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.

파리 준비 1.있는 분석한다 <ol><li> 문화 표준 당밀에 파리 / 25 ° C 및 65 % 습도에서 설정 인큐베이터에서 옥수수 가루 미디어. 유리 병을 혼잡하게하지 않도록주의하십시오. 예를 들어, 좁은 식품 유리 병에 8 명의 여성과 6 명의 남성을 넣어. 주 : 여기에서 사용 된 &quot;식품 좁은 병&quot;은 2.3 cm의 내경을 갖는다. 우리는 일반적으로 각각의 유리 병에 플라이 음식의 약 10 ml의 분배. 우리가 사용하는 플라이 음식의 조리법은 여기에 설명되어 있습니다 http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/molassesfood.html. </li><li> 2 - 그림 1E에 도시 된 바와 같이, 삼일 여성의 우화 후, 신선한 효모 붙여 넣기와 유리 병을 준비합니다. 0.5 % proprionic 산 10 ml의 활성 효모 6g을 혼합하여 신선한 효모 붙여 넣기를합니다. 식품 유리 병의 측벽에 효모 페이스트를 적용 할 주걱을 사용합니다. 유리 병에 25 명의 남성 - 20 35 eclosed 여성 함께 - 30 수집합니다. <ol><li> 일반적으로, 여성은 수집 동일한 튜브에서 수집 수컷(수 있도록 시간과 노력을 절약하기 위해). 수컷의 생식 능력에 대한 우려가있는 경우에는, WT 대신 수컷을 사용한다. 또한, 효모 붙여 넣기 신선한 매일을 준비 따라서 달걀 생산을 자극하는 것이 중요합니다. </li></ol></li><li> 파리 온도 민감하지 않는 한, 25 ° C, 65 % 습도에서 수집 된 여성 / 남성을 유지한다. </li><li> 4 ~ 후 - 오일, 암컷이 알을 낳는 실험 준비가되어 있는지 확인하기 위해 튜브를 확인합니다. 식품 매체 표면 적극적 음식내는 유충 젖은 때 준비 (- F도 1E 참조). 참고 : 식품 표면이 젖어 유충으로 크롤링하게되면 암컷은 일반적으로 누워 계란을 보류. 이 단계는 암컷이 그들이 원하는 텍스처와 기판가 발생할 때 많은 알을 낳기 할 준비가되어있을 것이라는 점을 보장한다 (예., ~ 1 % 아가로 오스). </li></ol> 2. 상공 회의소 건설, 조립, 분석 설정 <ol><li> (컴퓨터 가게 아크릴 알을 낳는 챔버를 구축 Fi 인터넷이gure의 1A - D). 다른 조각의 엔지니어링 도면을 보충 그림 1에 나타낸다 -. 3 고해상도 사진은 여기에서 찾을 수 있습니다 (http://www.rebeccayang.org/pdf/chamber%20design.pdf). </li><li> 도 1c에 도시 된 바와 같이 챔버의 로딩 (위) 부재에 플라스틱 시트를 삽입한다. 로드 개별 알을 낳는 경기장에 날아 동안이 바닥면 역할을합니다. </li><li> 공동 2 패드에 여성을 마취하고 각 알을 낳는 경기장에 개별적으로로드합니다. 파리는 CO 2를 복구하고 새로운 환경에 적응 될하기위한 ~ 30 분을 허용합니다. </li><li> 아가로 오스 기판을 준비합니다. <ol><li> 편의를 위해 55 ° C의 물을 욕조에 녹아 1 % 아가로 오스의 미리 만들어진 병을 유지합니다. </li><li> 50 ML 원뿔 튜브에 재고 자당 용액 (2 M)의 원하는 금액을 추가하고 아가로 오스의 적절한 양을 혼합. 예를 들어, 150 mM의 수크로오스 기판 PLA를 제조CE는 750 튜브에 2 M 자당 솔루션의 μL 후 10 ml의 표시로 아가로 오스와 튜브를 입력합니다. </li><li> 동일하게 일반 기판을 준비하는 대신 자당 용액에 증류수를 추가한다. 참고 :이 프로토콜의 아가로 오스의 최종 농도가 약간 1 % 미만이다. 1.1 % 및 두 기판이 같은 아가로 오스 농도의 것을 - ~ 0.9 이내로 제어 우리의 경험에 의하면, 아가로 오스의 정확한 농도는 너무 오래 중요하지 않습니다. </li></ol></li><li> 그림 1D에서 볼 수 있듯이 각각의 통에 챔버와 피펫 아가로 오스 기판의 1,000 μl를의 기판 (아래) 조각을 가져 가라. </li><li> 아가 ~ 30 분 동안 응고하도록 허용합니다. </li><li> 아가로 오스 기판과 파리 준비가되면 알을 낳는 챔버의 세 조각을 조립 한 후 플라스틱 시트를 꺼내. </li><li> 플라이 인큐베이터에있는 챔버를 놓습니다. 참고 : 알을 낳는 실험의 길이 expe의에 따라 달라질 수 있습니다rimental 필요. (- 16 시간 14) 우리는 일반적으로 실험 O / N을 실행합니다. 또한, 알을 낳는 환경에 주기성 타이밍의 유의 한 효과는 관찰되지 않았다. </li><li> 챔버 내에 이산화탄소를 주입하여 마취 여성. 챔버를 분해 플라이 시체 공시 소에 마취 파리를 폐기 (예. 빈 커피는 일부 옥수수 기름 가득 수 있습니다.) 기록 관리에 대한 결과의 사진을 촬영합니다 (그림 2 참조). </li><li> 수동으로 계란의 수를 계산 및 분석을위한 기본 인덱스를 계산합니다. 같은 환경 지표를 계산 (N A - B N) / (A + N의 N b) N N A 및 B는 각각의 사이트에 대 사이트 B를 알의 수를 나타내며. </li></ol>

<ol>
	<li>Perisse, E., Burke, C., Huetteroth, W., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking+revelations+and+saccharin+sweetness+in+the+study+of+Drosophila+olfactory+memory.">Shocking revelations and saccharin sweetness in the study of Drosophila olfactory memory.</a> Curr. Biol. 23 (17), 752-763 (2013).</li><li>Yamamoto, N. C., Koganezawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+circuits+of+courtship+behaviour+in+Drosophila+males.">Genes and circuits of courtship behaviour in Drosophila males.</a> Nat. Rev. Neurosci. 14 (10), 681-692 (2013).</li><li>Zwarts, L., Versteven, M., Callaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+neurobiology+of+aggression+in+Drosophila.">Genetics and neurobiology of aggression in Drosophila.</a> Fly. 6 (1), 35-48 (2012).</li><li>Joseph, R. M., Devineni, A. V., King, I. F., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviposition+preference+for+and+positional+avoidance+of+acetic+acid+provide+a+model+for+competing+behavioral+drives+in+Drosophila.">Oviposition preference for and positional avoidance of acetic acid provide a model for competing behavioral drives in Drosophila.</a> Proc. Natl. Acad. Sci. 106 (27), 11352-11357 (2009).</li><li>Miller, P. M., Saltz, J. B., Cochrane, V. A., Marcinkowski, C. M., Mobin, R., Turner, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+decision-making+behavior+in+Drosophila+melanogaster.">Natural variation in decision-making behavior in Drosophila melanogaster.</a> PLoS One. 6 (1), 16436 (2011).</li><li>Schwartz, N. U., Zhong, L., Bellemer, A., Tracey, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg-laying+decisions+in+Drosophila+are+consistent+with+foraging+costs+of+larval+progeny.">Egg-laying decisions in Drosophila are consistent with foraging costs of larval progeny.</a> PloS One. 7 (5), 37910 (2012).</li><li>Yang, C. H., Belawat, P., Hafen, E., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+egg-laying+site+selection+a+system+to+study+simple+decision-making+processes.">Drosophila egg-laying site selection a system to study simple decision-making processes.</a> Science. 319 (5870), 1679-1683 (2008).</li><li>Kacsoh, B. Z., Lynch, Z. R., Mortimer, N. T., Schlenke, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+flies+medicate+offspring+after+seeing+parasites.">Fruit flies medicate offspring after seeing parasites.</a> Science. 339 (6122), 947-950 (2013).</li><li>Wu, C. -. L., Fu, T. -. F., Chou, Y. -. Y., Yeh, S. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Single+Pair+of+Neurons+Modulates+Egg-Laying+Decisions+in+Drosophila.">A Single Pair of Neurons Modulates Egg-Laying Decisions in Drosophila.</a> PloS one. 10 (3), e0121335 (2015).</li><li>Yang, C. H., He, R., Stern, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+circuit+basis+of+sucrose+rejection+by+Drosophila+females+in+a+simple+decision-making+task.">Behavioral and circuit basis of sucrose rejection by Drosophila females in a simple decision-making task.</a> J. Neurosci. 35 (4), 1396-1410 (2015).</li><li>Gou, B., Liu, Y., Guntur, A. R., Stern, U., Yang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+neurons+on+the+internal+reproductive+tract+contribute+to+egg-laying+induced+acetic+acid+attraction+in+Drosophila.">Mechanosensitive neurons on the internal reproductive tract contribute to egg-laying induced acetic acid attraction in Drosophila.</a> Cell Rep. 9 (2), 522-530 (2014).</li><li>Stern, U., Zhu, E. Y., He, R., Yang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-duration+animal+tracking+in+difficult+lighting+conditions.">Long-duration animal tracking in difficult lighting conditions.</a> Sci. Rep. 5, 10432 (2015).</li><li>Zhu, E. Y., Guntur, A. R., He, R., Stern, U., Yang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg-laying+demand+induces+aversion+of+UV+light+in+Drosophila+females.">Egg-laying demand induces aversion of UV light in Drosophila females.</a> Curr. Biol. 24 (23), (2014).</li><li>Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nat. Methods. 6, 451-457 (2009).</li><li>Klatpoetke, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Optical+Excitation+of+Distinct+Neural+Populations.">Independent Optical Excitation of Distinct Neural Populations.</a> Nat. Methods. 11 (3), 338-346 (2014).</li></ol>

The authors have nothing to disclose.

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 between different experimental days and from different research groups. Third, the chambers can be fitted with attachments to accommodate additional analysis one wants to pursue (Supplementary Figure 4). For example, for behavioral analysis, video cameras can be attached to the top of the chamber to record the flies7,11-13. Most importantly, this approach is scalable. These chambers are relatively inexpensive to produce with the help of a machine shop. Also, it typically takes a skilled worker less than 2 hr to set up 5 chambers (150 single females) worth of egg-laying assays.
One of the most critical factors when studying egg-laying preference of single females is to ensure that each female is primed to lay many eggs. (A preference of &#34;50 vs. 0 egg&#34; is much more convincing than a preference of &#34;1 vs. 0 egg&#34;). This protocol, when properly executed, should allow regular wild-type flies (Canton S and w1118) to lay at least 50 eggs/per female O/N. Several factors contribute to producing females with high egg-laying rate. First, one should take care to not overpopulate the vials/bottles when culturing the females to be assayed. Overcrowding often produces smaller larvae, which would grow into smaller females that lay fewer eggs. Second, one should make sure that females to be assayed have access to plenty of yeast paste when mixed together with males in the food vials. It is recommended that 0.5% proprionic acid instead of water be used to prepare yeast paste because proprionic acid not only increases egg-laying but also reduces fungal infection in vials. Third, one should not assay the females until the larvae/food surface of the food vials look like the picture shown in Figure 1F; if not sufficiently egg-laying deprived, females would not lay as many eggs when assayed in the chambers. Also, note that the food vials used here are the &#34;narrow vials&#34;. If one uses vials with larger diameter, one needs to increase the number of females/males to be placed in the vial so that the females would be ready to be assayed in 4 - 5 days.
While the protocol described here specifically focuses on examining females&#39; preferences between sucrose and plain substrates, it can be adapted to study females&#39; preference in other conditions. For example, these chambers can be used to assay flies&#39; preference when choosing between harder and softer agarose (e.g., 1% vs. 1.5% agarose), as well as substrates that differ in other chemosensory cues (e.g., 3% acetic acid). If fitted with lid that contains LED to illuminate one or both substrates, the chamber may also provide an efficient platform to conduct optogenetic studies (Supplementary Figure 4).
Finally, it is worth noting that although this setup allows higher throughput for analyzing egg-laying preferences of Drosophila, it has some important limitations. First, the fixed dimension of the chambers limits the flexibility of the behavioral assay. For example, one will need to make new chambers in order to test how other parameters (e.g., distance between substrates) may affect the egg-laying preferences of flies. In addition, this system does not capture the complex environment Drosophila encounter in the wild. It is very rare that flies will have to decide between a pure sucrose substrate vs. a pure plain substrate for egg-laying in nature. Thus while our assay is efficient in studying the neural basis of a simple decision task, one needs to be aware that the specific &#34;decisions&#34; females make in our chambers may not be ethologically relevant.

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.

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">UltraPure Agarose&#160;</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Sucrose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Water bath&#160;</td>
			<td style="width:104px;">Fisher</td>
			<td style="width:119px;">15-462-6Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LifeCam Cinema webcam</td>
			<td style="width:104px;">Microsoft</td>
			<td style="width:119px;">H5D-00013</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:269px;">Red LEDs</td>
			<td style="width:104px;">Cree</td>
			<td style="width:119px;">C503B-RAN-CA0B0AA1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Egg-laying chambers</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Camera holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">LED holders</td>
			<td style="width:104px;">Custom Built</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Fly vials (narrow)</td>
			<td style="width:104px;">Genesee</td>
			<td style="width:119px;">32-116BC</td>
			<td></td>
		</tr>
	</tbody>
</table>

high throughput assay to examine egg-laying preferences of individual drosophila melanogaster

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 &quot;큰 두 가지 상품을.&quot; 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.

The egg-laying chambers are composed of several pieces: a substrate (bottom) piece, a divider (middle) piece, a loading (top) piece, and 2 sliding doors (Figure 1A - D). These pieces are used to independently setup flies and substrates before egg-laying experiments. Figure 1F shows how vials should look when female flies are ready for egg-laying. When flies are given a choice between a plain substrate and a sucrose-containing substrate, females robustly preferred the plain substrate for egg-laying as shown in Figure 2.
<img alt="Figure 1" src="/files/ftp_upload/53716/53716fig1.jpg" /> 
Figure 1. Egg-laying Chambers and Egg-laying Protocol. (A) Fully assembled egg -laying chamber. (B) Disassembled pieces. a: the loading piece (top), b: the divider piece (middle), c: the substrate (bottom) piece. Engineering drawings of these pieces are shown in Supplementary Figure 1 - 3. (C) The loading piece of the chamber. a&#39;: sliding doors, a&#34;: rails. The plastic sheets are inserted into the loading piece to serve as a floor to keep the loaded flies in place. We typically put color tapes onto the edge of the plastic sheets (red arrow). (D) The substrate (bottom) and the divider (middle) pieces of the chamber. Agarose is deposited into individual troughs to serve as egg-laying substrates (arrows). Quadrilateral outlines the egg-laying arena for a single fly. (E) Day 0 of collected females/males in a yeasted vial. (F) Day 4 - 5 of collected females/males in a yeasted vial. Note that larvae and adult females have eaten most of the yeast and the surface of the food has become occupied by larvae. The wet surface food crawling with larvae prevents females from laying more eggs in the vial. (G) Schematic depicting the protocol for setting up egg-laying behavior experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/53716fig1large.jpg" target="_blank">(Please click here to view a larger version of this figure)</a>
<img alt="Figure 2" src="/files/ftp_upload/53716/53716fig2a.jpg" /> 
Figure 2. Representative Egg-laying Results for Flies Choosing between Sucrose and Plain Substrates. (A) Side view of egg-laying results of wild type flies when they were given a choice between sucrose (150 mM) and plain substrates. White box outlines one egg-laying arena for a single fly. (B) Top view of egg-laying results. White box outlines one egg-laying arena for a single fly. (C) Preference index (PI) of wild type flies when asked to choose between a sucrose-containing substrate and a plain substrate. PI for each female is calculated as follows: (number of eggs on sucrose substrate - number of eggs on the plain substrates)/total number of eggs. Error bar indicates SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/53716fig2large.jpg" target="_blank">(Please click here to view a larger version of this figure)</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/53716/53716supfig1.jpg" /> 
Supplementary Figure S1. Engineering Drawings for the Divider and the Substrate Piece of the Chamber. (A - A&#34;) Different views of the substrate piece of the chamber. (B - B&#39;) Different views of the middle divider piece of the chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/Supplemental1.jpg" target="_blank">(Please click here to download this file)</a>
<img alt="Supplemental Figure 2" src="/files/ftp_upload/53716/53716supfig2.jpg" /> 
Supplementary Figure S2. Engineering Drawings for the Loading Piece of the Chamber. (A - A&#39;) Different views of the upper layer of the loading piece of the chamber. (B - B&#39;) Different views of the bottom layer of the loading piece of the chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/Supplemental2.jpg" target="_blank">(Please click here to download this file)</a>
<img alt="Supplemental Figure 3" src="/files/ftp_upload/53716/53716supfig3.jpg" /> 
Supplementary Figure S3. Engineering Drawing for the Sliding Door and the Rail on the Chamber. (A) The rail fixed onto the top piece of the chamber. (B) The sliding door for the chamber. (<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/Supplemental3.jpg" target="_blank">Please click here to download this file)</a>
<img alt="Supplemental Figure 4" src="/files/ftp_upload/53716/53716supfig4.jpg" /> 
Supplementary Figure S4. Accessories and Customization of Egg-laying Chambers. (A) Egg-laying chambers equipped with cameras to video record flies during egg-laying experiments13. Ctrax14 with extension12 is used to track the flies and custom MATLAB code is used to plot the trajectories. (B) Setup to illuminate egg-laying substrates with red light. LEDs (1) are connected to a LED driver (2). LED intensity is controlled by microcontroller (3). For more information about the camera, light delivery system and tracking animals in abnormal lighting conditions, see Zhu et al. and Stern et al. (C) Red LEDs (light off) attached to top of egg-laying chambers. (D) Red LEDs (light on) attached to top of egg-laying chambers. The LED intensity of red LEDs is typically set to be around 7 - 10 &#181;W/mm when CsChrimson15 is used to activate the neurons. (E) Egg-laying chambers equipped with cameras and LEDs to video record flies during experiments with red light (light off). (F) Egg-laying chambers equipped with cameras and LEDs to video record flies during experiments with red light (light on). (<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/Supplemental4.jpg" target="_blank">Please click here to download this file)</a>

Watch this Scientific Journal Video about High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster at JoVE.com

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

높은 처리량 분석은 개인의 알을 낳는 환경 설정을 검사합니다<em&gt; 노랑 초파리</em

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.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

high-throughput-assay-to-examine-egg-laying-preferences-individual

Nanyang Technological University

Research

JoVE Journal

Neuroscience

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

7.9K 조회수.  Duke University. 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.

비디오: High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

개발 마우스 망막의 생체내 Electroporation에

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

연구

신경과학

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

를 사용하여 화합물 Bioactivity위한 이점 Nanoinjection 및 화면에 대한 전기 생리학 분석 Drosophila melanogaster 거대 섬유 시스템

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

분석의 방법 Drosophila 행동

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

의 이스케이프 문제의 Optogenetic의 자극<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

사회 회피의 정량화에 대한 간단한 분석<em&gt; 초파리</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

사회적 공간에 영향을 미치는 조건<em&gt; 초파리</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

에서 냄새 유도 행동의 고해상도 정량화<em&gt; 초파리</em사용&gt;<em&gt; Flywalk</em&gt; 패러다임

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

의 Mechanosensation에 대한 행동 분석 클론의 MARCM은 기반<em&gt; 초파리</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

모세관 공급 분석은 식품 섭취를 측정<em&gt; 노랑 초파리</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

의 시냅틱 변조 분석<em&gt; 노랑 초파리</em&gt; 장시간 빛에 노출 후 광수

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

높은 처리량 분석은 개인의 알을 낳는 환경 설정을 검사합니다

요약

더 많은 비디오 탐색

이 비디오의 챕터

개발 마우스 망막의 생체내 Electroporation에

를 사용하여 화합물 Bioactivity위한 이점 Nanoinjection 및 화면에 대한 전기 생리학 분석 Drosophila melanogaster 거대 섬유 시스템

분석의 방법 Drosophila 행동

의 이스케이프 문제의 Optogenetic의 자극

사회 회피의 정량화에 대한 간단한 분석

사회적 공간에 영향을 미치는 조건

에서 냄새 유도 행동의 고해상도 정량화

의 Mechanosensation에 대한 행동 분석 클론의 MARCM은 기반

모세관 공급 분석은 식품 섭취를 측정

의 시냅틱 변조 분석