High Throughput Assay til at undersøge æglæggende præferencer af Individuel<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
For nylig har æglægning præference for Drosophila opstået som en genetisk medgørligt model til at studere det neurale grundlag af simple beslutningsprocesser. Når du vælger steder at deponere deres æg, kvindelige fluer er i stand til at rangordne de relative tiltrækningskraft deres muligheder og vælge de "største af to varer." Men de fleste æglæggende præference analyser er ikke praktisk, hvis man ønsker at tage en systematisk genetisk screening tilgang til at søge efter kredsløbet grundlag bag denne simple beslutningsproces, da de er populationsbaseret og møjsommelig at sætte op. For at øge gennemløbet af at studere af æglæggende præferencer enkelt hundyr, vi udviklede brugerdefinerede kamre, der hver især kan samtidig assay æglæggende præferencer på op til tredive individuelle flyver samt en protokol, der sikrer hver hun har en høj ægproduktion sats (så deres præference er let mærkbar og mere overbevisende). Vores tilgang er enkel at udføreog producerer meget ensartede resultater. Derudover kan disse kamre udstyres med forskellige redskaber til at tillade videooptagelse de æglæggende dyr og levere lys til optogenetics studier. Denne artikel indeholder planerne for opdigte disse kamre og proceduren for udarbejdelse fluer, der skal analyseres i disse kamre.
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, der er beskrevet her, har flere forbedringer i forhold til tidligere æglæggende analyser. Første, de øger gennemløbet til analyse præferencer af enkelte dyr betydeligt. Hvert kammer kan analysere 30 enlige kvinder, og det tager mindre end en time at sætte op. For det andet, de øger sammenhængen i de æglæggende præferencer i forhold til tidligere metoder. Standardiseringen af dimensionerne af arenaen, størrelsen af de æglæggende substrater, og afstanden mellem underlag g…
Disclosures
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 |
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