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?...

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?...

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

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.

在气味引导行为的高分辨率定量

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在昆虫单感器记录果蝇按蚊

从查明的神经元和取食行为的钙信号的同时记录果蝇

高分辨率实时成像发展中的神经上皮细胞的行为

Optogenetic刺激的逃避行为

在自发活动的测定

简单的检测方法的社交回避的量化

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

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

监测果蝇中朊病毒样蛋白聚集物的细胞间传输