我们感谢丹尼尔·维特技术援助和佩德罗戈维亚在Electricidade EM宝（electricidadeempo.net）定制的跟踪软件为我们的要求。我们也感谢汤姆Retzke支持在拍摄过程。这项研究是由马克斯 - 普朗克协会的支持。

Flywalk的结构和技术细节已描述在别处26（如果是建立这一设置，进一步的信息可以从MK获得的任何问题）。在这里，我们重点的模式，这将有助于获得可靠的结果的处理的详细说明。 1.飞处理<ol><li>后飞低到下一个12小时的食品中27中密度培养:12小时暗条件在23-25℃，相对湿度70％。为此，让20-30新出现的成蝇在一个大的食品小瓶重现1周，然后丢弃成蝇，等待后代出现。 </li><li>收集30-40新兴（年龄&lt;24小时），成蝇和年龄他们在新的含食物中27 3-5天小瓶。 </li><li>二十四小时的行为实验开始前:传输所有30-40以前收集3-5日龄（见2.2）飞往新VI含有人潮湿橡胶泡沫塞或使用吸气湿润薄纸。 注意:不要麻醉苍蝇用CO 2。 </li></ol> 2.制备Flywalk设置的<ol><li>使用250毫升瓶作为加湿器。填加湿器用100ml蒸馏水。 </li><li>准备气味瓶。 <ol><li>制备500μl的10 -3稀释液的纯气味乙酸乙酯，丁酸乙酯，乙酸异戊酯和2,3-丁二酮在溶剂中的矿物油。 </li><li>附加每气味瓶两分球止回阀。需要注意的是单向阀只允许单向气流。因此，连接止回阀在空气能够进入所述小瓶在一侧并使其保持在另一侧这样的方式。 </li><li>除去200μl的PCR反应管的盖子。吸液管将100μl每气味稀释到一个单独的反应管，并将管在分开的气味小瓶中。还要准备一种气味瓶中只含有溶剂的矿物OI湖</li><li>紧紧通过关闭他们使用不锈钢插头和橡胶垫圈密封气味小瓶。 </li><li>连接5气味小瓶（4含有气味和含有1矿物油中）的气味传递系统。请务必将其连接在正确的流向。一个错误的连接，不仅损害计划实验，但它也有可能污染递送系统。 </li></ol></li><li>通过密封刺激装置的混合室的出口检查是否泄漏。确保，该刺激装置前，所有现在的气流逐步降为零。如果不是，检查它现在可以通过空气中的嘶嘶声使系统来识别泄漏。 </li><li>小心地使用传输双方使用相应的适配器吸气，靠近玻璃管15个人苍蝇至15个人的玻璃管。 注:由于系统必须密封成功的实验，确保适配器适合玻璃管紧并注意玻璃管可以在这一步打破。小心避免受伤戴防护手套和护目镜。 </li><li>连接玻璃管的Flywalk设置，并从这里开始，等待至少15分钟开始实验，让果蝇使其习惯新环境之前。 </li><li>附玻璃管后:检查计算机1上的下行数字流量计的读出，如果玻璃管后16气流加起来进入系统的气流。还检查计算机1上，如果湿度为60％和80％。 </li><li>设计激励协议​​控制呈现给苍蝇的刺激气味的顺序和定时。同时获得例如所描述的数据，本4气味和控制（矿物油）单独使用及所有可能的三元和气味的季混合物为每个40次。设定脉冲持续时间，以500毫秒，在90秒的刺激间隔和随机激励序列。 </li><li>接通李GHT源（LED集群;λ= 630）。确保提供足够的光线进行有效跟踪，而不增加玻璃管内的温度。 </li><li>通过拖动帧跨过区域中，所有15玻璃管包含这样的方式被监视和大约5毫米的管的边缘被排除设置的跟踪系统的感兴趣区域。 </li><li>成立于跟踪系统各个管之间的平行14分线，通过改变它们的Y位置在相应的脚本，以保持个人的整个实验过程中苍蝇识别。务必将其以这样的方式定位总是有两个这样隔线之间的一个玻璃管中，因为只有一个飞将任何一组的两行之间进行跟踪。 </li><li>确保在苍蝇可靠地跟踪整个玻璃管这样的方式设置相机参数。如果蝇丢失在感兴趣区域的边缘，增加亮度的跟踪s或增益oftware。避免机械跟踪系统的振动。跟踪根据制造商的方案使用商业软件。 </li><li>通过启动经济刺激方案开始实验。记录flies&#39; XY坐标在20 FPS（每秒帧数），并登录在文本文件中的气味阀门状态组合。 </li></ol> 3.数据分析注:在数据分析下面的步骤是使用在R编程自定义编写例程automatized因为这些步骤是关键，得到分析将仍然在一步一步地被呈现有意义的结果。的原始数据的分析是含有上气味阀状态，脉冲数在实验和15飞的x位置，以cm，并持续一个刺激气味周期共同的时间轴同步信息的.csv-文件。根据要求可以提供数据分析的自定义代码。 <ol><li>开放的.csv文件，发现时间点阀的开度由茶所指的NGE在代表阀门状态列。 </li><li>计算的形式的气味位置的线性函数 F（T）= S * T + I 其中t是时间的刺激循环，s是风速（此处20个厘米/秒），我可以利用时间点的气味进入管在位置0（开阀加上延迟）来计算截距。 </li><li>寻找在该气味的时间点和飞行x位置相交，每飞行，并将此时间点为0。注:这种方式飞行位置对准每个individual&#39;s遇到的气味。 </li><li>排除蝇坐在感兴趣区域的非常边缘。 </li><li>通过将沿x轴位移的时间间隔（100毫秒），并重复步骤为每个刺激周期来计算速度从X位置。 </li><li>以获得速度时道，如图2E计算平均速度的时间-过程为每个蝇和气味，并从那些平均时间过程对于给定的气味。 </li><li>气味脉冲对每个跟踪事件，之后每苍蝇和气味的平均净位移后，得到净位移，如图3C计算4秒钟内的净位移。 </li></ol> 4.清洗程序<ol><li>清洁玻璃管<ol><li>从玻璃管中取出的苍蝇和适配器泡的玻璃管洗涤剂。 </li><li>冲洗玻璃管下运行的蒸馏水，用压缩空气干燥。 </li><li>热玻璃管，在200℃8小时。 </li></ol></li><li>清洁气味输送系统<ol><li>从中央混合室中取出所有的气味瓶和管道。 </li><li>从混合室中取出管道适配器。 </li><li>清洁混合室通过用实验室清洗溶液和溶剂（如乙醇，丙酮）漂洗。执行实验室引擎盖下这些步骤。 </li><li>利用干式混合室的加压空气和加热它在200℃8小时。 </li></ol></li><li>清洁气味瓶和止回阀<ol><li>卸下钢塞（丢弃橡胶垫圈），并检查从气味瓶阀门，浸泡所有组件在实验室的清洁液。 </li><li>在超声浴中超声处理元件和冲洗它们用蒸馏水。 </li><li>清洗除了用乙醇和丙酮止回阀所有组件。执行实验室引擎盖下这些步骤。 </li><li>使用加压空气和干燥部件加热它们在200℃下8小时。 </li><li>上的内部的清洁单向阀通过用乙醇和使用注射器丙酮冲洗它们（考虑流动方向）。执行实验室罩戴护目镜实验室根据这些步骤。由于丙酮用压缩空气冲洗他们攻击橡胶件，立刻干止回阀。 </li><li>通过脉冲空气通过单向阀几天清除残留异味。此清洗步骤中使用的培养箱，在60℃，1秒的空气上/ 1秒的空气断制度。 </li></ol&gt; </li></ol>

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作者宣称，他们有没有竞争的财务权益。

虽然Flywalk系统似乎相当复杂乍看之下，一旦建立并运行它易于使用，并产生非常强大的结果。强调与生物测定所产生的结果的一致性，可以说的是，这里示出的代表性的结果差不多2年后一些与使用新的跟踪软件和光源的变形设置示出在先前的研究29的结果获得的。尽管如此，引诱反应是 - 尽管略高反应振幅 - 非常类似于以前发表的关于他们的动态。 有哪些应被视为尤其为了得到使用Flywalk高质量效果一些关键方面。重要的是，湿度不实验的过程中，低于60％。一个典型的实验性会议持续大约8小时。合理大小的女性CS野生型果蝇除权前禁食2​​4小时periment至少12小时的实验提供设置湿度容易生存足够高。为了避免对湿气，最好是安装一个湿度传感器在一个空的玻璃管（图1B），并避免将流量调节器中的气流的加湿器和玻璃管之间的任何相关的问题。它也是该系统是密封的数据质量绝对必要的。离开系统的玻璃管后气流应该总结到进入系统的气流。最失败的实验可以归因于在系统中，并非常小心泄露应采取实验开始前，要确保该系统是气密。最后，正如用在嗅觉研究中使用的任何设置，主要的日常问题之一是，以避免污染。大部分部件进入与气味接触的是由玻璃，钢，铁弗龙或PEEK，因此可以被加热到至少200℃，这是足以除去大多数öDORS除了那些以特别高的沸点，例如长链信息素。因为止回阀含有橡胶部件它们不能被加热为高，因此，是污染的主要来源，这就是为什么一个特定的洗涤协议被设计用于它们。尽管如此，最好是跟踪特定止回阀已接触到的气味。在有关其洁净度有疑问时更换。 作为系留测定和实验队列之间的折衷，当然Flywalk也有一些缺点相比其他方法。范式 是非常有效的，当向多个不同刺激的行为已经进行评估和比较。值得注意的是，由于响应时间过程15个人只有一个脉冲一个给定的气味强烈类似于在一个完整的数据集获得的响应时间，课程（ 即 30苍蝇和40介绍每一个;图2D，2E </str翁&gt;），有可能通过使用不同的刺激的方法，例如偶合系统的气相色谱仪，以进一步增加其在给定的实验提出的不同刺激的数目。因为实验会话的持续时间。然而，只有一个实验是每天可能的，并且一个给定的实验应至少重复一次，以获得可靠的结果。因此，阱测定法或T型迷宫是当例如只有一个气味的享乐化合价需要研究更有效的。此外，视觉跟踪系统的时间分辨率通常比最快生物测定如系留飞行或跑步机的下部。报道的果蝇气味引导行为最短响应延迟远低于气味相遇后100毫秒在系留范式20,23并且因此落入的时间窗以10Hz分析数据时，不能得到解决。然而，在Flywalk引诱反应通常在第100-300开始毫秒（图3B），这是很好的，在自由飞行的苍蝇5观察到逆风浪涌延迟线。因此，仍然需要确定该差值在响应延迟在系留范式是否相比风洞和Flywalk是造成在视觉跟踪范式或苍蝇的系留情况更高觉醒状态在空间和/或时间分辨率的差别。 总之，Flywalk是一个没有选择的生物测定，它结合了拴系测定的高度控制刺激呈现与队列实验在果蝇神经行为经常使用的效率。因为同一组的个体可以用多种不同的刺激受到挑战，其具体的强度比较朝不同的刺激反应时在于统计功率。此外，它利用了逆风飞波涛把一个有吸引力的气味相遇的事实，这种方式脱开气味评价从无需梯度作为定向线索气味源定位。因此，应当非常适合利用光遗传学工具箱果蝇可用 。

任何神经行为学研究的总体目标是建立单个神经元或神经元电路的活动状态和生物体的行为之间的因果关系。为了实现这一目标的神经元活动和行为应该在相同的激励条件被监控并且这些刺激条件下理想地应当类似于受到审查神经系统发展到使感。特别是当它涉及到行为的生物测定，这些需求在历史上被证明相当苛刻果蝇嗅觉神经行为学。 一旦从源释放，气味羽状迅速分解成薄细丝造成空气运动是气味分布1的主要决定因素湍流扩散。其结果是，昆虫导航朝向气味源经历间歇刺激与穿插的洁净空气可变间隔气味包。都步行和飞行昆虫-包括果蝇 -已被证实通过在羽遭遇逆风飙升，主要是移动的侧风在没有气味2利用这一间歇刺激制度导航- 5。而在生理实验刺激程序基本上模仿那些昆虫可以通过提供单个抽吸气味穿插的洁净空气或动态刺激序列6长时间在其天然环境体验- 11，许多行为的生物测定在果蝇神经行为使用，诸如阱测定，旷场阿里纳斯或T型迷宫依靠气味梯度的12 - 15。但是，由于气味梯度的定义是在浓度可变根据来自气味源的距离，特定行为不能归因于使用这些范例精确臭气浓度。另外，该斜率气味梯度关键取决于臭气物质的物理化学性质。高挥发性化合物的梯度将比由低挥发性化合物创建并因此也更难跟踪生物体依靠测量浓度差在空间导航16的唯一手段较浅- 20，这可能导致一个曲解嗅觉喜好特别是在选择检测。这个效果也非常有害调查朝气味的混合物时的行为，因为它导致不同混合组分比率在空间中的每个点，因此，再次排除生理和行为之间有明显的相关性。 虽然醋蝇倾向于聚集在发酵的水果，它们是独居在他们对食品来源和产卵场所导航。然而，用在果蝇 neuroetholo而不是测试个别动物的许多行为范式gy的检查蝇同伙的气味引导行为和吸引力的得分为苍蝇选择气味过控制刺激的分数。这些人群实验极大地促进了飞神经行为学的理解，许多使用他们提出的意见可以在单飞行实验来证实。然而，已经观察到，苍蝇可以影响每个other&#39;s 第 21和在极端情况下的气味的评价，可以从漠不关心切换撤销取决于人口密度22。此外，从这类实验结果往往提供的行为决定，而不是观察什么苍蝇做，而它是这样做，试图与相关神经元活动的行为时，这将是可取的序列的只有终点。这些相当低解析度人群实验由高分辨率的单飞的方法，例如允许系留飞行竞技场和跑步机对比在当时直接观察的行为反应刺激呈现20,23,24。然而，队列实验仍然很受欢迎，因为他们是非常有效的，并提供强大的结果，即使在相对较低的样本大小，因为个体间和审间的变化被部分平均出由于观察人口过长时间。而系留飞行和跑步机可能提供关于刺激呈现和时间分辨率的金标准，所使用的领域被设计用于单一动物并且因此费时，得到样品大小所需的统计分析。若干其他方法最近已开发出允许一个高效采集高分辨率行为数据的结合定义良好的刺激机制。这些包括无监督3D跟踪多个醋蝇在风洞中组合与气味羽5的精确的3D模型</SUP&gt;，多个单独的苍蝇在气流从两侧25和 Flywalk范例26供应商会选择跟踪。 在Flywalk，15个人的苍蝇都坐落在小玻璃管，并通过高架摄像头下的红色光条件下连续监测。气味被添加到20厘米/秒的连续气流，并通过玻璃管以恒定的速度行进。气流是通过250毫升瓶进入气味输送系统之前，含蒸馏水（加湿器）传递给它加湿。所述flies&#39;位置的感兴趣区（ROI）包围大部分的气味管的长度（但不包括管子（大约5毫米处每侧的外边缘的正方形区域），其中所述蝇不能移动进一步上调或内记录顺风）周围气味介绍（图1A，B）的时间。飞身份保持恒定由跟踪系统吨hroughout与其Y位（即他们的玻璃管限制）的基础上进行实验。刺激气味是使用一种多组分的刺激装置，该装置允许多达8个单气味和所有可能的混合物26,29（图1B）的介绍实现。一个实验的过程是由计算机调节气味递送系统以及收集的温度和湿度信息（计算机1， 图1C）来控制。这台计算机还控制数据记录器（开始/停止录音）它连续不断地跟踪每秒20帧（电脑2）飞行位置的第二台计算机上。飞位置，气味阀门状态（ 即时间点阀门开度），气味ID，温度和湿度周围的气味刺激周期登录计算机2这样的信息对气味和飞位置是同步的，并出口为.csv文件，文件，这可进一步加工，并使用自定义编写分析程序进行分析。因为整个系统是计算机控制的，没有人的干预的实验会话期间是必要的。

<table border="0" cellpadding="0" cellspacing="0" width="866">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flywalk setup</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td style="width:207px;">details available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stimulus device</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td>details available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LED cluster</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td>details available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HD Pro Webcam C920</td>
			<td style="width:325px;">Logitech, Lausanne, Switzerland</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 Computers</td>
			<td style="width:325px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:216px;">Flywalk Reloaded v1.0 software</td>
			<td style="width:325px;">Electricidade Em P&#243; (electricidadeempo.net)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Labview 11.0 software</td>
			<td style="width:325px;">National Instruments, Austin, TX</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard fly food</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:216px;">Standard fly vials</td>
			<td style="width:325px;">Greiner bio-one GmbH, Frickenhausen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard fly vials</td>
			<td style="width:325px;">Greiner bio-one GmbH, Frickenhausen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">aspirator</td>
			<td style="width:325px;">Custom</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mineral oil</td>
			<td style="width:325px;">Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">odors</td>
			<td style="width:325px;">Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:216px;">200 &#181;l PCR reaction tubes</td>
			<td style="width:325px;">Biozym Scientific GmbH, Oldendorf, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution quantification of odor-guided behavior in drosophila melanogaster using the flywalk paradigm

在自然环境中，昆虫等的飞醋果蝇轰炸了大量的不同化学气味物质。为了进一步使问题复杂化，由昆虫的神经系统检测到的气味通常不是单一化合物而是混合物，其成分和浓度比例有所不同。这导致具有由神经系统进行评估不同嗅觉刺激的几乎无限量。 为了理解其中的刺激气味的各方面通过飞确定其评价，因此，希望能够有效地检查气味引向许多增味剂和气味的混合物的行为。要直接关联的行为神经元的活动，行为应该量化可比较的时间框架，并在相同的刺激条件下在神经生理学实验。然而，在果蝇神经行为学许多目前使用的嗅觉生物测定相当斯佩恰lized无论是对效率或实现的分辨率。 Flywalk，一个自动气味递送和跟踪系统，桥梁效率和分辨率之间的差距。它允许什么时候气味包刺激了行走自如飞行，并确定宠动态行为反应的决心。

因为果蝇被允许通过玻璃管气味脉冲和气味脉冲行进之间自由分发他们的玻璃试管内以恒定的速度苍蝇遇到气味在不同的时间取决于它们的x位置处的刺激的时间。其结果是，通过一个有吸引力的10 -3稀释的乙酸乙酯的500毫秒脉冲诱发逆风轨迹的起始点是由约1秒，在他们的玻璃管的顺风端相比，这些苍蝇坐在接近的延迟为蝇逆风端以20厘米/秒和20厘米的玻璃管长度（图2A）的风速。校正在气味遭遇用于根据它的x位置在气味上介绍的时间每一个个体的时间差表明，向乙酸乙酯响应延迟是不同个体（图2B）一致。 相应地，平均迎风不改正邻轨迹多尔行程由大约0.5秒延迟相比，平均轨迹与校正（ 图2C;请注意，校正的气味需要输入玻璃管两个轨迹进行的逆风端的时间）。另外，修正后的平均迎风轨迹为一个单一的气味脉冲还显示更陡的斜率（即更高的步行速度）比未校正1（图2D）。类似的意见用于单个气味脉冲，省略了校正的气味旅行导致在一个完整的数据集，由两个实验会议（即30蝇）与40展示气味脉冲的增加的延迟和低响应振幅每个（图2E） 。 反复刺激的吸引力10 -3稀释丁酸乙酯的500毫秒脉冲（ETB），异戊基乙酸（IAA），乙酸乙酯（ETA）和2,3-丁二酮（BEDN）引发后外径逆风浪涌或饥饿雌性果蝇中相遇，而刺激与溶剂矿物油（MOL）唤起没有或仅有微弱的回应。单独的机械刺激以前已显示诱导以类似的范例28增加的运动。但是，由于刺激气味的Flywalk模式不会改变总的气流，并增加运动大多是在没有使用MOL控制的情况下，这些上风潮反映真实气味的反应。平均响应时间课程，跨越个人（ 图3A）和气味特异的潜伏期千篇一律，幅度和持续时间（ 图3A，B）。响应乙酸乙酯显示急剧发作，高幅度和一个持续时间短。与此相反，应对2,3-丁二酮通常显示一个略晚发病，低级幅度和一个持续时间较长。丁酸乙酯和乙酸异戊酯引发类似时间动态如乙酸乙酯，但反应是在较低的幅度。 Correspondingly，所有4气味气味遭遇比该溶剂和阴性对照矿物油（图3C）后引起4秒钟内较高的逆风位移。 使用相同的4引诱，它以前曾表明，引诱二元混合物是至少有吸引力的更具吸引力的混合物构成29。在这里，这个观察是通过测试所有可能的三元混合物，并充分混合所有4引诱延长。类似于先前观察与二元混合物，所有这些更复杂的共混物的至少有吸引力的最有吸引力的单一化合物（图4A）。最有吸引力的共混物是那些含有乙酸乙酯和2,3-丁二酮。响应这些3掺合物不显著彼此不同，也是响应动力学惊人地相似（ 图4A，B）。与此相反，省略乙酸乙酯从全BLENð导致最大逆风速度的降低，同时省略2,3-丁二酮缩短了响应（图4C）。由于醋酸乙酯引发短期的高振幅响应，而丁二酮引起幅度较低，但持续时间较长（ 图3B，4D）的反应，这些意见是让人想起我们以前的发现，即响应时间的课程对引诱的混合物倾向于遵循一个最佳的响应时间，路线从混合组成的响应时间，课程29创建的。在此数据集的所有4引诱的最佳可能的响应时间菜朝乙酸乙酯和2,3-丁二酮的基础上构造。丁酸乙酯和/或异戊基乙酸是必要的，除了达到最大行走速度在朝向充分混合（图4D）的反应观察到。因此，从2至3或4的组件的增加的复杂混合物增加了混合物e的吸引力VEN进一步比会从以前的观察可以预期，这对引诱的混合物的反应是朝着混合成分的反应的最佳。然而，一般的结论是，组分化合价是保守的气味混合物保持有效也为这些更复杂的混合物29。 <img alt="图1" src="/files/ftp_upload/53394/53394fig1.jpg" /> 图1.原理和Flywalk设置布局。（A）的原理示意图。黄方:刺激气味通过管子移动并导致飞上风运动;黑色物体:摄像机跟踪行为反应。通过用活性炭过滤空气设置的气流（B）的示意图被加湿并分成8个信道，在进入气味递送系统26,30之前。吹胀图:1，三通电磁阀通<html>荷兰国际集团的气流或者通过一个空药瓶（C;补偿性流），或通过含有气味来源小瓶（O;气味流量）; 2，球形止回阀，以限制空气在一个方向流动，并避免系统污染;混合室:定制盒，收集的空气从所有电磁阀并且转移到分裂式板，其中空气被分离为装有个别苍蝇和1管配备有温度和湿度传感器15的玻璃管。注:流量调节器和流量计玻璃管后保证相同的流动在所有管。蓝方代表的跟踪系统感兴趣区域（ROI）。跟踪摄像，电脑跟踪和气味输送系统之间的信息流的（C）示意图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53394/53394fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> OAD / 53394 / 53394fig2.jpg"/&gt; 图2.程序和后面的数据分析的理由。（A）的个别苍蝇遇到气味在不同的位置，因此，不同的时间。左图:示意图的气味阀切换时可能飞的位置。右板:大约10 -3稀释的乙酸乙酯的500毫秒脉冲的呈现15苍蝇的x位置的原始数据。注意在不同的时间逆风​​走路个别苍蝇取决于它们的x位置。左虚线:气味阀切换时间。个别蝇气味遭遇被移位由一个系统固有延迟的气味达到玻璃管（d）和风速（w）的。因此，气味碰到单独计算每个基于其x位飞。右下:15苍蝇对准X位置（灰色）和平均x位置（黑色粗体）。 （B）相同的数据在A，但对时延和风速的修正。博特OM:对齐的X位置的15苍蝇（灰色）和平均X位置（红色粗体）修正后的。 15苍蝇的乙酸乙酯1 500毫秒脉冲具有和不具有校正气味旅行引起的平均逆风进展（C）的比较。需要注意的是未校正的（黑）的跟踪校正的延迟，但不适合旅行的气味。 （D）的平均数迎风速度15苍蝇的乙酸乙酯1 500毫秒脉冲具有和不具有校正气味旅行引起的。虚线表示的速度从C中所示上风进度值计算的，大胆的线条使用1级9点Savitzky -格雷滤波器滤波后显示逆风速度。 （ 五）未过滤的意思是逆风速度并没有修正为30苍蝇和40个脉冲乙酸乙酯每个（ 即完整的数据集）的气味旅行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53394/53394fig2large.jpg" target="_blank">请点击此处查看这是一个更大的版本</a>数字。 <img alt="图3" src="/files/ftp_upload/53394/53394fig3.jpg" /> 图3.示例响应为10 -3稀释4诱人的气味（A）彩色编码的平均响应时间课程，30个人蝇4诱500毫秒脉冲和溶剂矿物油（MOL; ETB:丁酸乙酯; IAA:乙酸异戊酯; ETA:乙酸乙酯; BEDN:2,3-丁二酮）。每一行代表的平均响应时间，当然一个人飞。每飞提出，每个气味的40倍 - 因为苍蝇被允许自由地分发和可能留下的跟踪系统感兴趣的区域 - 平均响应时间课程，每飞所有完整的轨迹计算（N = 7-39轨迹每飞和气味）。黄条代表的气味脉冲。 （B）响应时间的课程，4引诱溶剂矿物油（N = 30苍蝇;平均+/- SEM）。 （C）内气味后遭遇4秒的逆风净排量（相同的数据，在（A）和（B），N = 30苍蝇）。填充框表示统计学显著上风运动相比，阴性对照矿物油（P &lt;0.05; Wilcoxon符号秩检验）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53394/53394fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53394/53394fig4.jpg" /> 图4.响应对引诱的三元和四元混合物（一 ）净迎风排量4引诱和所有三元和四元混合物排序他们的中位缓解。不同的字母表示的反应（P &lt;0.05统计学显著差异;签署秩和检验秩和检验和事后的Wilcoxon秩和检验; N = 30苍蝇）。下面的黑色框表示含有乙酸乙酯和2,3butanedione混合物。 （二）响应时间，课程含醋酸乙酯和2,3-丁二酮的混合物（平均值+/- SEM; N = 30苍蝇）。注意类似的响应动力学。的响应时间菜混合物（C）的比较而不乙酸乙酯或2,3-丁二酮和反应动力学通过充分混合诱发（平均值+/- SEM;每组30蝇）。注意较低的幅度没有ETA和无BEDN更短的响应。最佳时间过程（虚线）从ETA（红色）和BEDN（绿色）构建和全混合（D）的比较。阴影表示的时间 - 过程由不同混合物组分解释的部分。注意，在先前的研究中，已经证明，对引诱二元混合物的反应可以从一个最佳的时间-过程的混合物构成时道29的基础上创建的预测。这个最佳的时间 - 过程为ETA和BEDN被示为虚线。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53394/53394fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm at JoVE.com

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

自动跟踪系统Flywalk是用于在果蝇气味引导行为高分辨率定量。

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 ...

high-resolution-quantification-odor-guided-behavior-drosophila

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

9.2K 次观看.  Max Planck Institute for Chemical Ecology. 自动跟踪系统Flywalk是用于在果蝇气味引导行为高分辨率定量。 

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

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

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

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 ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

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 ...

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 ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.