個々の産卵環境を調べるために、ハイスループットアッセイ<em>キイロショウジョウバエ</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
最近、 ショウジョウバエの産卵選好は、単純な意思決定プロセスの神経基盤を研究するために、遺伝的に扱いやすいモデルとして登場しました。その卵を堆積するサイトを選択する際、女性のハエは、そのオプションの相対的な魅力をランク付けし、選択することが可能である」以上の2品を。」一つは、彼らが集団ベースとセットアップが面倒であるため、この単純な意思決定プロセスの基礎となる回路の基礎を検索するために体系的な遺伝子スクリーニングアプローチを取りたい場合は、ほとんどの産卵選好アッセイは実用的ではありません。単一の雌の産卵選好の勉強のスループットを向上させるために、我々は、最大30個々のハエの各缶を同時にアッセイ産卵選好だけでなく、それぞれの女性が高い産卵率が保証するプロトコルカスタムチャンバーを開発しました（自分の好みが容易に識別可能な、より説得力があるように）。我々のアプローチは、実行するのは簡単ですそして、非常に一貫性のある結果を生成します。さらに、これらのチャンバは、ビデオは、産卵動物を記録し、光遺伝学研究のための光を提供することを可能にするために、異なるアタッチメントを装備することができます。この記事では、これらのチャンバでアッセイするハエを製造するためのこれらのチャンバと手続きを製造するための青写真を提供します。
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
ここで説明するチャンバーとプロトコルは、以前の産卵アッセイにいくつかの改良を持っています。まず、彼らは大幅に単一の動物の嗜好をアッセイのスループットを向上させます。各チャンバは、30単一の雌をアッセイすることができる、それがセットアップに時間未満かかります。第二に、彼らは以前の方法に比べて産卵選好の一貫性を高めます。アリーナの寸法の標準化は、基板間の?…
Declarações
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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