Name: high throughput assay to examine egg-laying preferences of individual drosophila melanogaster
Uploaded: 2016-03-24T13:00:00
Description: Watch this Scientific Journal Video about High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster at JoVE.com

We thank the Duke Physics Shop, especially Phil Lewis, for helping us build the behavioral apparatus and attachments and creating the drawing. This work is funded by the National Institutes of Health under award number R01GM100027.

1. Preparación de moscas a ensayar <ol><li> Cultura vuela de melaza estándar / media de harina de maíz en incubadora a 25 ° C y 65% ​​de humedad. Tenga cuidado de no saturar los viales. Por ejemplo, poner 8 mujeres y 6 hombres en un vial de alimentos estrecho. Nota: El &quot;estrecho vial comida&quot; usado aquí tiene un diámetro interior de 2,3 cm. Nosotros generalmente pueden obtenerse unos 10 ml de alimentos mosca en cada vial. La receta de comida mosca hemos utilizado se describe aquí: http://fly...

<ol>
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Las cámaras y los protocolos descritos aquí tienen varias mejoras con respecto a los ensayos de la puesta de huevos anteriores. En primer lugar, que aumentan el rendimiento del ensayo de las preferencias de los animales individuales de manera significativa. Cada cámara puede ensayar 30 hembras individuales y se tarda menos de una hora de configurar. En segundo lugar, aumentan la consistencia de las preferencias de la puesta de huevos respecto a los métodos anteriores. La estandarización de las dimensiones de la are...

Drosophila melanogaster is a powerful genetic model organism to study the neural basis of behaviors. The rapid developments of genetic tools to manipulate neurons in a targeted manner and the emergence of sophisticated behavioral analysis tools have significantly improved our ability to dissect the circuit mechanisms that underlie the sensory-motor transformation processes of several innate and learned behaviors1-3.
Drosophila egg-laying is a suitable model to study the neural basis of simple decision making processes. In particular, Drosophila females have been shown to possess the ability to compare and rank their options before "committing" to depositing an egg onto a given option4-8. For example, when given only a plain (sucrose-free) substrate or only a sucrose-containing substrate, females readily accept either option for egg-laying. However, when presented with both options, females robust reject the sucrose substrate in some contexts7,9,10. Relatively little is known about the neural mechanisms that allow females to "choose the greater of two goods", however. A major obstacle has been the lack of an efficient method to assay egg-laying preferences such that one can use a systematic genetic screening approach to study this problem.
In this report, we describe the protocol we developed that allows egg-laying preferences of females to be assayed at single-animal resolution and with substantially improved throughput and consistency over previous methods. Specifically, we provide the blueprints for constructing the chambers we designed, the protocol for preparing the females so that each is primed to lay many eggs, and the protocol for using the chambers.

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			<td height="20" style="height:20px;width:269px;">UltraPure Agarose&#160;</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Sucrose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Water bath&#160;</td>
			<td style="width:104px;">Fisher</td>
			<td style="width:119px;">15-462-6Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LifeCam Cinema webcam</td>
			<td style="width:104px;">Microsoft</td>
			<td style="width:119px;">H5D-00013</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:269px;">Red LEDs</td>
			<td style="width:104px;">Cree</td>
			<td style="width:119px;">C503B-RAN-CA0B0AA1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Egg-laying chambers</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Camera holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LED holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Fly vials (narrow)</td>
			<td style="width:104px;">Genesee</td>
			<td style="width:119px;">32-116BC</td>
			<td></td>
		</tr>
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</table>

high throughput assay to examine egg-laying preferences of individual drosophila melanogaster

Recientemente, la preferencia de la puesta de huevos de Drosophila se ha convertido en un modelo manejable de forma genética para estudiar la base neural de los procesos de toma de decisiones simples. Cuando la selección de sitios para depositar sus huevos, las moscas hembras son capaces de clasificar el atractivo relativo de sus opciones y elegir el &quot;mayor de dos bienes.&quot; Sin embargo, la mayoría de los ensayos de preferencia que ponen huevos no son prácticos si se quiere adoptar un enfoque sistemático para la detección genética buscar la base de circuito que subyace a este sencillo proceso de toma de decisiones, ya que son laboriosos y configurar basado en la población. Para aumentar el rendimiento de estudiar las preferencias puesta de huevos de las hembras individuales, desarrollamos cámaras personalizadas que cada uno puede al mismo tiempo de ensayo preferencias de la puesta de huevos de hasta treinta individuo moscas, así como un protocolo que asegura cada mujer tiene una alta tasa de puesta de huevos (de modo que su preferencia es fácilmente discernible y más convincente). Nuestro enfoque es sencillo de ejecutary produce resultados muy coherentes. Además, estas cámaras pueden equiparse con diferentes accesorios para permitir la grabación de vídeo a los animales ovíparos y para suministrar luz para los estudios de la optogenética. Este artículo proporciona los planos para la fabricación de estas cámaras y el procedimiento para la preparación de las moscas para ser ensayadas en estas cámaras.

Las cámaras de la puesta de huevos se componen de varias piezas: una pieza de sustrato (parte inferior), una pieza divisora ​​(centro), una pieza de carga (parte superior), y 2 puertas correderas (Figura 1 A - D). Estas piezas se utilizan para moscas de forma independiente de configuración y los sustratos antes de los experimentos que ponen huevos. Figura 1F muestra cómo viales deben buscar cuando las moscas hembras están listas para la puesta de...

Watch this Scientific Journal Video about High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster at JoVE.com

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Ensayo de Alto Rendimiento para el Examen de Preferencias de la puesta de huevos individual<em&gt; Drosophila melanogaster</em

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Research

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Neuroscience

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

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