Este trabajo fue apoyado en parte por los Institutos Nacionales de Salud (NIH) otorgar AI063433 a Laurence G. Rahme, de los Institutos Nacional de Salud (NIH) Centro de Donación (P50GM021700) a Ronald G. Tompkins (A. Aria Tzika, Director de el núcleo de RMN), y el Hospital Shriner una beca de investigación para la Infancia (# 8893) para A. Aria Tzika. Damos las gracias a Dionyssios Mintzopoulos Ph.D. de asistencia en las fases iniciales del desarrollo de este protocolo y Ovidiu C. Andronesi Ph.D. para obtener ayuda con la secuencia de pulsos TOBSY.

Parte 1: Preparación para las mediciones de Drosophila HRMAS <ol><li> Utilizando procedimientos estándar flylab 1, recoger nuevas eclosing vuela por 3 días y la transferencia a volar viales que contienen los alimentos frescos volar. Incubar las moscas recogidas por 5 días para que las moscas se convertirán en 5-8 días de edad, justo antes del experimento. Un solo sexo (por lo general hombres) se utiliza para los experimentos. </li><li> Uso saludable, de tipo salvaje intacta vuela a comparar con el tratado, por ejemplo, traumatizados o alterados genéticamente moscas 1. </li><li> Lugar vuela solo en tubos de 2 ml que contiene un trozo (~ 0,2 ml) de la mosca de los alimentos 24 horas antes del experimento. Estos tubos tienen un agujero en la copa de tubo hecho con una aguja de insulina de la llama calienta. </li><li> Pesar los tubos inmediatamente antes y después de la inserción de volar con una balanza de alta precisión y definir el peso de cada vuelo s restando el primer valor de este último. Una mosca macho pesa generalmente 0,7-1 mg. </li></ol> Parte 2: Preparación HRMAS Rotor. <ol><li> Coloque un solo tubo en hielo durante menos de un minuto para anestesiar el interior volar. </li><li> Poner la mosca en una capa de papel de aluminio colocado en hielo y empuje de la mosca suavemente en el espacio semiesférico hueco de la pieza del rotor de RMN con un cepillo suave para asegurar una inserción completa volar evitando lesiones volar. </li><li> Coloque el inserto en el óxido de zirconio (ZrO 2) del tubo del rotor (4 mm de diámetro, 50 l) para localizar las moscas entre el inserto de rotor y la parte inferior del rotor (ver figura 1). </li><li> Cerca de la inserción de un tornillo y se cubre con parafina (ver figura 1) para evitar el contacto entre la mosca y la solución de TSP estándar (TSP: trimetilsilil-propiónico-2 ,2,3,3-d4 ácido, Mw = 172, δ = 0.00 ppm, 50 mM en agua deuterado-D O 2, que funcionan como una referencia tanto para los desplazamientos químicos de resonancia y cuantificación). </li><li> Añadir 8 l de solución de TSP de serie en la parte superior de la pieza del rotor. (Ver figura 1). </li><li> Seguro y apretar el instituido por los rotores con una tapa superior (ver figura 1). </li></ol> Parte 3: HRMAS Adquisición de Datos <ol><li> Introducir el rotor en la sonda HRMAS y colóquelo en el ángulo mágico 54,7 ° (ver figura 1). </li><li> Ajuste la temperatura a 4 ° C por una unidad de BTO-2000 en combinación con la unidad neumática MAS. Las moscas se mantienen a 4 ° C, mientras que en el espectrómetro para mantener la anestesia. </li><li> Establecer el HRMAS una tasa de H MRS girando a dos MAS kHz. </li><li> Estabilizar la frecuencia de rotación del MAS en el 2,0 ± 0.001 kHz por un controlador de velocidad MAS (frecuencia de rotación). </li><li> Para la preparación de imán: sintonía y que coincida con la bobina para un rendimiento óptimo y calza el imán para una calidad óptima de los espectros. </li><li> Adquirir una dimensión un espectro de H con rotor sincronizado Carr-Purcell-Meiboom-Gill (CPMG) spin echo secuencia de pulsos 2, [90 ° - (τ-180 º - τ) n-adquisición], que funciona como un filtro para T 2 eliminar el ensanchamiento espectral 1 H RMN de una sola mosca de espectros para adquirir una dimensión de datos en todas las muestras. Sincronizar el retardo entre pulsos (τ = 500μs) a la frecuencia de rotación MAS (2 kHz). Establecer el número de transitorios a 256, con 32.768 (32K) puntos de datos. Tiempo de adquisición de 9 min. </li><li> La adquisición de dos dimensiones (2D) 1 H-1 H RMN HRMAS solo vuelan los espectros de todas las muestras usando una secuencia de pulsos adiabáticos TOBSY con 3. Parámetros de adquisición son: dimensión 2k puntos de datos directos (11 ppm ancho espectral), el agua 1 s antes de la saturación durante el tiempo de relajación, 8 lecturas por el incremento de 128 aumentos, el tiempo de la repetición total de 2 s, 45 ms de tiempo de mezcla, y un total de adquisición tiempo de 29 min. </li></ol> Parte 4: Tratamiento de datos / Análisis <ol><li> Analizar el MRspectra de especímenes con MestReC software (Mestrelab Investigación, www.mestrec.com) </li><li> Utilice una línea de ampliar la función de apodización de 0,5 Hz y se aplican a todos los HRMAS un FID H antes de la transformación de Fourier. </li><li> Referencia de los espectros de RM con respecto a TSP a δ = 0.0 ppm (estándar externo). </li><li> Fase de espectros y aplicar manualmente un estimador de referencia Whittaker para restar los componentes de gama de la línea de base antes de que los cálculos del área del pico. </li><li> Estimar las áreas de pico utilizando software MestReC. Escala de alturas de los picos con respecto a TSP para cada espectro adquirido (TSP altura del pico = 1). Utilice la prueba t (dos colas, p &lt;0,05) para comparar dentro del grupo de relaciones entre grupos. </li><li> 2D parámetros del proceso TOBSY son: QSINE = 2 la función ventana en ambas dimensiones, FT, con puntos de 2k en la dimensión directa y cero para el llenado de 1k en la segunda dimensión, la corrección de fase en las dos dimensiones y la corrección de línea de base en la segunda dimensión. </li><li> Producir los espectros 2D con Sparky programa (TD Goddard y Kneller DG, Sparky 3, USCF, <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/</a>Inicio / sparky /) </li></ol> Parte 5: Espectros Representante de Live moscas Drosophila melanogaster Los procedimientos descritos anteriormente permiten recoger espectros reproducibles de moscas Drosophila melanogaster vivir. Figuras 2 y 3 muestran representante MR espectros adquiridos de tipo salvaje (wt) de Oregon-R moscas. La figura 2 presenta 1D 1 H HRMAS espectros CPMG. Los principales componentes lípidos [C H 3 (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)], el glicerol (1,3-C H 4,10 ppm y 4,30 ppm, 2-CH 2 5.24 ppm), y pequeños metabolitos: β-alanina (β-Ala , 2,57 ppm), el acetato (Ac 1,97 ppm), fosfocolina (PC, 3,22 ppm) y phophoetanolamine (PE, 3.23ppm) fueron detectados y clasificados de acuerdo con informes anteriores 4, 5. Señales a 2.02 ppm fueron asignados a los protones de metileno de la C H2-CH = CH mitad de los mono-ácidos grasos insaturados (palmitoleico es decir). Los ácidos grasos insaturados fueron identificados por una señal a 5,33 ppm producido por protones de la-CH = CH-fracción. Metabolitos pequeños que no pueden ser asignados o no visible en el espectro 1D fueron detectados utilizando 2D 1 H-1 H TOBSY HRMAS (ver figura 3). <img src="/files/ftp_upload/1710/1710fig1.jpg" alt="Figura 1" /> Figura 1. Experimental puesta en marcha de in vivo HRMAS 1H MRS para la investigación de Drosophila en vivo en el 14,1 T. externo estándar trimetilsilil-propiónico-2 ,2,3,3-d4 ácido en el agua deuterado (TSP / D 2 O). En la plaza: la posición del rotor en el ángulo mágico en HRMAS sonda. <img src="/files/ftp_upload/1710/1710fig2.jpg" alt="Figura 2" /> Figura 2. 1D En vivo HRMAS espectros de 1 H CPMG de vivir en peso mosca Drosophila melanogaster. Componentes lipídicos: C H 3 (0,89 ppm), (C H 2) n (ppm 1,33), C H 2 C-CO (1.58ppm), acetato (Ac), C H 2 C = C (2,02 ppm), C H 2 C = O (2,24 ppm), β-alanina (β-Ala), fosfocolina (PC) y phophoetanolamine (PE), glicerol (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="Figura 3" /> Figura 3. In vivo 2D 1H-1H TOBSY espectro HRMAS de vivir en peso mosca Drosophila melanogaster en el 14,1 metabolitos T. pequeñas y componentes lipídicos fueron identificados. Metabolitos: alanina (Ala), β-alanina (β-Ala), arginina (Arg), glutamina (Gln), el glutamato (Glu), fosfocolina PC (PC), phophoetanolamine (PE), taurina (Tau), α-glucosa (α-Glc) y glicerol. Lípidos componentes: C H 3 (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>

Los experimentos con animales se realizaron de acuerdo con las directrices y regulaciones establecidas por el Massachusetts General Hospital de Investigación Institucional Animal Comité de Revisión de la Junta.

Con la excepción del reciente informe de la viabilidad de la resonancia magnética in vivo en moscas de la fruta 6, los estudios in vivo MRS en Drosophila aún no han sido reportados. En el presente Protocolo, se describe la implementación de una novela en vivo HRMAS 1 H-MRS enfoque para la detección de moléculas biológicamente importantes. En concreto, se detectaron metabolitos de los lípidos y los pequeños viven en moscas Drosophila en ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

De alta resolución Magic Angle Spinning (HRMAS) espectroscopia de resonancia magnética de protones (<sup&gt; 1</sup&gt; H-MRS) es una novela técnica no destructiva que mejora espectral anchos de línea y permite a los espectros de alta resolución que se obtiene a partir de extractos, las células intactas, cultivos celulares y de tejidos más importante intacta para investigar las relaciones entre los metabolitos y los procesos celulares.<em&gt; En vivo</em&gt; HRMAS<sup&gt; 1</sup&gt; H-MRS estudios aún no se han reportado en la mosca de la fruta en vivo<em&gt; Drosophila melanogaster.</em&gt;<em&gt; Drosophila,</em&gt; Como un organismo más simple genética, permite las múltiples funciones biológicas y conservado evolutivamente distintas vías de señalización que se examinarán en el nivel de todo el organismo y es un modelo útil para la investigación genética y la fisiología. Con este fin, hemos desarrollado e implementado un<em&gt; In vivo</em&gt; HRMAS<sup&gt; 1</sup&gt; H-MRS método para investigar en vivo<em&gt; Drosophila</em&gt; En el 14,1 T. A continuación, se describe un HRMAS<sup&gt; 1</sup&gt; H-MRS protocolo para la caracterización molecular de<em&gt; Drosophila</em&gt; Con un espectrómetro convencional de RM equipado con una sonda HRMAS. Esta técnica es una novela,<em&gt; In vivo,</em&gt; No destructivos<em&gt; Drosophila</emEnfoque&gt; medición de metabolitos, lo que permite la identificación de biomarcadores de enfermedades y por lo tanto puede contribuir al desarrollo de terapéuticas novedosas.

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

Esta técnica permite el uso de ángulo mágico de alta resolución girar espectroscopia de resonancia magnética (HRMAS 1H-MRS) para la caracterización molecular de vivir<em&gt; Drosophila melanogaster</em&gt; Con un espectrómetro convencional de 14,1 teslas equipado con una sonda HRMAS.

Espectroscopia de Resonancia Magnética de vivir Drosophila melanogaster Utilizando Spinning Magic ángulo

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 vistas.  Massachusetts General Hospital, Harvard Medical School. Esta técnica permite el uso de ángulo mágico de alta resolución girar espectroscopia de resonancia magnética (HRMAS 1H-MRS) para la caracterización molecular de vivir Drosophila melanogaster Con un espectrómetro convencional de 14,1 teslas equipado con una sonda HRMAS.

Video: 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.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

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

Investigación

Neurociencias

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.

Optogenético estimulación de comportamiento de escape 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;

Determinación de la actividad locomotora espontánea en<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.

El uso de la espectroscopia de resonancia magnética como herramienta para la medición de Bi-hemisféricos Transcraneal eléctricos Efectos de estimulación sobre la corteza primaria Motor Metabolismo

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.

Imágenes en vivo de<em&gt; Drosophila</em&gt; larvales neuroblastos

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).

Análisis de Metabolómica de cerebro de rata por Alta Resolución Nuclear Espectroscopía de Resonancia Magnética de Extractos de Tejidos

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.

Condiciones que afectan Espacio Social en<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.

De alta resolución de cuantificación del comportamiento guiado Olor en<em&gt; Drosophila melanogaster</em&gt; Uso de la<em&gt; Flywalk</em&gt; Paradigma

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.

Forrajeo Protocolo Ruta de longitud de<em&gt; Drosophila melanogaster</em&gt; Las larvas

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.

Análisis metabólico del cerebro adulto y Larval de Drosophila melanogaster

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