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.

1.准备苍蝇待测定<ol><li>培养蝇在标准糖蜜/在培养箱玉米面媒体设定为25℃和65％的湿度。小心不要过度拥挤的小瓶。例如，将8名女性和男性6成窄食瓶。 注意:这里使用的"窄食小瓶"有2.3厘米内径。我们通常分配大约10毫升飞食品到每个小瓶中。我们用的飞食品配方说明如下:http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/molassesfood.html。 </li><li> 2 - 3天后雌羽化， 如图1E制备用新鲜酵母膏小瓶中。通过混合6克活性酵母用10ml 0.5％丙酸使鲜酵母膏。用刮刀酵母膏施加到食品小瓶的侧壁。 20 35 eclosed女性在一起 - - 25雄性变成小瓶收集30。 <ol><li>一般情况下，收集来自那里的女性被收集在同一小瓶男性（以便节省时间和精力）。然而，如果有关于男性的生育能力的关注，使用WT男性代替。此外，酵母泥是刺激产蛋重要的，因此每日新鲜准备。 </li></ol></li><li>保持所收集的女性/男性在25℃，湿度65％，除非苍蝇温度敏感。 </li><li>经过4〜 - 5天，检查瓶，看看女性是准备好产蛋实验。它们是准备当食物介质的表面是从幼虫在食品积极挖洞湿（ 见图1E - F）。 注:通常女性扣留产卵一次食品表面变湿和幼虫爬行。此步骤可确保女性都准备就绪时，他们遇到所需的纹理基板打好的鸡蛋（ 例如 ，〜1％琼脂糖）。 </li></ol> 2.硐室施工，组装，实验设置<ol><li>有一个加工车间建设丙烯酸产蛋室（ 网络连接古尔1A - D）。不同部分的工程制图示于图补充1 - 3高分辨率照片也可以在这里找到（http://www.rebeccayang.org/pdf/chamber%20design.pdf）。 </li><li>插入塑料片成装载（顶部）片的腔室， 如图1C所示 。这可作为底面而载入飞入个人产蛋阿里纳斯。 </li><li>麻醉于二氧化碳垫女性，单独加载它们到每个产蛋舞台。允许〜30分钟，苍蝇从二氧化碳中恢复，并成为适应新的环境。 </li><li>准备琼脂糖基板。 <ol><li>为方便起见，继续融化的1％琼脂糖预制瓶在55℃水浴。 </li><li>添加的库存蔗糖溶液（2M）的所需量到50毫升锥形管，并与琼脂糖适量混合。例如，以制备150mM的蔗糖基板，PLACE 2M的蔗糖溶液进入管750微升，然后用琼脂糖填充管10 ml刻度线。 </li><li>制备中相同的方式在滑动基底但添加代替蔗糖溶液蒸馏水。 注意:在这个协议琼脂糖的最终浓度略小于1％。 1.1％，而在两个基板是相同的琼脂糖浓度 - 在我们的经验，琼脂糖的确切浓度不只要其被控制为〜0.9内无关紧要。 </li></ol></li><li>就拿基板（底）片室和吸管1000μl琼脂糖基板到每个槽， 如图1D所示。 </li><li>允许琼脂糖巩固约30分钟。 </li><li>一旦琼脂糖基板和苍蝇都准备好了，组装所有三块产蛋室，然后取出塑料片。 </li><li>放置在室内飞孵化器。 注:产蛋实验长度可根据EXPE变化rimental需求。我们通常运行实验O / N（14 - 16小时）。此外，观察昼夜定时上产卵的喜好没有显著影响。 </li><li>通过注入的 CO 2进入室麻醉雌性。拆卸腔，放弃麻醉苍蝇飞入太平间（ 即 ，一个空的咖啡能充满了一些玉米油）。就拿结果记录保存的照片（ 见图2）。 </li><li>手动计数卵的数目，并计算偏好指数分析。计算偏好指数（N A - N B）/（N A + N B），其中N 一和N B上分别代表蛋的数量在现场与网站B，。 </li></ol>

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The authors have nothing to disclose.

该商会在这里描述的协议具有比以前产蛋测定了一些改进。首先，它们提高显著测定单个动物的偏好的吞吐量。每个房间都可以分析30单身女性，它需要不到一个小时来设置。第二，它们增加超过以前的方法产蛋偏好的一致性。竞技场的尺寸的标准化，产蛋基板尺寸，和基片之间的距离可以更容易地不同实验天之间，并从不​​同的研究小组比较结果。第三，腔室可以配备附件以容纳更多的分析人想追求（ 补充 图4）。例如，对于行为分析，摄像机可以被附连到所述腔室的顶部，以记录苍蝇7,11-13。最重要的是，这种方法是可扩展的。这些腔室相对INEX沉思生产与加工车间的帮助。此外，它通常需要技术工人不到2小时，成立5室（150单身女性）值得产蛋化验。 其中一个研究单身女性的产卵偏好时的最关键因素是确保每一位女性被引打好的鸡蛋。 （"50对0蛋"的优先级高于"1对0蛋"的偏好更加有说服力）。该协议，妥善执行时，应该让普通野生型果蝇（广州S和w1118）每个女性O / N打下至少50粒/。几个因素导致了生产的女性具有较高的产蛋率。首先，我们应该照顾培养女性时，要分析不overpopulate小瓶/瓶。过度拥挤常常会产生较小的幼虫，它会发展成更小的女性，再就是减少鸡蛋。其次，应确保当混合在一起的智慧女性被检测有机会获得大量的酵母膏ħ男性在食品小瓶中。建议在0.5％的丙酸代替水被用于制备酵母膏因为丙酸不仅增加产卵，但也减少了在小瓶中的真菌感染。第三，应该不测定雌直到食物小瓶类似于图1F中所示的图像的幼虫/食品表面上;如果没有足够的产蛋剥夺，在室内检测时女性不会躺在尽可能多的鸡蛋。此外，请注意，这里使用的食品小瓶"窄瓶"。若用小瓶直径较大，需要增加被放置在小瓶女性/男性的数量，使得女性将准备在4待测定 - 第5天。 而这里具体描述的协议的重点检查女性的蔗糖和滑动基板之间的偏好，它可以适合于研究雌&#39;在其它条件下的偏好。例如，这些室可以被用来阿萨较硬和较软琼脂糖之间选择ý果蝇的偏好时（ 例如 ，1％和1.5％的琼脂糖），以及在其他化学感受线索不同底物（ 例如 ，3％乙酸）。如果装配有盖即包含LED照亮的一个或两个衬底，腔室还可以提供一种有效的平台进行光遗传学研究（ 补充 图4）。 最后，值得注意的是，虽然此设置允许更高的吞吐量，用于分析果蝇的产蛋偏好，它有一些重要的限制。首先，腔室的固定尺寸限制了行为测定的灵活性。例如，一种需要，以测试如何其他参数（ 例如 ，基板之间的距离）可能影响蝇产卵偏好做出新的腔室。此外，该系统不捕获在野外复杂的环境中遇到果蝇。这是非常罕见的，苍蝇将有一个纯蔗糖基板与对大自然的产蛋一个纯色基板之间来决定。因此，虽然我们的分析是研究一个简单的决策任务的神经基础有效的，需要注意的具体"决定"使女性在我们商会可能不会ethologically有关。

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

近日， 果蝇产卵偏好已经成为一个听话的基因模型，研究简单的决策过程的神经基础。当选择的网站存入他们的蛋，雌蝇能居其选择的相对吸引力和选择的"较大的两种商品。"然而，如果一个人想采取系统的遗传筛选方法的搜索电路基础底层这个简单的决策过程，因为它们是基于人群的和费力的建立最产蛋偏好测定法是不实际的。为了增加单个雌性产卵偏好学习的吞吐量，我们开发了长达三十个人的每个可以同时测定产蛋偏好蝇以及确保每个女性具有高产蛋率的协议自定义腔室（使他们的偏好是容易辨别和更有说服力）。我们的方法是简单的执行并产生非常一致的结果。此外，这些室可以配备不同的附件，以允许视频记录产蛋动物，并为光遗传学研究提供光。本文提供了制造这些商会和程序准备在这些商会进行检测苍蝇的蓝图。

产蛋室由几个部分组成:一基底（底）片，一个分频器（中间）片，装载（顶部）片，和2滑动门（ 图1A - D）。这些作品被用于前产蛋实验独立设置苍蝇和基板。 图1F显示了当雌性果蝇准备好产蛋瓶的外观。当苍蝇给出一个普通基材和含有蔗糖的衬底之间的选择，女性鲁棒优选平原基板产蛋如图2。 <img alt="图1" src="/files/ftp_upload/53716/53716fig1.jpg" /> 图1.产蛋钱伯斯和产蛋协议。（A）完全组装鸡蛋-laying室。 （ 二 ）反汇编作品。答:装件（上图），B:在除法器片（中），C:在基板（底）片。这些条的工程图纸示于补充图1 - 3（C）的装载件的腔室的。一个&#39;:推拉门，一个":导轨的塑料片被插入装载块以用作地板，以保持装载苍蝇代替我们通常把彩色磁带到塑料片材（红色箭头）的边缘。 （D）将基板（底部）和分频器（中）的腔室的部分。琼脂糖沉积到各个槽以用作产卵底物（箭头）。四边形概述了单个飞产蛋竞技场。（E ）收集女性/在一个yeasted小瓶男性0天（F）的第4天- 5收集女性/在一个yeasted小瓶的男性注意，幼虫和成年女性已经吃大部分酵母和食品的表面已成为幼虫占据。湿润的表面的食物的幼虫爬行防止女性在小瓶铺设更多的鸡蛋。 （G）示意图描绘了协议设置产蛋行为实验。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/53716fig1large.jpg" target="_blank">（请点击此处查看该图的放大版本）</a> <img alt="图2" src="/files/ftp_upload/53716/53716fig2.jpg" /> 图2.代表产蛋结果苍蝇蔗糖和平原基板之间选择。（A）野生型的产蛋结果侧视图苍蝇，当他们被给予蔗糖（150毫米）和滑动基板之间的选择。白盒概述了一只苍蝇一个鸡蛋铺设舞台。 （B）的产蛋结果的顶视图。白盒概述了一只苍蝇一个鸡蛋铺设舞台。 （C）偏好指数（<html>当问及含有蔗糖基板和普通基材之间做出选择野生型PI）苍蝇。 - /总蛋号（在平原基板卵数蔗糖基板上的蛋的数目）:PI每个女性计算如下。错误条表示SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/53716fig1large.jpg" style="font-size: 14px; line-height: 28px;" target="_blank">（请点击此处查看该图的放大版本）</a> <img alt="参考图1" src="/files/ftp_upload/53716/53716supfig1.jpg" /> 补充图S1 工程图纸的分频器和商会的衬底块。（A - A"）不同意见衬底片室的。（B - B&#39;）的中间分隔片室的不同意见。D / 53716 / Supplemental1.jpg"的风格="FONT-SIZE:14px的;行高:28px;"目标="_空白"&gt;（请点击此处下载该文件） <img alt="补充图2" src="/files/ftp_upload/53716/53716supfig2.jpg" /> 补充图S2 工程图纸商会的加载件。（A - A&#39;）装载片室的上层不同的看法。 （B - B&#39;）装载片室的底部层的不同意见<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/Supplemental2.jpg" style="font-size: 14px; line-height: 28px;" target="_blank">（请点击此处下载该文件）</a> <img alt="补充图3" src="/files/ftp_upload/53716/53716supfig3.jpg" /> 补充图S3。 <st荣&gt;工程制图的推拉门和商会的轨道。（A）固定在顶片室的轨道。 （B）中的腔室的滑动门。 （ <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53716/Supplemental3.jpg" style="font-size: 14px; line-height: 28px;" target="_blank">请点击此处下载该文件）</a> <img alt="补充图4" src="/files/ftp_upload/53716/53716supfig4.jpg" /> 补充图S4。配备了摄像头，录像产蛋钱伯斯配件和定制。（A）产蛋室期间产蛋实验13苍蝇。 Ctrax 14与扩展12用于跟踪苍蝇和定制MATLAB代码用于绘制轨迹。 （ 二 ）设置来照亮红色二极管灯产蛋基板<html>吨。发光二极管（1）被连接到一个LED驱动器（2）。 LED强度是由微控制器控制（3）。关于相机跟踪动物异常照明条件下的更多信息，光传输系统，并见到朱等人 。和斯特恩等人 。 （C）红色发光二极管（灯灭）连接到的产蛋室顶部。 （ 四 ）红色发光二极管（光）连接到的产蛋室顶部。的红色LED的LED强度通常设置为大约10 - 10μW/毫米时CsChrimson 15被用来激活神经元。 （E）产蛋期间与红光（灯灭）的实验配备摄像头和LED视频记录苍蝇室。 （F）产蛋室期间与红光（指示灯亮）实验配备摄像头和LED视频记录苍蝇。 （T:28px;"目标="_空白"&gt;请点击这里下载此文件）

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

高通量检测，以检查个体产蛋首<em&gt;果蝇</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 次观看.  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.

视频: 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.

在发展中国家小鼠视网膜体内电穿孔

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

科研

神经科学

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.

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

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.

对检测方法果蝇行为

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.

Optogenetic刺激的逃避行为<em&gt;果蝇</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.

简单的检测方法的社交回避的量化<em&gt;果蝇</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.

在条件影响社会空间<em&gt;果蝇</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.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

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.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</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.

毛细管馈线分析测量的摄食<em&gt;果蝇</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.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

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