这项工作是由研究经费JWW从美国国立卫生研究院（R01DK092640）和美国国家科学基金会（0920668）的支持。

1。粉煤灰收集和饥饿<ol><li>后面的实验苍蝇在控制温度和湿度条件下（ 如 21℃，50〜60％相对湿度）在12小时亮/暗周期。 </li><li>收集雌蝇在羽化的日子，把它们摆放，伴随着4-5的男性，成为新的食品小瓶（最多30个每小瓶）。年龄过得2-5天。 </li><li>准备室的苍蝇饥饿。 <ol><li>推单一组织（4.8×8.4英寸）下降到一个空的塑料小瓶的底部。完全浸泡的组织用蒸馏水冲洗。使用对象向下推纸巾，轻轻挤出多余的水分。 </li><li>倒置小瓶丢弃多余的水。应该有足够的水，以保持肌肤的苍蝇和饥饿室湿润，但不能足以淹没苍蝇。 </li></ol></li><li>从食品小瓶传送苍蝇进入饥饿室，然后将小瓶约18-24小时，在实验开始之前。存储在受控的温度和湿度条件下的小瓶过夜，直至实验开始的第二天。 </li></ol> 2。食物气味的制备<ol><li>加入0.1克低熔点琼脂糖在一个玻璃烧瓶中加入10ml蒸馏水制备1％琼脂糖溶液。热火在微波琼脂糖溶液只是直到它开始沸腾，但沸腾了好之前。 <ol><li>停止微波和摇动烧瓶一次。重复此步骤两次，直至琼脂糖完全溶解。通过保持烧瓶温暖的加热板上设置在50℃保持琼脂糖溶液在液体状态</li></ol></li><li>加入990微升1％琼脂糖溶液和10μl的苹果醋来1.5ml离心管中，制成1％苹果醋的解决方案。涡旋该溶液直至混合，并装在一个干浴恒温箱设定为50℃。 </li></ol> 3。测试室和行为商会设置<ol><li&gt;将测试室到所需的条件（ 如温度和湿度）。 </li><li>打开LED面板（660纳米）上。 </li><li>冲洗筛和用热水的测试板和加热它们在干燥炉中，直到所有的湿气蒸发。实验开始前，冷却筛和板块下降到检测室温度。 </li><li>定位在扩散板的顶部上的浅盘中并灌满水，以增加局部湿度和掩膜的水在琼脂糖液滴。 </li><li>定位筛过水菜。 </li></ol> 4。飞装载到测试板图与规格测试板可以在补充文件一节中找到。该测试板是由透明的丙烯酸和由6测试领域。一个简单的滑块包含控股室，允许动态加载，暂时的遏制，并同时发布6苍蝇到他们各自在室在实验的开始。十字蚀刻在每个舞台在板中央指示在那里的气味应该吸取。 <ol><li>将滑块到丙烯酸测试板。 </li><li>轻轻吸气泵滑入小瓶过去的棉塞，并允许约6苍蝇走进aspirator.it关键是要尽可能轻柔处理它们。有人可能会采取的趋光飞行为好处，以诱使苍蝇指向一个昏暗的光源小瓶开放抓取朝着吸引。如果有必要，还可以申请温柔的吸力吸出约6雌性果蝇。 </li><li>插入抽吸器的尖端插入测试板的第一孔中。允许单个飞通入牢房，轻轻推动滑块向前加载一只苍蝇进入下一洞。继续，直到苍蝇占据板的所有6个控股细胞。 </li><li>吸取5微升1％，苹果醋琼脂糖溶液直接喷在CR的中心在测试板的内表面OSS-毛发。 </li></ol> 5。定位板测试<ol><li>到中心的检测板，打开名为"定位Tool.vi."文件"LabVIEW的VI的定位工具。六，可以在补充文件一节中找到。通过点击白色箭头在屏幕的左上角运行该文件。 </li><li>将测试板在筛上的顶部，使得竞技场开口面对筛地板和气味的目标是在板的顶。对准蚀刻到测试板的背面与显示器屏幕上的十字线的十字星。 </li><li>当校准完毕后，通过点击红点靠近显示器的左上角中止执行。 </li></ol> 6。记录飞的位置在实验<ol><li>每个食品搜索审讯过程中跟踪和记录苍蝇的坐标，打开采集软件文件"楼Ÿ跟踪 - 六Zones.vi"的LabVIEW VI为"飞跟踪 - 六Zones.vi"可以在补充文件一节中找到通过点击白色箭头在显示器的左上角运行该文件。 。 </li><li>指定文件名，然后单击"确定"。 </li><li>推动滑块在测试室释放苍蝇进入测试领域。要小心，不要移动检测室，因为这会导致不正确的对齐与分析软件坐标。 </li><li>点击"开始"（开始记录），并确保光在测试室的唯一来源是660 nm的LED面板。 </li><li>当试验结束后，取出筛子和行为室。从筛提起的测试板和淹没板冰取出苍蝇。轻轻擦拭板用热水和删除任何琼脂糖碎片。将测试板在干燥炉中以除去水分。 </li><li>通过打开一个SMaL通风试验区升风扇为约2分钟。关闭风扇和负载苍蝇下一组进入下一个测试板。 </li></ol> 7。使用自定义软件数据分析 "对飞跟踪，六个区域的数据分析"，可以在补充文件一节中找到。在数据采集，采集软件记录个人动态位置坐标在文本文件中的每个时间点。一个单一的数码相机定位在所述检测板获得的图像在0.5 Hz的帧速率。该分析软件程序"的飞跟踪，六个区域的数据分析"中提取信息，从该文本文件一）计算出平均车速，b）决定的时间点了一只苍蝇成功地找到气味源，和c）建立图形窗口允许用户查看:飞位置，距离飞从气味源随着时间的推移和平均飞行速度随着时间的推移。它也能格式化，方便的数据导出到一个SPREadsheet程序。在这个宏，食品搜索延时被定义为在其拥有舞台的中心5毫米的半径范围内的花费至少5秒的时间点。 <ol><li>打开分析软件文件"为飞跟踪数据分析 - 六区"。在"窗口"选项卡，点击"创建新表。"直到已建立六个表重复此步骤。 </li><li>在"宏"选项卡，点击"Foodfinding。" A主面板应该会出现以下选项:打开原始数据文件进行布局;打开原始数据文件的数据文件;飞位置，距离，速度，布局; FormatDataFile。 </li><li>要查看原始数据，而不附加值到一个文本文件，点击"打开原始数据文件的布局。"找到并选择在浏览器窗口中显示的实验数据文件。点击"打开"。 </li><li>点击"飞的位置"在所有六个领域（6 XY坐标图描绘每个飞"中查看每个苍蝇的位置随着时间的推移地位应该出现在屏幕上）。 </li><li>点击"距离"，从气味源（6宗地块从气味源随着时间的推移描绘了苍蝇的距离应该出现在屏幕上）查看每个飞的距离。水平线在y =5毫米表示其上飞被认为是定位在食物源的阈值。 </li><li>点击"速度"的审判（6宗地块描绘了苍蝇的速度随着时间的推移应该会出现在屏幕上）中查看每个苍蝇的平均速度。 </li><li>点击"布局"与所有除了平均速度（第50秒时），并找到气味源的每个飞（图1）的延迟蝇位置，距离和速度的图表显示的布局。以正确观赏的布局，它可能有必要调整边界。要做到这一点，首先单击布局窗口。在"文件"选项卡，点击"为布局页面设置"。重置边距0.2英寸并单击"确定"。马上离开的每个位置情节是一张小桌子与标题"速度"和"延迟"。每个标题下输入的数值表示在毫米/秒，并在几秒钟内食品搜索延迟的平均速度。潜伏在空白项表示飞未能找到气味源。食品搜索延迟定义为时间点的苍蝇花了至少5秒从腔的中心有5 mm的半径之内。 </li><li>若要打印布局，点击布局区域（更新布局，当前文件）。点击"文件"，然后点击"打印布局"。 </li><li>要查看下一个文件，只需点击"打开原始数据文件的布局。"单击要查看和点击下原始数据文件"确定"。单击布局窗口上，用新的数据文件更新窗口。 </li><li>该设置可以保存供以后使用的实验文件。 </li></ol> 8。从出口数据数据分析软件到电子表格程序<ol><li>要导出速度和延迟数据为每个文件，点击"打开原始数据文件的数据文件。"选择一个实验数据文件并单击"打开"。将出现一个新的浏览器窗口。 </li><li>在新的浏览器窗口中，在"新建"单击鼠标右键，然后单击"文本文档"。命名新的文本文档。选择新命名的文本文件并单击"打开"。该存储数据从原始数据文件转换成文本文件。 </li><li>若要从另一个文件导出数据，点击"打开原始数据文件的数据文件。"单击另一个文件，然后单击"打开"。选择从步骤8.2文本文件）。继续这个过程要导出剩余的数据文件。 </li><li>一旦所有所需的数据文件已被添加到文本文件，单击"格式数据文件。"选择用于存储从前面的步骤数据，然后单击"打开"（新windo的文本文件W将自动打开）。 </li><li>在窗口中新建一个文本文件，指定文件的名称，然后单击"保存"。这将创建一个包含文件名，平均车速，和延迟对于每个飞，可导入到电子表格程序的文本文件。 </li><li>累积曲线是从上苍蝇到达食物气味靶作为时间（图3）的函数的总数量数据构成。 </li><li>实验数据集被用于使用z检验对比例的统计显着性分析。 </li></ol>

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作者宣称没有竞争的经济利益。

在这个协议中，我们描述了食物搜索行为分析一步一步的过程。除了食物有关的气味，它也可适于用于苍蝇的定位其他气味物体的能力的研究。例如，它可能会朝雄蝇3在队友的本地化行为的研究应用还有几个额外的考虑这个协议，我们将在这里提到有关此过程: 首先，饲养温度决定实验苍蝇多久应该进行老化测试之前。建议年龄的范围进行检查，以确定最合适的年龄为实验。例如，在我们的经验，当在21℃下饲养，喂养饥饿和苍蝇的反应之间的差异是他们已经老化4-5天之后最强大的。 其次，LED灯照亮的玻璃扩散板供应创建常量，日下方均匀照明Ë亚克力室。充分均匀，恒定的，明亮的照明是苍蝇的运动自动跟踪的关键。光线不均匀或闪烁的光源，可能会导致在，要么导致间歇性无法检测果蝇的位置或使软件误以为光文物为飞蝇自动跟踪误差。我们发现这两种市售的LED背光源或定制的LED阵列的工作同样在满足照明需要这个实验。 第三，如果该软件错误地检测到光线的微小变化或琼脂糖作为额外的对象，在"飞跟踪，六个区域的"采集软件的物体检测的设置可对阈值，以及对象的大小调整。调整检测设置确保只有一个对象中的每个检测到的竞技场。要查看被跟踪在每个舞台上的对象的数量，点击"飞跟踪六个区域生态的"阈值"选项卡ES"采集软件。如果超过一个对象被追踪，人们可以调整闵大小，最大，最小阈值，或最大阈值，直到检测到神器消失。 四，飞在这些实验中使用的股票应isogenized。在这种模式的行为表演，是在遗传背景的差异非常敏感。交配的雌性用于降低与配套的状态或性别相关联的潜在行为的变化。我们没有理由相信这个实验不会是同样有效学习的男性或女性的处女之行为。 第五，筛应略高于光扩散板暂停，以防止气味梯度（暂停与我们的商业购买的型号大约2公分）的气味饱和度。我们使用市售的筛子来创建测试板下方的多孔层。 最后，为了产生可靠的数据套，一致性，需要在饲养试验和试验条件。倘未能看到控制苍蝇喂养饥饿和反应之间显著的差异可以通过检查来确保1）苍蝇稳定的温度和湿度条件下饲养得到解决，2）苍蝇喂以新鲜的食物和拥挤的条件不饲养， 3）新eclosed飞暴露于CO 2被最小化，4）苍蝇经历饥饿的长度相同，5）蝇稳定的温度和湿度条件下进行试验，和6）的测试环境和腔室不沾染从以前的试验气味或实验。除了上述参数，蝇股isogenization是重要的，因为不同的遗传背景可以在该测定中影响动态性能。而且，如果醋作为气味源，必须小心，以确保它不会被保持紧密地密封，并保存在4℃下失去其效力6或四场嗅觉4,5飞测定。

饥饿的国家促进两种欲求行为:寻找食物和食品消费1。这个简单的行为测定法是与觅食2,3相关的趋化行为的研究非常有用。具体来说，它跟踪飞位置，行走速度和延迟定位食物气味的目标。食品发现的延迟作为度量测量的变化在苍蝇的气味探测系统的变化在其内部欲求状态的下游的灵敏度。此法的手动版是以前用来显示GABA-B受体信号是很重要的气味本地化行为，成蝇3。检测的电流自动版本是有助的在多短神经肽F（sNPF）信令重塑地图嗅觉的果蝇和影响欲求行为2的研究。 测试是在黑暗的，温度和湿度控制的房间进行。数字上面的透明亚克力板的测试设置摄像机跟踪苍蝇由660 nm的LED照明背光。从相机信息是实时由驻旁边的测试区域的计算机进行处理。我们使用的数据采集软件在测试期间录制和保存苍蝇位置的坐标。 在这个范例中，受试者被释放到包含在该中心食品气味的舞台;气味对象创建诱导苍蝇的食物搜索行为竞技场内食品串味梯度。类似的气味搜索协议已对chemosensation在单一果蝇幼虫7的研究应用。而其他行为分析，如四场嗅觉4,5或T型迷宫6评估厌恶的气味或退房的行为，这种模式是最适合评估嗅觉的灵敏度和趋化行为。 几个关键的优势伴随着这个ASSA年。首先，它允许快速获取大数据集，因为数据收集和分析大多是自动化的。第二，该测定分离和测量单个苍蝇的行为，从而消除社会嗅觉可能影响他们的行为。三，协议和简单的实验设计的简单性使检测效率，容易教别人。 另外，此法可用于进一步探讨神经回路基本食品的搜索行为由它提供给果蝇 8的广泛的遗传工具箱结合。转基因的靶向表达，沉默或激发的神经元可以与如GAL4-UAS系统，以及无人机，shibire TS1，UAS-破伤风毒素，和UAS-TRPA1（B）工具来实现转基因9-12。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Apple Cider Vinegar</td>
			<td>Spectrum</td>
			<td></td>
			<td>commercially available</td>
		</tr>
		<tr>
			<td>Agarose, Type VII</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td>low gelling temperature agarose</td>
		</tr>
		<tr>
			<td>Acrylic Testing Plate</td>
			<td></td>
			<td>custom</td>
			<td>Plate contains 6 arenas. Each arena is 60 mm in diameter 6 mm in height. See testing plate diagrams for specific measurements.</td>
		</tr>
		<tr>
			<td>LabVIEW V.8.5</td>
			<td>National Instruments</td>
			<td>776670-09</td>
			<td>platform for programs: PositioningTool.vi, FlyTracking--Six Zones.vi NOTE: &#34;elapsed time.vi&#34;, &#34;time into file.vi&#34;, and &#34;two object detect.vi&#34; are included subroutines that must be available in order for the main data acquisition program &#34;FlyTracking--Six zones.vi&#34; to run.</td>
		</tr>
		<tr>
			<td>LabVIEW Vision 8.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Vision Acquisition Software 8.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Vision Builder AI 3.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Igor Pro V.6</td>
			<td>Wavemetric, Inc.</td>
			<td></td>
			<td>platform for macro: Data Analysis for Fly Tracking--Six Zones</td>
		</tr>
		<tr>
			<td>Basler scA1390-17fm</td>
			<td>National Instruments</td>
			<td>779980-01</td>
			<td>Digital Camera NOTE: driver for camera available at Baslerweb.com</td>
		</tr>
		<tr>
			<td>8 mm lens</td>
			<td>National Instruments</td>
			<td>780024-01</td>
			<td>Lens for Basler Digital Camera</td>
		</tr>
		<tr>
			<td>Ground Glass Diffuser Plate</td>
			<td>Edmund Optics</td>
			<td>custom</td>
			<td>Diffuses light, 25 cm x 30 cm</td>
		</tr>
		<tr>
			<td>US Std. No. 100</td>
			<td>Fischer Scientific</td>
			<td>04-881X</td>
			<td>Sieve with nominal opening of 150 &#956;m</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Lighting Option 1</td>
		</tr>
		<tr>
			<td>LED backlight 660 nm (20 cm x 20 cm)</td>
			<td>Spectra West</td>
			<td>BL47192</td>
			<td>a simpler but more expensive lighting option.</td>
		</tr>
		<tr>
			<td>Power Supply for LED Backlight</td>
			<td>Spectra West</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Lighting Option 2</td>
		</tr>
		<tr>
			<td>660 nm LEDs</td>
			<td>Superbrightleds</td>
			<td>RL5R1330</td>
			<td>Wavelength 660 nm (approximately 7 x 7 LED array for a 14.7 inch x 9.75 inch panel)</td>
		</tr>
		<tr>
			<td>Linear DC Power Supply</td>
			<td>GW Instek</td>
			<td>GPS-1830D</td>
			<td>Power supply for LED Panel</td>
		</tr>
		<tr>
			<td>Solderless Breadboard</td>
			<td>Digikey</td>
			<td>922354-ND</td>
			<td>Breadboard for LEDs</td>
		</tr>
	</tbody>
</table>

a single-fly assay for foraging behavior in drosophila

对于许多动物，饥饿促进了有利于寻找适当的食物来源的方式变化的嗅觉系统。在这个视频文章中，我们描述了一个自动检测测量饥饿或过饱的嗅觉依赖食物搜索行为的影响在成年果蝇果蝇 。在一个不透光的盒子由红灯是看不见的果蝇照明，链接到自定义数据采集软件的摄像机同时监控6苍蝇的位置。每个飞局限于走在包含在该中心食物的气味个别领域。测试领域休息多孔层的功能，以防止气味的积累。延迟来定位气味源，它反映在不同的生理状态嗅觉灵敏度的度量，是通过软件分析确定。在这里，我们讨论运行此行为范式的关键力学和涉及有关飞LOADI具体问题纳克，气味污染，检测温度，数据质量和统计分析。

数据分析软件和布局，其中的一个例子可在​​图1中可以看出，是根据一组数据的标准使用其10分钟试验期间，以评估每个蝇的效能。下列标准用于确定是否从每个苍蝇的数据将被用于数据分析，其目的是消除这些苍蝇是无法执行食品搜索任务因受伤，疾病，压力，或缺乏动力。 苍蝇不活动的时间超过300秒被认为是"无效"，并从数据集将被拒绝，除非它们一）已经成功地在寻找食物源或b）表现出平均车速&gt; 10毫米/秒，至少100秒下列非活跃期间（图2a，2b，2c）的 。 健康的苍蝇后，立即从他们的容纳腔释放表现出强大的搜索行为。因此，只选择那些苍蝇在积极寻找气味的早期阶段表现出健康的速度，只苍蝇这一举动在一定的速度范围内第一个50秒的审判之内被接受的数据分析。该标准是基于我们的观察是:a）苍蝇靠近气味源，它们的速度降低以及b）几只苍蝇到达第一个50秒的检测范围内的异味目标。速度标准是通过评估至少100控制蝇类在给定的试验温度下的平均速度来确定。标准偏差分别为 - 上/下的车速限制是由平均速度+ /套。例如，在21℃下，只苍蝇在第一个50秒的审判3.5-10.5毫米/秒之间移动用于数据分析。此规则的例外是苍蝇的成功定位的前50秒内的气味源，因而比低限速慢做。 苍蝇不动，通过所有四个象限中的舞台上那些和头直为试验后的食物源开始将被拒绝（图2d）。 该编织朝向和远离10mm的半径为最小的50秒内的食物来源蝇被认为已经成功地找到的食物来源。苍蝇从气味源随时间推移的情节描绘的距离可以用来评估这种罕见的情况。这是一个成功的搜索（图2e）的唯一实例不会自动由当前的数据分析宏观检测，并且必须手动检测阿里纳斯在飞位置跟踪中可见的文物将被拒绝。工件可以通过其中的数据采集软件检测对象比苍蝇其他任何情况下被创建。他们经常出现跨越整个舞台或从它的中心（图2F）辐射为长而直的线条。 在图3中，成蝇饿死18-24小时表现出较高的Ôlfactory敏感性与食物有关的气味比他们的同行喂养1。苍蝇的累计百分比，成功地找到食物的气味源的图形曲线显示在10分钟的窗口都饿死苍蝇的30％成功，相反，只有7％的喂苍蝇这样做（ 图3）。此升高的嗅觉行为反应以前已经证明需要完整的天线1。不遵守美联储和饿死控制苍蝇有着明显的区别在这个实验可以通过检查饲养环境和测试条件加以解决。 故障排除测试条件之一有用的策略是研究苍蝇是否被吸引到除异味目标通过测量吸引苍蝇的气味车辆，琼脂糖等附加线索。饥饿的苍蝇应该表现出显著更大的吸引力，醋，而不是单独的琼脂糖车辆（ 图4b）。图4a展品结果来自于32℃下用野生型果蝇表现为35％的环境湿度，一个食品搜索实验。在这组数据，检测苍蝇吸引醋和琼脂糖控制之间没有显著差异。这可能是由于增加的吸引力的水在琼脂糖液滴中找到下暖测试温度。通过提高测试湿度为50-60％，我们能够纠正这种行为的转变，恢复吸引力之间的显著差异，以醋和琼脂糖车辆（ 图4b，*表示p值&lt;0.05）。 <img alt="图1" fo:content-width="4.5in" src="/files/ftp_upload/50801/50801fig1.jpg" /> 图1。一个典型的分析软件数据布局说明了飞的位置随着时间的推移，飞行距离气味源随着时间的推移，以及飞速度随着时间的推移。在左上角的2列的表格每个赛场角落的前50秒（第1列），并以秒为单位的食品发现（第2列）的潜伏期过程中显示平均速度（毫米/秒）。此外，在打开的文本文件的名称被附加到左下角（图示为，"OZ120807_ORCODTKRi_1％_S4"）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50801/50801fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50801/50801fig2.jpg" /> 图2。不同类型的痕迹的例子在讨论分析的标准，一个 ）。飞了一直无所作为300 +秒被拒绝。 乙 ）飞一个已经无效的定位成功后的食物被接受。 角 ）飞已经处于非活动状态300 +秒，但显示了至少100秒健壮活动后无效时段被接受。Ð。）飞日<html>在直接前往释放后的食物来源被拒绝的 E）。苍蝇在接近和远离10毫米半径最小的50秒内的食物来源编织被认为已经成功地找到了食物来源和被接受。F 。） -阿里纳斯在布局可见的文物将被拒绝。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50801/50801fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="2in" src="/files/ftp_upload/50801/50801fig3.jpg" /> 图3。图形化的情节，显示美联储和饥饿的苍蝇说找到气味源随着时间的推移使用的食品发现潜伏期的累积百分比 。 （N = 88-96蝇; *表示p值&lt;0.05，**表示p值&lt;0.01）。 ad/50801/50801fig4.jpg"/&gt; 图4。故障排除测试条件A）图形显示阴谋苍蝇的发现1％的醋或琼脂糖车辆随着时间的累积百分比。在苍蝇吸引到任何气味的目标中没有检测到显著差异，当测试条件为32℃和35％湿度（N = 62-94苍蝇），B）图形图，显示的苍蝇说发现1％食醋或琼脂糖车辆累计百分比随着时间的推移。测试条件为32℃和50-60％的湿度。在这些条件下，苍蝇显著比琼脂糖车辆更吸引到1％醋（N = 55-71蝇; *表示p值&lt;0.05）。

Watch this Scientific Journal Video about A Single-fly Assay for Foraging Behavior in Drosophila at JoVE.com

A Single-fly Assay for Foraging Behavior in Drosophila

在这个视频文章中，我们描述了一个自动检测测量饥饿或过饱的嗅觉依赖食物搜索行为的影响在成年果蝇果蝇。

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

a-single-fly-assay-for-foraging-behavior-in-drosophila

Nanyang Technological University

Research

JoVE Journal

Neuroscience

A Single-fly Assay for Foraging Behavior in Drosophila

13.1K 次观看.  University of California-San Diego. 在这个视频文章中，我们描述了一个自动检测测量饥饿或过饱的嗅觉依赖食物搜索行为的影响在成年果蝇果蝇。 

视频: A Single-fly Assay for Foraging Behavior in Drosophila

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

染色昆虫行为检测:麻醉的交配行为<em&gt;果蝇</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

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 ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

觅食路径长度协议<em&gt;果蝇</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

口味偏好测定成人<em&gt;果蝇</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

果蝇美容行为的量化

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

单苍蝇检测方法的觅食行为的

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此视频中的章节

在昆虫单感器记录果蝇按蚊

对检测方法果蝇行为

Optogenetic刺激的逃避行为

一个程序，植入脊柱商会纵

染色昆虫行为检测:麻醉的交配行为

一个行为分析对机械感觉MARCM为基础的克隆

觅食路径长度协议

口味偏好测定成人

一个简单的方法来衡量在改建悬赏寻求行为使用

果蝇美容行为的量化