我们感谢有益的讨论T. Huber和TP Sakmar。这项工作得到了来自美国国立卫生研究院的资助LBV和比利时，美国教育基金会和雷夫森基金会ML

1。设备和试剂气味稀释： <ul><li>石蜡油（Sigma公司，CAS编号：8012-95-1） </li><li>气味，纯度最高可引出吸引果蝇幼虫1。在这个视频文章中，醋酸异戊酯（Sigma公司，CAS编号：123-92-2）进行了测试与动物。 </li><li>聚四氟乙烯第1.5毫升玻璃瓶（安捷伦科技） </li><li>按重量计算精确的稀释数字测量规模</li></ul>含量舞台设置： <ul><li>琼脂糖（Promega公司） </li><li>盖的96孔板（BD猎鹰，目录号：353071）。我们的行为的舞台由三个叠盖。 </li><li>单通道1-20μLpipetter或多通道pipetter加载到堆栈的顶部盖气味液滴（S）。要设置多个源检测，我们使用RAININ 5-50μL吸取- Lite的多渠道pipetter。 </li></ul>制备果蝇幼虫的行为测试： <ul><li> 15％（W / W）蔗糖溶液</li><li>定期培养皿（BD猎鹰）清洗和存储幼虫</li><li>油漆刷（4 - 5mm的猪鬃长度）操纵和传输的幼虫</li></ul>记录幼虫的议案： <ul><li>转移到舞台上的幼虫的一个平头的小引脚</li><li>产钳或删除记录后的幼虫的画笔</li><li>同质的非加热光源照明的阶段，“修身边缘”光片生产洛根电</li><li>内阁环绕舞台和控制光照条件下的行为</li><li>视频跟踪系统记录动物的运动。我们在使用Ethovision软件（荷兰Noldus，）。 </li><li>购买Ethovision软件的CCD相机和图像采集卡视频卡（EureCard短笛基本） </li><li>温度计和湿度记录仪，监测大气条件下，在行为测试（可选） </li><li> MathWorks的MATLAB进行定制的数据分析（可选） </li></ul> 2。行为的舞台行为上由三个叠96孔培养皿盖子。底部的盖子是用来隔离系统的其余部分从光垫，并减少在赛场上的对流。第二个盖子，3％的琼脂糖凝胶25毫升覆盖，作为一个阶段的幼虫上行走。顶盖持有的气味，保持悬浮液滴表面张力。 气味水滴吸管直接进入96孔盘盖的水井。可以使用一个或多个气味的来源，产生梯度分明，核查的型材2。为了测试嗅觉丧失和幼虫的趋化缺陷，我们建议使用以下两种检测。 2.1。单气味源检测：一个单一的气味液滴，奠定了良好＃E7（标准96孔板参考系统）。 <ul><li>刺激：加入10μl石蜡油稀释气味</li><li>初始条件：一个单一的幼虫是根据气味源（或在其密切邻里）推出。 </li><li>基本意见：有吸引力的刺激（如醋酸异戊酯），野生型幼虫下方源积累。 Anosmic嗅觉缺陷的动物或动物迅速漫步远离液滴。 </li><li>试用时间：3分钟。一个给定的动物的运动记录，直到试用时间已过或尽快动物接触的钢板墙。 </li><li>行为量化指标为：在一个小圆形上的气味源的中心区的动物所花费的时间的百分比;气味源的平均距离;时间距离的过程中气味的来源等。 </li></ul>这有效地检测测试突变幼虫的基本嗅觉丧失。它也可以用来可靠地确定检测阈值的2个特别的气味。 2.2。多气味源检测顶盖以及一些气味水滴被解雇。在目前的视频文章，我们设立了共6液滴沿中央行E.井＃E2，E4＃，＃E6，＃E8＃E10和E12的介绍（标准96孔板参考系统梯度）。在培养皿中，单一的气味源分析与对比，多个气味源检测允许沿长度和宽度的舞台建立了渐变几何的定性控制。此实验是用来描述感官机制，使果蝇幼虫chemotax 2 。 <ul><li>刺激：加入10μl气味滴在交替沿着一排井敷设。这种安排导致创造渐变为中心选定的行。要建立一个同质梯度沿舞台的宽度，几行可以载入多个来源。 </li><li>初始条件：一个单一的幼虫介绍下水井＃E3和＃之间的气味源E4。 </li><li>基本表型：从起点随机漫步anosmic幼虫。有吸引力的刺激（如醋酸异戊酯），就能闻到异味浓度增加的方向沿导航的动物。动物上升的气味线索的准确性，相关性与意识和能力过程中的气味刺激。弯路往往造成感官缺陷。一旦动物已经达到了最高浓度在试验点，但它仍然在这个位置附近。 </li><li>审讯时间：最大3分钟（跟踪持续时间长，往往引入噪声，作为幼虫失去兴趣，最终放弃的梯度）。在这个视频文章说明的梯度，个别幼虫的议案，直到动物达到的最高浓度源（以及＃E12）附近。录音是尽快停止动物达到的目标区域或接触任何竞技场的墙壁。 </li><li>可能的行为量化的度量：由动物下度过气味线（蓝色矩形）的时间的百分比;意味着尊重当地的气味梯度方向的标题;一个评分相结合的趋化作用，测量动物的全局倾向，按照气味第2行。 </li></ul> 3。梯度配置文件验证测试在一个特定的梯度的幼虫之前，重要的是，随着时间的推移，以确定其稳定。实验者可以在此基础上，确定每个审判足够的时间和幼虫，可以在同一舞台上连续跟踪。为了测试的稳定的梯度，我们已经开发出基于红外光谱2的方法。这种方法是本协议的范围之外，并不会进一步阐述。 4。准备实验的议定书 4.1。琼脂糖凝胶板的制备和气味稀释： <ol><li> 1。 96孔盘盖平顶倒到3％琼脂糖25毫升;允许琼脂糖冷静下来，巩固（约30分钟）。 重要事项 ：确保96孔盖一个平坦的表面上存在的一个斜坡上凝固的琼脂糖凝胶琼脂糖浇筑前，可能会影响到幼虫的行为。浇筑时的凝胶，去除任何表面上形成的泡沫。 </li><li> 2。石蜡油所需的浓度，使用电子秤精确地测量石蜡油气味，并在每个稀释度的数额稀释气味。 </li></ol> 注：当使用低气味的浓度，最好是准备了一套基于连续稀释的源浓度。开始，例如，一个1.0M的解决方案，并进行1:2稀释，以获得一个解决方案0.5M。出发的循环，获得了系列稀释以下几何级数1.0M，0.5M，0.25米，0.12M，0.06M，0.03M，0.015M。 重要提示：由于许多有机气味与塑料发生反应，气味稀释，必须在与铁氟龙瓶盖的玻璃瓶中。理想的情况下，气味应准备新鲜的每次实验前，以减少在整个实验的实际异味浓度的波动。当使用高挥发性化学物质，这是特别真实。 4.2。幼虫的准备： <ol><li> 1。准备在蒸馏水中15％的蔗糖溶液。 注：15％的蔗糖溶液比幼虫的密度和将导致他们能够支撑到溶液的表面。然而，不溶飞食品块有更大的密度比的解决方案，并迅速沉入水底。因此，使用这种蔗糖溶液提供了幼虫的食物分开的有效手段。蔗糖溶液是一个优秀的中型生物污染物，如果解决方案变得混浊，它可以过滤消毒，去除污染。 </li><li>获取一小瓶6日龄幼虫或幼虫发育阶段所需。 注：我们所有的实验都进行了6日龄幼虫（三龄发育阶段的中间），在这一发展阶段的幼虫有足够大的，为便于我们的跟踪软件的检测，但也非常活跃。 </li><li>倒入小瓶幼虫的食物含有蔗糖。 </li><li>使用抹刀，打破和解散飞食品，轻轻地释放幼虫。发布幼虫会漂浮表面。 </li><li>允许飞的食物块几分钟下沉到瓶底部，然后，倒入培养皿中的蔗糖和成幼虫。如果预选的幼虫是必要的，动物可以收集通过一个过滤器浇蔗糖。幼虫可以被转移到一碟选择画笔。 </li><li>离开约30分钟的幼虫在sucrose解决方案进行行为测试前让动物habituate蔗糖溶液。由于幼虫不会出现蔗糖饲料，动物经验的饥饿状态，从而提高了趋性能。 </li><li>培养皿的盖子，可以装满水（或蔗糖）和行为测试后丢弃动物的插座。 </li></ol> 5。议定书“的执行行为的录音 5.1。竞技场设置： <ol><li> 1。套用气味水滴到一个新的96孔盘盖底面（盈满方）。 重要事项：为了避免不同的气味，气味浓度的污染，必须为每个加载设置一个新的盖子。含有气味来源的盖子没有被回收后进行实验。根据气味的浓度范围内使用，气味梯度已经发现，在一个封闭的系统，仅适用于10至20分钟稳定。因此重要的是尽量减少之间的梯度设置和实际行为测试开始的时间。 </li><li>倒置的盖子，上面涂有3％的琼脂糖层第二个盖子和堆栈。 重要事项 ：反转盖子小心，以防止气味蔓延到相邻井水滴。 </li><li>琼脂糖表面的顶盖后倒置，使气味弥漫推行幼虫前30秒。 </li></ol> 5.2。载入幼虫： <ol><li>提起顶盖含有的气味滴就足以让加载幼虫。 </li><li>将幼虫从蔗糖溶液琼脂糖层。取决于所使用的实验动物的起始位置： 对于单一的气味源检测，幼虫被释放的气味下液滴。 对于多气味源检测 ，幼虫释放井＃E3和E4＃之间。 重要事项：东方新引进的动物以一致的方式。我们东方的动物，在渐变的方向，确保动物的身体和头部面临的最高浓度。在梯度方向相反的动物发起搜索行为。我们认为，这个初始搜索期间不必要的噪音源。控制头部方向，减少动物的数量，接触舞台上墙，在他们的探索行为。这种做法并不总理动物的运动方向或chemotaxing偶然的气味沿线anosmic动物的数量显着增加（即假阳性气味测试）。 </li><li>更换顶盖，并立即开始录音。 注：健康幼虫他们在舞台上推出后立即开始抓取，可能会覆盖在第一个10秒的距离为1cm 。如果实验者是缓慢的启动录音，初步数据点都将丢失。出于这个原因，我们建议实验者变得熟悉软件命令启动，然后再尝试运行实验记录。 </li><li>允许录制的期限（一般为3分钟）的幼虫的行为。 注：录音3分钟结束前终止的，如果幼虫打竞技场的墙壁或达到的目标区域（如在梯度检测）。 </li><li>在录制结束，及时解除盖子，取出产钳或画笔的幼虫。放置在培养皿盖子含有水（或蔗糖）的跟踪动物。 重要信息：记录动物是不重用。水盘仅仅是一个方便的插座，单独运行时的实验记录和联合国录制的动物。 </li><li>如果下一个舞台上的多个动物跟踪设置，及时加载下的幼虫，开始录制，如上图所示。 重要事项 ：设置在同一舞台上进行的试验数量应执行任何行为实验2之前，应验证的渐变稳定的基础上决定。 </li><li>一旦所有的设置已被跟踪，并从舞台上删除舞台下的测试幼虫，丢弃的顶盖和琼脂糖层。中间盖（即琼脂糖盖）可以回收利用。 请注意：我们建议Ethovision用户选择的背景减法“的检测方法。 </li></ol> 6。视频文章中表现出的样品实验 6.1。单气味源1.0M乙酸异戊酯的测定。 我们一味地测试了10个Or83b - / - 突变体和10个野生型（W1118）的幼虫，其中每一个数字被分配。跟踪实验呃没有收到任何有关每种动物的基因型信息。每个记录后，被分配了一个预测表型的每只动物测试：smellers，如果积累源下观察整个审判;非smellers，如果路径迅速离开的气味源附近。后录得的20只动物的行为，按表型路径。这个分组结果，然后用较幼虫的实际基因型：发现所有的作业是正确的。 6.2。乙酸异戊酯的指数和线性渐变多气味源检测。 在这个实验中，我们比较了野生型chemotaxing幼虫沿着一条直线和指数梯度的表演。趋化行为均录得两个相同的最高浓度为0.5m的梯度。正如在文章中解释，指数渐变的气味来源为0.015和0.5M之间。介于0.25和0.5米（与常见的差异0.05M算术级数）之间的线性渐变来源。 线性渐变代表一个更具挑战性的趋化环境，在集中的地方差异常数和相对较小的，浓度范围窄（0.25米）。相比之下，指数渐变的浓度范围内增加径沿途当地的浓度差异较大。 十五幼虫均录得每两个梯度的几何形状和一个图形叠加的路径。一两个图形的视觉检查表明，野生型幼虫能够可靠地登上为几何结构的梯度，但蜿蜒量为线性渐变条件更大。这表明沿渐变长度的信号目前是更可靠的检测指数比线性形状。 6.3。多气味，在“屋顶”的几何形状的乙酸异戊酯梯度源的检测。 在这里，我们核实介绍幼虫的气味梯度敏感的气味浓度的变化，而不是仅仅存在或没有气味。成立一个异戊梯度的气味滴的应用与以下浓度：0.06，0.12，0.25，0.5，0.25，0.12M。随后的梯度的几何形状类似于一个不对称的“屋顶”峰值在0.5M。 0.06M和0.12M的气味液滴之间的幼虫被释放。我们的假设是，如果幼虫只是计算存在的气味，他们将没有特别的偏爱浓度最高的整个渐变。相反，如果幼虫他们会积累下的0.5M液滴浓度变化非常敏感。十五幼虫，它是发现，一个简短的探索时期，所有的幼虫底浓度较高的积累。这表明，当地的气味浓度的变化计算并告知果蝇幼虫的趋化行为。

<ol>
	<li>Fishilevich, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+behavior+mediated+by+single+larval+olfactory+neurons+in+Drosophila.">Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila.</a> Curr Biol. 15, 2086-2096 (2005).</li><li>Louis, M., Huber, T., Benton, R., Sakmar, T. P., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilateral+olfactory+sensory+input+enhances+chemotaxis+behavior.">Bilateral olfactory sensory input enhances chemotaxis behavior.</a> Nature Neuroscience advance online publication. , (2007).</li></ol>

The authors have nothing to disclose.

high-resolution measurement of odor-driven behavior in drosophila larvae

传统上一直在果蝇幼虫的嗅觉反应研究中的Petri菜组成一个单一的外设气味源。在这种行为范式，实验者通常假定的气味分子，从源头上迅速扩散，导致创造一个稳定的菜梯度。要建立一个感觉输入和行为反应之间的定量关系，这是必要的气味刺激条件下实现更彻底的表征。在这个视频文章中，我们描述了一种新的方法，让嗅觉梯度与稳定，可控的几何图形的建设。我们简要地说明这些梯度如何可用于嗅觉缺陷（完全和部分嗅觉丧失）屏幕和趋化行为的研究更微妙的功能。

Watch this Scientific Journal Video about High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae at JoVE.com

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

在这个视频文章中，我们描述了一种新的方法，让嗅觉梯度与稳定，可控的几何图形的建设。我们简要地说明这些梯度如何可用于嗅觉缺陷（完全和部分嗅觉丧失）屏幕和趋化行为的研究更微妙的功能。

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

high-resolution-measurement-odor-driven-behavior-drosophila

Research

JoVE Journal

Biology

10.7K 次观看.  Rockefeller University. 在这个视频文章中，我们描述了一种新的方法，让嗅觉梯度与稳定，可控的几何图形的建设。我们简要地说明这些梯度如何可用于嗅觉缺陷（完全和部分嗅觉丧失）屏幕和趋化行为的研究更微妙的功能。

视频: High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

从3龄果蝇幼虫的成虫光盘夹层

科研

生物学

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...