em&gt; Drosophile,</em&gt; Comme un simple organisme génétique, qui permet aux fonctions multiples et diverses biologiques évolutivement conservés voies de signalisation qui sera examiné à l&#39;échelle de l&#39;organisme entier et il est un modèle utile pour étudier la génétique et la physiologie. À cette fin, nous avons développé et mis en œuvre une<em&gt; In vivo</em&gt; HRMAS<sup&gt; 1</sup&gt; H-MRS méthode pour étudier en direct<em&gt; Drosophile</em&gt; À 14,1 T. Ici, nous présentons une HRMAS<sup&gt; 1</sup&gt; H-MRS protocole pour la caractérisation moléculaire des<em&gt; Drosophile</em&gt; Avec un spectromètre conventionnel MR équipé d&#39;une sonde HRMAS. Cette technique est un roman,<em&gt; In vivo,</em&gt; Non destructif<em&gt; Drosophile</em&gt; Approche de mesure métabolite, qui permet l&#39;identification de biomarqueurs liés aux maladies et peut donc contribuer au développement de thérapeutiques nouvelles.

Watch this Scientific Journal Video about Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning at JoVE.com

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

Cette technique permet l&#39;utilisation de l&#39;angle magique à haute résolution filer spectroscopie par résonance magnétique protonique (HRMAS 1H-MRS) pour la caractérisation moléculaire de vivre<em&gt; Drosophila melanogaster</em&gt; Avec un spectromètre de 14,1 teslas conventionnelle équipée d&#39;une sonde HRMAS.

Spectroscopie par résonance magnétique du direct Drosophila melanogaster En utilisant Magic Angle Spinning

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

magnetic-resonance-spectroscopy-live-drosophila-melanogaster-using

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Neuroscience

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

12.4K vues.  Massachusetts General Hospital, Harvard Medical School. Cette technique permet l'utilisation de l'angle magique à haute résolution filer spectroscopie par résonance magnétique protonique (HRMAS 1H-MRS) pour la caractérisation moléculaire de vivre Drosophila melanogaster Avec un spectromètre de 14,1 teslas conventionnelle équipée d'une sonde HRMAS.

Vidéo: Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-Mouse Imaging neuroanatomiques par résonance magnétique

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Recherche

Neurosciences

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Stimulation optogenetic de comportement de fuite en<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Détermination de l&#39;activité locomotrice spontanée,<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

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.

L&#39;utilisation de la spectroscopie par résonance magnétique, un outil pour la mesure de la Bi-hémisphériques transcrânienne électriques effets de stimulation sur le moteur primaire Cortex métabolisme

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

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Imagerie en direct de<em&gt; Drosophila</em&gt; larves Neuroblastes

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

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.

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).

Analyse métabolomique de cerveau de rat par la Résolution nucléaire Haute spectroscopie par résonance magnétique d&#39;extraits de tissus

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

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

Conditions affectant espace social<em&gt; Drosophila melanogaster</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

Haute résolution La quantification du comportement Odor-guidée dans<em&gt; Drosophila melanogaster</em&gt; Utilisation de la<em&gt; Flywalk</em&gt; Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Foraging Protocole Chemin de longueur pour<em&gt; Drosophila melanogaster</em&gt; larves

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Analyse métabolique des larves de Drosophila melanogaster et un cerveau adulte

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...