높은 처리량 분석은 개인의 알을 낳는 환경 설정을 검사합니다<em> 노랑 초파리</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
최근 초파리의 알을 낳는 환경 설정은 간단한 의사 결정 과정의 신경 기초를 공부하는 유전자 다루기 쉬운 모델로 떠오르고있다. 알을 증착하는 사이트를 선택하면, 여성의 파리는 옵션의 상대적 매력도 순위와 선택 할 수있다 "큰 두 가지 상품을." 하나는 그들이 인구 기반 및 설정하기 힘든만큼,이 간단한 의사 결정 과정의 기초가되는 회로의 기초를 검색 할 체계적인 유전자 검사 방법을 원하는 경우, 대부분의 알을 낳는 환경 분석은 실용적이지 않다. 한 여성의 알을 낳는 환경의 공부의 처리량을 증가시키기 위해, 우리는 최대 30 개인의 각 수를 동시에 분석 알을 낳는 환경 설정은 각 여성은 높은 알을 낳는 속도가 보장하는 프로토콜뿐만 아니라 파리 정의 챔버를 개발 (그래서 선호 쉽게 식별 할 더 설득력 있음). 우리의 접근 방식은 실행이 간단매우 일관된 결과를 얻을 수 있습니다. 또한 이러한 챔버는 비디오가 알을 낳는 동물을 기록하고 optogenetics의 연구에 빛을 제공 할 수 있도록 다른 첨부 파일을 장착 할 수 있습니다. 이 문서에서는 이러한 챔버에서 분석 할 수있는 파리를 준비 이러한 챔버 및 절차를 제조하기위한 청사진을 제공합니다.
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 |
Referências
- Perisse, E., Burke, C., Huetteroth, W., Waddell, S. Shocking revelations and saccharin sweetness in the study of Drosophila olfactory memory. Curr. Biol. 23 (17), 752-763 (2013).
- Yamamoto, N. C., Koganezawa, M. Genes and circuits of courtship behaviour in Drosophila males. Nat. Rev. Neurosci. 14 (10), 681-692 (2013).
- Zwarts, L., Versteven, M., Callaerts, P. Genetics and neurobiology of aggression in Drosophila. Fly. 6 (1), 35-48 (2012).
- Joseph, R. M., Devineni, A. V., King, I. F., Heberlein, U. Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila. Proc. Natl. Acad. Sci. 106 (27), 11352-11357 (2009).
- Miller, P. M., Saltz, J. B., Cochrane, V. A., Marcinkowski, C. M., Mobin, R., Turner, T. L. Natural variation in decision-making behavior in Drosophila melanogaster. PLoS One. 6 (1), 16436 (2011).
- Schwartz, N. U., Zhong, L., Bellemer, A., Tracey, W. D. Egg-laying decisions in Drosophila are consistent with foraging costs of larval progeny. PloS One. 7 (5), 37910 (2012).
- Yang, C. H., Belawat, P., Hafen, E., Jan, L. Y., Jan, Y. N. Drosophila egg-laying site selection a system to study simple decision-making processes. Science. 319 (5870), 1679-1683 (2008).
- Kacsoh, B. Z., Lynch, Z. R., Mortimer, N. T., Schlenke, T. A. Fruit flies medicate offspring after seeing parasites. Science. 339 (6122), 947-950 (2013).
- Wu, C. -. L., Fu, T. -. F., Chou, Y. -. Y., Yeh, S. -. R. A Single Pair of Neurons Modulates Egg-Laying Decisions in Drosophila. PloS one. 10 (3), e0121335 (2015).
- Yang, C. H., He, R., Stern, U. Behavioral and circuit basis of sucrose rejection by Drosophila females in a simple decision-making task. J. Neurosci. 35 (4), 1396-1410 (2015).
- Gou, B., Liu, Y., Guntur, A. R., Stern, U., Yang, C. H. Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying induced acetic acid attraction in Drosophila. Cell Rep. 9 (2), 522-530 (2014).
- Stern, U., Zhu, E. Y., He, R., Yang, C. H. Long-duration animal tracking in difficult lighting conditions. Sci. Rep. 5, 10432 (2015).
- Zhu, E. Y., Guntur, A. R., He, R., Stern, U., Yang, C. H. Egg-laying demand induces aversion of UV light in Drosophila females. Curr. Biol. 24 (23), (2014).
- Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. High-throughput ethomics in large groups of Drosophila. Nat. Methods. 6, 451-457 (2009).
- Klatpoetke, N. C., et al. Independent Optical Excitation of Distinct Neural Populations. Nat. Methods. 11 (3), 338-346 (2014).
