Høy gjennomstrømning analyse for å undersøke Eggleggingen Preferences av Individuell<em> Drosophila melanogaster</em
Summary
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.
Abstract
Nylig har egg-legging preferanse for Drosophila dukket opp som en genetisk medgjørlig modell for å studere nevrale grunnlaget for enkle beslutningsprosesser. Når du velger steder å sette eggene sine, kvinnelige fluer er i stand til vurdering den relative attraktiviteten av sine alternativer og velge de "største av to goder." Men de fleste egg-legging preferanse analysene er ikke praktisk hvis man ønsker å ta en systematisk genetisk screening tilnærming for å søke etter kretsen grunnlaget bak denne enkle beslutningsprosessen, som de er populasjonsbasert og arbeidskrevende å sette opp. For å øke gjennomstrømningen av å studere av egg-leggende preferansene enkelt kvinner, utviklet vi tilpassede kamre som hver kan samtidig analyse egg-leggende preferanser på opp til tretti enkelte fluer, samt en protokoll som sikrer at hver hunn har en høy eggleggingen hastighet (slik at deres preferanser er lett merkbar og mer overbevisende). Vår tilnærming er enkel å utføreog produserer svært konsistente resultater. I tillegg kan disse kamrene være utstyrt med ulike vedlegg for å tillate videoopptak egg-legging dyr og å levere lys for optogenetics studier. Denne artikkelen inneholder skissene for tilvirkning av disse kamre og fremgangsmåten for fremstilling av fluene som skal bli undersøkt i disse kamrene.
Introduction
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.
Protocol
Representative Results
Discussion
Kamrene og protokoller som er beskrevet her har flere forbedringer fra forrige egg-legging analyser. For det første øker de gjennomstrømningen for å analysere preferanser av enkelt dyr betydelig. Hvert kammer kan analysere 30 enslige kvinner, og det tar mindre enn en time å sette opp. For det andre øker de konsistensen av egg-legging preferanser over tidligere metoder. Standardisering av dimensjonene på arenaen, størrelsen på egg-legging underlag, og avstanden mellom underlag gjør det lettere å sammenligne re…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| UltraPure Agarose | Invitrogen | 16500-500 | |
| Sucrose | Sigma | S0389 | |
| Water bath | Fisher | 15-462-6Q | |
| LifeCam Cinema webcam | Microsoft | H5D-00013 | |
| Red LEDs | Cree | C503B-RAN-CA0B0AA1 | |
| Egg-laying chambers | Custom Built | ||
| Camera holders | Custom Built | ||
| LED holders | Custom Built | ||
| Fly vials (narrow) | Genesee | 32-116BC |
Riferimenti
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