Assay תפוקה גבוהה לבחינת העדפות הטלה של פרט<em> תסיסנית</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
לאחרונה, העדפת הטלה של תסיסנית התפתחה כמודל צייתן גנטי ללמוד את הבסיס העצבי של תהליכי קבלת החלטות פשוטים. בעת בחירת אתרים מטילים את ביציהן, נקבות זבוב מסוגל בדירוג האטרקטיביות היחסית של האופציות שלהם ובחירת "יותר משני מוצרים." עם זאת, מבחני ההעדפה ביותר ההטלה אינם מעשיים אם רוצה לנקוט בגישת סריקה גנטית שיטתית לחיפוש הבסיס המעגל שבבסיס תהליך קבלת החלטות פשוט זו, כפי שהן מבוססות על אוכלוסייה ומייגעת להקים. כדי להגדיל את התפוקה של הלימוד של העדפות הטלה של נקבות אחת, פתחנו תאי מנהג שכל יכול העדפות assay בו זמנית הטלה של עד שלושים פרט עפו כמו גם פרוטוקול המבטיח לכל נקבה יש שיעור גבוה הטלה (כך העדפתם היא להבחין בקלות ועוד משכנע). הגישה שלנו היא פשוטה לביצועומייצר תוצאות מאוד עקביות. בנוסף, החדרים האלה יכולים להיות מצוידים עם קבצים מצורפים שונים כדי לאפשר ווידאו הקלטת חי ההטלה, וכדי לספק אור ללימודי optogenetics. מאמר זה מספק את השרטוטים בודים תאים אלה ואת הליך הכנת הזבובים להיות assayed בתאים אלה.
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
The chambers and protocols described here have several improvements over previous egg-laying assays. First, they increase the throughput of assaying preferences of single animals significantly. Each chamber can assay 30 single females and it takes less than an hour to set up. Second, they increase the consistency of the egg-laying preferences over previous methods. The standardization of the dimensions of the arena, size of the egg-laying substrates, and distance between substrates makes it easier to compare the results …
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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