Diese Arbeit wurde teilweise durch einen National Institutes of Health (NIH) gewähren AI063433 zu Laurence G. Rahme, ein National Institute Institutes of Health (NIH) Center Grant (P50GM021700) Ronald G. Tompkins (A. Aria Tzika, Direktor der unterstützten der NMR-Kern) und eine Shriner Hospital for Children Forschungsstipendium (# 8893) an A. Aria Tzika. Wir danken Dionyssios Mintzopoulos Ph.D. für die Unterstützung in den ersten Phasen der Entwicklung dieses Protokolls und Ovidiu C. Andronesi Ph.D. für die Unterstützung bei der TOBSY Pulssequenz.

Teil 1: Vorbereiten Drosophila für HR-MAS-Messungen <ol><li> Verwendung von Standard flylab Verfahren 1, sammeln neu schlüpfende Fliegen für 3 Tage und sie auf Flaschen mit frischen Lebensmitteln zu fliegen fliegen. Inkubieren Sie die gesammelten Fliegen für 5 Tage, so dass die Fliegen wird 5-8 Tage alt just vor dem Experiment. Nur ein Geschlecht (meist Männer) ist für Experimente verwendet. </li><li> Verwenden Sie gesunden, fliegt intakten Wildtyp mit behandelten zB traumatisierte oder genetisch veränderte fliegt 1 zu vergleichen. </li><li> Legen Sie einzelne fliegt in 2ml Röhrchen mit einem Stück (~ 0,2 ml) von essen fliegen 24 Stunden vor dem Experiment. Diese Rohre tragen ein Loch in das Rohr Tasse erfolgt über eine Flamme erwärmt Insulin Nadel. </li><li> Wiegen Sie die Rohre unmittelbar vor und nach der Fliege Einsetzen mit einer hohen Präzision Balance definieren und jeder fliegen s Gewicht durch Subtraktion der erste Wert aus der zweiten. Ein einzelnes Männchen fliegen wiegt in der Regel 0,7-1 mg. </li></ol> Teil 2: HR-MAS-Rotor Vorbereitung. <ol><li> Legen Sie eine einzelne Röhre auf Eis weniger als einer Minute auf die Fliege im Inneren zu betäuben. </li><li> Lay the fly auf einer Schicht aus Aluminiumfolie auf Eis gelegt, und drücken Sie die Fliege sanft in die halbkugelförmigen Hohlraum des NMR Rotoreinsatz mit einer weichen Bürste in Anspruch zu fliegen Einstecken zu gewährleisten bei gleichzeitiger Vermeidung fliegen Verletzungen. </li><li> Platzieren Sie den Einsatz in der Zirkonoxid (ZrO 2) Rotor Rohr (4 mm Durchmesser, 50 ul), um die Fliegen zwischen den Rotor einlegen und den Boden des Rotors (siehe Abbildung 1) zu finden. </li><li> Schließen Sie den Einsatz mit einer Schraube und bedecken Sie es mit Parafilm (siehe Abbildung 1), um den Kontakt zwischen dem Fliegen und der TSP-Standardlösung (TSP zu verhindern: trimethylsilyl-Propionsäure-2 ,2,3,3-d4-säure, Mw = 172, δ = 0,00 ppm, 50 mM in deuteriertem Wasser-D 2 O, diese Funktion als Referenz für beide Resonanz chemische Verschiebung und Quantifizierung). </li><li> Add 8 ul TSP-Standardlösung auf der Oberseite des Rotors einzufügen. (Siehe Abbildung 1). </li><li> Sichere und ziehen das ganze bis in die Rotoren mit einer Kappe (siehe Abbildung 1). </li></ol> Teil 3: HR-MAS-Datenerfassung <ol><li> Einführung des Rotors in die HR-MAS-Sonde und positionieren Sie es im magischen Winkel 54,7 ° (siehe Abbildung 1). </li><li> Stellen Sie die Temperatur um 4 ° C durch einen BTO-2000-Einheit in Kombination mit dem MAS Pneumatikeinheit. Fliegen sind bei 4 ° C, während in das Spektrometer Anästhesie beizubehalten gehalten. </li><li> Stellen Sie die HR-MAS-1 H MRS Spinngeschwindigkeit bei 2 kHz MAS. </li><li> Stabilisieren Sie die MAS Drehfrequenz bei 2,0 ± 0,001 kHz mit einem MAS Drehzahlregler (Drehfrequenz). </li><li> Für Magnet Zubereitung: Melodie und passen die Spule für optimale Leistung und Shim der Magnet für die optimale Qualität der Spektren. </li><li> Erwerben Sie eindimensionalen 1 H-Spektren mit Rotor synchronisiert Carr-Purcell-Meiboom-Gill (CPMG) Spin-Echo-Pulssequenz 2, [90 ° - (τ-180 ° - τ) n-Übernahme], die als T 2 Filter werden Entfernen Sie die spektrale Verbreiterung 1 H-NMR-Spektren single-fly zu erwerbenden eindimensionale Daten zu allen Proben. Synchronisieren Sie die inter-Puls-Verzögerung (τ = 500μs), um die MAS Drehfrequenz (2 kHz). Legen Sie die Anzahl von Transienten bei 256 mit 32.768 (32k) Datenpunkten. Acquisition Zeit 9 min. </li><li> Erwerben Sie zweidimensionale (2D) 1 H-1 H HR-MAS-NMR-Spektren single-fly bei allen Proben mit einem TOBSY Sequenz mit adiabatischer Impulse 3. Acquisition Parameter sind: 2k Datenpunkte direkten Dimension (11 ppm spektrale Breite), 1 s Wasser pre-Sättigung während des Relaxationsdelay, 8 Scans pro Inkrement, 128 Stufen, 2 s insgesamt Wiederholzeit, 45 ms Mischzeit und insgesamt Übernahme Zeit von 29 min. </li></ol> Teil 4: Data Processing / Analyse <ol><li> Analysieren Sie die MRspectra von Proben mit MestReC Software (Mestrelab Research, www.mestrec.com) </li><li> Verwenden Sie eine Linienverbreiterung Apodizierungsfunktion von 0,5 Hz und gelten für alle HR-MAS-1 H FIDs vor der Fourier-Transformation. </li><li> Verweisen Sie auf die MR-Spektren in Bezug auf δ = 0,0 ppm (externer Standard) TSP. </li><li> Phase-Spektren manuell und wenden Sie einen Whittaker Baseline Schätzer der breiten Komponenten der Ausgangswert vor den Peakfläche Berechnungen subtrahieren. </li><li> Estimate Peakflächen mit MestReC Software. Scale-Peakhöhen in Bezug auf für jede erworbene Spektrum (TSP peak height = 1) TSP. Verwenden Sie t-Tests (zweiseitig, p &lt;0,05), um innerhalb der Gruppe, zwischen Gruppen-Kennzahlen zu vergleichen. </li><li> 2D TOBSY Prozessparameter sind: QSINE = 2 Fenster-Funktion in beiden Dimensionen, FT mit 2k Punkte in der direkten Dimension und Null-Füllung auf 1k in der zweiten Dimension, Phasenkorrektur in beiden Dimensionen und Baseline-Korrektur in der zweiten Dimension. </li><li> Produzieren Sie das 2D-Spektren mit Sparky Programm (TD Goddard und DG Kneller, SPARKY 3, USCF, <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/</a>home / sparky /) </li></ol> Teil 5: Repräsentative Spectra von Live Drosophila melanogaster Flies Die oben beschriebenen Verfahren erlauben eine reproduzierbare Spektren von lebenden Drosophila melanogaster Fliegen zu sammeln. Abbildungen 2 und 3 zeigen repräsentative MR-Spektren in Wildtyp-(wt) Oregon-R erworben Fliegen. Abbildung 2 zeigt 1D 1 H HR-MAS-Spektren CPMG. Auftraggeber Lipidkomponenten [C H 3 (0,89 ppm), (C H 2) n (1,33 ppm), C H 2 C-CO (1.58ppm), C H 2 C = C (2,02 ppm), C H 2 C = O (2,24 ppm), C H = C H (5,33 ppm)], Glycerin (1,3-C H 4,10 ppm und 4,30 ppm; 2-CH 2 5,24 ppm), und kleine Metaboliten: β-Alanin (β-Ala wurden 2,57 ppm), Acetat (Ac, 1,97 ppm), phosphocholin (PC, 3,22 ppm) und phophoetanolamine (PE, 3.23ppm) erkannt und zugeordnet in Übereinstimmung mit früheren Berichten 4, 5. Signale bei 2,02 ppm wurden Methylenprotonen der C H2-CH = CH-Einheit von einfach ungesättigten Fettsäuren (zB Palmitoleinsäure) zugeordnet. Die ungesättigten Säuren wurden durch ein Signal am 5,33 ppm durch Protonen der-CH = CH-Einheit produziert identifiziert. Kleine Metaboliten, die nicht zugeordnet werden konnten oder nicht sichtbar waren mit dem 1D-Spektrum wurden unter Verwendung von 2D-1 H-1 H TOBSY HR-MAS (siehe Abbildung 3). <img src="/files/ftp_upload/1710/1710fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Versuchsaufbau aus in vivo HR-MAS-1H MRS für die Untersuchung von lebenden Drosophila mit 14,1 T. Externer Standard trimethylsilyl-Propionsäure-2 ,2,3,3-d4 Säure in deuteriertem Wasser (TSP / D 2 O). Auf dem Platz: Rotorlage um den magischen Winkel in HR-MAS-Sonde. <img src="/files/ftp_upload/1710/1710fig2.jpg" alt="Abbildung 2" /> Abbildung 2. In-vivo-1D HR-MAS-1 H CPMG Spektren von einer Live-Drosophila melanogaster wt fliegen. Lipid-Komponenten: C H 3 (0,89 ppm), (C H 2) n (1,33 ppm), C H 2 C-CO (1.58ppm), Acetat (Ac), C H 2 C = C (2,02 ppm), C H 2 C = O (2,24 ppm), β-Alanin (β-Ala), phosphocholin (PC) und phophoetanolamine (PE), Glycerin (1,3-C H 4,10, 4,30 ppm; 2-C H 2 5,22 ppm) , C H = C H (5,33 ppm). <img src="/files/ftp_upload/1710/1710fig3.jpg" alt="Abbildung 3" /> Abbildung 3. In vivo 2D-1H-1H TOBSY HR-MAS-Spektrum von einer Live-Drosophila melanogaster wt fliegen mit 14,1 T. Klein Metaboliten und Lipid-Komponenten wurden identifiziert. Metaboliten: Alanin (Ala), β-Alanin (β-Ala), Arginin (Arg), Glutamin (Gln), Glutamat (Glu), PC phosphocholin (PC), phophoetanolamine (PE), Taurin (Tau), α-Glucose (α-Glc) und Glycerin. Lipids Komponenten: C H 3 (0,89 ppm), (C H 2) n (1,33 ppm), 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 H = C 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>

Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von Massachusetts General Hospital Institutional Animal Research Review Board Ausschusses durchgeführt.

Mit Ausnahme des jüngsten Berichts über die Durchführbarkeit der in-vivo-MRT in Fruchtfliegen 6 haben in vivo MRS-Studien in Drosophila noch nicht berichtet worden. In der vorliegenden Protokoll, beschreiben wir die Implementierung eines neuartigen In-vivo-HR-MAS-1 H-MRS-Ansatz zum Nachweis von biologisch wichtigen Molekülen. Insbesondere wiesen wir Lipiden und kleinen Metaboliten in lebenden Drosophila-Fliegen mit 14,1 T in etwa 45 min, die eine ang...

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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 (HR-MAS-) Protonen-Magnetresonanz-Spektroskopie (<sup&gt; 1</sup&gt; H-MRS) ist ein neuartiges zerstörungsfreies Verfahren, das spektrale Linienbreiten verbessert und ermöglicht hochauflösende Spektren von Extrakten, intakte Zellen, Zellkulturen, und vor allem intakte Gewebe gewonnen werden, um Beziehungen zwischen Metaboliten und zellulären Prozesse zu untersuchen.<em&gt; In-vivo-</em&gt; HR-MAS-<sup&gt; 1</sup&gt; H-MRS Studien stehen noch in der Live-Fruchtfliege gemeldet werden<em&gt; Drosophila melanogaster.</em&gt;<em&gt; Drosophila,</em&gt; Als einfachere genetische Organismus, ermöglicht die vielfältigen biologischen Funktionen und verschiedenen evolutionär konservierte Signalwege auf den gesamten Organismus-Ebene untersucht werden und es ist ein nützliches Modell für die Untersuchung von Genetik und Physiologie. Zu diesem Zweck entwickelten wir und umgesetzt<em&gt; In vivo</em&gt; HR-MAS-<sup&gt; 1</sup&gt; H-MRS-Methode zu untersuchen, zu leben<em&gt; Drosophila</em&gt; Mit 14,1 T. Hier skizzieren wir ein HR-MAS-<sup&gt; 1</sup&gt; H-MRS-Protokoll für die molekulare Charakterisierung von<em&gt; Drosophila</em&gt; Mit einem herkömmlichen MR-Spektrometer mit einer HR-MAS-Sonde ausgestattet. Diese Technik ist ein neuartiges,<em&gt; In vivo,</em&gt; Nicht-destruktiven<em&gt; Drosophila</em&gt; Metabolit Messung Ansatz, der die Identifizierung von krankheitsrelevanten Biomarkern ermöglicht und somit können neue therapeutische Entwicklung beitragen.

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

Diese Technik ermöglicht den Einsatz von hochauflösenden magic angle spinning Protonen MR-Spektroskopie (HR-MAS-1H-MRS) für die molekulare Charakterisierung von Live-<em&gt; Drosophila melanogaster</em&gt; Mit einem herkömmlichen 14,1-Tesla-Spektrometer mit einer HR-MAS-Sonde ausgestattet.

Magnetic Resonance Spectroscopy von Live- Drosophila melanogaster Verwendung von 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

Research

JoVE Journal

Neuroscience

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

12.4K Aufrufe.  Massachusetts General Hospital, Harvard Medical School. Diese Technik ermöglicht den Einsatz von hochauflösenden magic angle spinning Protonen MR-Spektroskopie (HR-MAS-1H-MRS) für die molekulare Charakterisierung von Live- Drosophila melanogaster Mit einem herkömmlichen 14,1-Tesla-Spektrometer mit einer HR-MAS-Sonde ausgestattet.

Serie: 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-Maus Neuroanatomische 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 ...

Forschung

Neurowissenschaften

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.

Optogenetische Stimulation von Escape Verhalten in<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;

Bestimmung der spontanen lokomotorischen Aktivität in<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.

Die Verwendung von magnetischen Resonanzspektroskopie als Werkzeug für die Messung der Bi-hemisphärische transkranielle elektrische Stimulation Auswirkungen auf primären motorischen Kortex Metabolism

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.

Live-Imaging von<em&gt; Drosophila</em&gt; Larven Neuroblasten

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).

Metabolomische Analyse von Rattenhirn Hohe Auflösung Kernresonanzspektroskopie von Gewebeextrakte

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.

Bedingungen, die sozialen Raum, in<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.

Hochaufgelöste Quantifizierung der Geruchsgelenkten Verhalten in<em&gt; Drosophila melanogaster</em&gt; Verwenden der<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.

Futter suchen Pfad-Länge-Protokoll für<em&gt; Drosophila melanogaster</em&gt; Larvae

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.

Stoffwechselanalyse von Drosophila Melanogaster Schildzecken und erwachsenen Gehirn

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