Name: an automated method to determine the performance of drosophila in response to temperature changes in space and time
Uploaded: 2018-10-12T14:00:00
Description: Watch this Scientific Journal Video about An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time at JoVE.com

这项工作部分由格罗宁根大学的行为和认知神经科学计划的奖学金和来自墨西哥的委员会 Ciencia y 国家 (国家科学技术委员会) 的研究生奖学金, 授予安德烈帕迪拉, 以及 Hedderik Rijn 和吉恩-克里斯托弗. 比耶泰的时间研究奖。我们也感谢彼得 Gerrit 峨参与开发FlySteps跟踪器。
脚本TemperaturePhases、FlySteps和FlyStepAnalysis可以被发现为补充信息, 并在以下临时和公开可用的链接: https://dataverse.nl/privateurl.xhtml？token=c70159ad-4d92-443d-8946-974140d2cb78

1. 飞行食品培养基的制备
<ol><li>将 1 l 的自来水倒入2升玻璃杯中, 加入一个磁力搅拌棒。将烧杯放在300摄氏度的磁性热板上, 直到达到沸腾温度。</li>
	<li>搅拌在500发/分钟, 并添加以下内容:10 克琼脂, 30 克葡萄糖, 15 克蔗糖, 15 克玉米粉, 10 克小麦胚芽, 10 克大豆面粉, 30 克糖蜜, 和 35 g 活性干酵母。</li>
	<li>当混合泡沫剧烈时, 将热板温度降低到120摄氏度, 同时继续搅拌。</li>
	<li>将热板温度进一步降...

<ol>
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在这里, 我们提出了一个自动化的温度控制竞技场 (图 1), 在时间和空间上产生精确的温度变化。这种方法允许单独的果蝇暴露, 不仅是预先编程的逐渐增加的温度 (图 2和图 3), 但也对动态温度挑战, 其中每个瓷砖的飞行竞技场被加热独立于不同的温度 (图 4和 图 5)。</...

温度是一个持续的环境因素, 影响生物体的功能和行为1。纬度和海拔高度的差异导致气候有机体类型的差异, 这导致了他们对温度2,3反应的进化选择。生物体通过形态学、生理和行为适应来响应不同的温度, 从而在其特定环境下实现最佳性能4。例如, 在果蝇中, 不同地区的人群在不同温度下有不同的温度偏好、体型、发育时间、长寿、繁殖力和行走性能2 ,5,6,7。不同起源蝇之间的多样性由遗传变异和塑性基因表达8,9部分解释。同样, 不同区域的果蝇种类在温度梯度上分布不同, 并显示耐极端热和冷试验的差异10、11、12。
果蝇最近也成为了选择的模型, 了解温度知觉的遗传和神经基础13,14,15,16,17。一般来说, 成人苍蝇通过在天线和通过温度传感器在大脑13,14,15,16的冷和热外围温度传感器感知温度,17,18,19,20. 热温度的外围受体表示Gr28b16或发热21, 而外围冷受体的特征是Brivido14。在大脑中, 温度由表达TrpA115的神经元处理。对这些途径突变体的行为研究正在提高我们对温度如何处理的理解, 并对不同地区果蝇种群的变化机制进行深入的了解。
在这里, 我们描述一个温度控制的竞技场, 产生快速和精确的温度变化。调查人员可以对这些变化进行预编程, 这样就无需人工干预即可实现标准化和可重复的温度操纵。飞行记录和跟踪与专门的软件, 以确定他们的位置和速度在实验的不同阶段。本协议中提出的主要测量方法是在不同温度下的行走速度, 因为它是一种生态学相关的生理性能指标, 可识别个体的热适应性5。与温度受体突变体一起, 这种技术可以帮助揭示细胞和生化水平的热适应机制。

<table border="0" cellpadding="0" cellspacing="0" width="965">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Arduino Due</td>
			<td style="width:223px;">Arduino</td>
			<td style="width:189px;">A000062</td>
			<td style="width:221px;">Software RUG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electronics Board</td>
			<td>Ruijsink Dynamic Engineering</td>
			<td>FF-Main-02-2014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power supply Boost</td>
			<td>XP-Power 48. V 65 W</td>
			<td>ECS65US48</td>
			<td>Set to 53 Volt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power supply Tile Heating</td>
			<td>XP-Power 15. V 80 W</td>
			<td>VFT80US15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power supply Cooling</td>
			<td>XP-Power 15. V 130 W</td>
			<td>ECS130U515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peltier elements</td>
			<td>Marlow Industries</td>
			<td>RC12-4</td>
			<td>2 Elements, controlled DC feed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat sink</td>
			<td>Fisher Technik</td>
			<td>LA 9/150-230V</td>
			<td>Decoupled for vibration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature sensors</td>
			<td>Measurement Specialties</td>
			<td>MCD_10K3MCD1</td>
			<td>Micro Thermistor Probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Copper block/tiles</td>
			<td>Ruijsink Dynamic Engineering</td>
			<td>FF-CB-01-2014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Auminum ring</td>
			<td>Ruijsink Dynamic Engineering</td>
			<td>FF-RoF-02-2015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tesa 4104 white tape 25 x 66 mm</td>
			<td>RS Components</td>
			<td>111-2300&#160;</td>
			<td>White conductive tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red LEDs</td>
			<td>Lucky Ligt</td>
			<td>ll-583vc2c-v1-4da</td>
			<td>Wavelength between 625 nm, 20 mAmp and 6 V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Warm white LED strip</td>
			<td>Ledstripkoning</td>
			<td>HQ-3528-SMD</td>
			<td>60 LEDs per meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Switch Power Supply</td>
			<td>Generic</td>
			<td>T-36-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Logitech c920</td>
			<td>Logitech Europe S.A</td>
			<td>PN960-001055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickTime Player</td>
			<td>Apple Computer</td>
			<td></td>
			<td>Recording program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tracking analysis software</td>
			<td>R</td>
			<td></td>
			<td>Packages: pacman</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tracking analysis software</td>
			<td>MATLAB</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Imaging</td>
			<td>FLIR T400sc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graphs and Statisticts Software</td>
			<td>Graph Pad Prism</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigmacote</td>
			<td>Sigma-Aldrich</td>
			<td>SL2-100ML</td>
			<td>Siliconising agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fly rearing bottles</td>
			<td>Flystuff</td>
			<td>32-130</td>
			<td>6oz Drosophila stock bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flypad</td>
			<td>Flystuff</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fly rearing vials</td>
			<td>Dominique Dutscher</td>
			<td align="right">789008</td>
			<td>Drosophila tubes narrow 25x95 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Sanyo</td>
			<td>MIR-154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic hot plate</td>
			<td>Heidolph</td>
			<td>505-20000-00</td>
			<td>MR Hei-Standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Caldic Ingredients B.V.</td>
			<td>010001.26.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Gezond&#38;wel</td>
			<td align="right">1019155</td>
			<td>Dextrose/Druivensuiker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Van Gilse</td>
			<td></td>
			<td>Granulated sugar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cornmeal</td>
			<td>Flystuff</td>
			<td>62-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wheat germ</td>
			<td>Gezond&#38;wel</td>
			<td align="right">1017683</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soy flour</td>
			<td>Flystuff</td>
			<td>62-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molasses</td>
			<td>Flystuff</td>
			<td>62-117</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Active dry yeast</td>
			<td>Red Star</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tegosept</td>
			<td>Flystuff</td>
			<td>20-258</td>
			<td align="right">100%</td>
		</tr>
	</tbody>
</table>

an automated method to determine the performance of drosophila in response to temperature changes in space and time

温度是影响物种分布和行为的普遍环境因素。不同种类果蝇对温度变化的生理耐受性和适应性有特定的反应。果蝇还拥有一个温度传感系统, 已成为了解变温动物温度处理的神经基础的基础。我们在这里提供一个温度控制的竞技场, 允许快速和精确的温度变化与时间和空间控制, 以探索个别苍蝇对变化的温度的反应。单独的苍蝇被放置在竞技场和暴露在预编程的温度挑战, 如均匀逐渐增加的温度, 以确定反应规范或空间分布的温度在同一时间确定偏好。自动跟踪个人, 允许量化速度或位置偏好。此方法可用于快速量化在大范围温度下的响应, 以确定果蝇或其他类似大小的昆虫的温度性能曲线。此外, 它还可用于基因研究, 以量化突变体或野生型苍蝇的温度偏好和反应。该方法可以帮助揭示热形态和适应的基础, 以及温度处理后的神经机制。

温度控制的竞技场 (图 1A) 由三个铜瓦组成, 其温度可通过可编程电路单独控制。每块铜瓦都有一个温度传感器, 可以向可编程电路提供反馈。电路激活电源以增加每个磁贴的温度。被动式热电元件充当恒定的加热元件, 以保持所需的温度, 而风扇冷却的散热片提供恒定的冷却。温度变化的幅度以非线性的方式决定了过程的速度。增加2摄氏度只需要0.1 ?...

Watch this Scientific Journal Video about An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time at JoVE.com

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

在这里, 我们提出了一个协议, 自动确定果蝇的运动性能在变化的温度使用可编程温度控制竞技场, 产生快速和准确的温度变化的时间和空间。

一种自动测定果蝇在空间和时间温度变化中的性能的方法

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

这种方法可以帮助我们回答关于苍蝇对温度变化的反应的关键问题，例如基因型之间的差异、与其他感官提示的相互作用，或者不同温度受体的功能。该技术的主要优点是，它允许以自动化的方式进行多个快速、精确的温度变化，控制时间和空间。首先将20只雄蝇和20只雌蝇放入含有45毫升飞食介质的饲养瓶中，并将瓶子放入12小时光、12小时黑暗周期的25摄氏度培养箱内。10天后，在二氧化碳垫上麻醉新近的苍蝇最多4分钟。并使用画笔收集处女苍蝇在2.5由9.5厘米飞饲养小瓶含有6.5毫升新鲜苍蝇食物介质，按性别分成每瓶20只苍蝇组。然后将小瓶返回孵化器五至七天。要设置温度控制竞技场，请打开竞技场并在控制计算机中打开温度阶段脚本。验证温度序列是否正确设置，并检查每个实验阶段的持续时间是否设置为 60 秒。在起始实验块部分下，确认这些设置：所需的相位数、指示性红色发光二极管的迭代开/关设置、每个相位温度升高 2 摄氏度和起始温度 16 摄氏度。接下来，运行温度阶段脚本。软件将初始化五秒钟，然后停止。对于温度行为实验，在竞技场的铜砖顶部放置一串白色导电胶带，确保所有边缘都覆盖。在铜片周围放置一个加热的铝环，并使用干净的纸巾擦拭玻璃盖。将盖子放在铝环的顶部，留下一个缝隙，通过该缝隙可以吹飞，然后点击一个适应的苍蝇小瓶两次，迫使苍蝇到小瓶的底部。使用吸嘴器，捕获一只苍蝇并关闭小瓶，然后再把它放回孵化器。通过玻璃盖和铝环之间的间隙将苍蝇吹入竞技场，并立即推动玻璃三叶草以缩小间隙。在竞技场周围放置一帧灯光，以确保对称照明，并开始录制视频录制程序。然后按控制计算机的空格键开始运行实验阶段。要跟踪视频，请打开飞行步骤跟踪软件，并在飞行跟踪器文件夹中打开配置文件。设置视频文件夹中的视频位置以及视频文件中的视频名称。根据竞技场边缘多个点的 x/y 像素坐标，在竞技场设置中指定飞行竞技场的边界。根据 LED 中心位置的 x/y 像素坐标，在 LED 设置中指定非活动红色 LED 的位置。要检查飞行竞技场边框的位置，请在竞技场设置中将调试设置为 true，单击"保存"并在终端中运行脚本。视频的屏幕截图将显示为蓝色正方形，该方块由竞技场设置中输入的坐标组成。然后将竞技场设置中的调试更改为 false。单击"保存"，然后再次在终端中运行屏幕以开始跟踪过程。在典型的温度行为实验中，暴露在16、20或24摄氏度的单个苍蝇在实验开始时表现出比5分钟后更高的运动。温度控制竞技场还可用于比较不同遗传背景的苍蝇行为反应与动态温度变化。例如，在这个实验中，所有被测试物种的速度都根据它们自己的反应曲线随着温度的增加而增加，直到达到最高性能点，之后它们的速度会变衰，苍蝇会灭亡。当个别苍蝇暴露在40摄氏度的中间和一侧瓷砖，另一侧的瓷砖在舒适的22摄氏度野生型苍蝇迅速停止沿竞技场移动，并保持在舒适的位置。相比之下，经典的记忆突变邓斯苍蝇不断探索竞技场，花更少的时间比控制在舒适的位置。此外，测试温度和位置的组合也可用于了解不同温度受体在动态温度变化期间的功能。正如这个实验所示，在实验中，单个黑色素突变体暴露在不断升高的温度下，同时提供了在22摄氏度下移动的舒适位置。执行这些过程时，请记住按顺序快速执行这些步骤，以便捕获尽可能多的苍蝇行为。在此过程旁边，其他方法（如负地轴或热扼流圈测定）可用于回答其他问题，例如苍蝇在什么地方正常移动或具有正常的耐热性。这种技术的影响延伸到我们了解神经紊乱或突变，其中温度感知或疼痛发挥作用。一般来说，对这种方法的新鲜人会挣扎，因为每个步骤本身都很简单，但需要完美协调，所以需要练习这项技术。

Research

JoVE Journal

Behavior

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior

Use of the Operant Orofacial Pain Assessment Device OPAD to Measure Changes in Nociceptive Behavior

使用操作性口面疼痛评估设备（OPAD）测量伤害行为的变化

We present an operant  system for the detection of pain in awake, conscious rodents. The Orofacial  Pain Assessment Device (OPAD) assesses pain behaviors ...

科研

行为学

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

一致性的任务作为一种手段来获取目标行为的措施中的橡胶手错觉范式Crossmodal

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

鼠类精神运动警觉测试（rPVT）：一个在大鼠和小鼠的神经行为评估性能的方法

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

行为障碍：一个创新的方法来监控保健品饮食的调节作用

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

测试环境线索对交配行为的影响的方法<em&gt;黑腹果蝇</em

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

可逆冷却诱导 Deactivations 对行走猫的障碍记忆的皮层贡献研究

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

旋钮旋任务: 一种半自动评估大鼠前肢功能的方法

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

一种自动快速迭代阴性 Geotaxis 分析方法在神经的果蝇模型中的成人攀爬行为

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...