我们感谢斯坦尼斯拉夫·切尔 Lazopulo 对视频内容的帮助。

1. 使用果蝇活动监测器 (大坝) 测量飞行运动 注意: 有关详细信息, 请参阅参考 5 。
<ol> <li> 在一端和棉花上准备单独的苍蝇管。食物的末端应密封, 以防食物干燥。
	<ol> <li> 在50毫升的烧杯中放入5-6 克的飞行食物。把食物切成小块, 使它更容易融化. </li>
		<li> 连接32个单独的玻璃管和一个橡皮筋. </li>
		<li> 将烧杯中的食物加热到微波炉中10-15 秒. 每5秒停止微波炉, 并小心摇动烧杯, 以确保食物的均等融化. </li>
		<li> 当食物仍然是液体时, 把准备好的单独的管子放入烧杯中, 然后用食物。把管子上下移动, 这样它们就同样充满了. </li>
		<li> 允许食物冷却并固化约1小时. </li>
		<li> 食物固体后, 用烧杯中的食物取出管子. </li>
		<li> 用蜡封住含有食物的末端。首先, 用纸巾小心地清洗管子, 然后按下管子对蜡。目视检查密封件的质量, 如有必要, 再次重复密封. </li>
		<li> 用棉把管子的另一端关上; 棉花将允许空气通过, 同时保持一只苍蝇被锁在管子里. </li>
	</ol> </li> <li> 在每个单独的管道中放置一只苍蝇, 并将管道放置在大坝中. </li>
	<li> 将监视器放置在恒温恒湿的恒温箱中。根据实验, 设置适当的光/暗条件如下。
	<ol> <li> 用于进行光/暗实验以使苍蝇在光/暗循环中进行整个实验。不要在分析中使用测量的第一天. </li>
		<li> 对于恒定的黑暗实验, 首先保持苍蝇在光/黑暗的条件下, 夹带和同步时钟, 然后切换到恒定的黑暗。不要在分析中使用恒定黑暗的第一天的测量. </li>
	</ol> </li> <li> 收集可在分析中使用的至少四天的数据. 
	注: 大坝系统将输出一个单一的文件与移动记录的所有苍蝇在显示器. </li>
</ol> 2。数据分析 <ol> <li> 将监视器输出文件拆分为多个单飞活动文件; 每个文件都应是单个列和 #39; .txt 和 #39; 文件与个人飞行运动测量. </li>
	<li> 运行和 #39; ModelFitPS3 #39; 在 Matlab 命令窗口中使用以下输入参数: <ol> <li> For samplingrate , 设置数据采样时间间隔 (以秒为单位)。例如, 如果每分钟测量一次活动, 则输入60作为 samplingrate. </li>
		<li> for bin_interval , 设置搁置数据的时间间隔 (以分钟为单位), 以便更好地进行可视化; 建议的 bin 间隔为 20-30 min. </li>
		<li> For 趋势 , 输入 和 #34; 1 和 #34; 如果数据显示基线趋势和 #34; 0 和 #34; 否则; 趋势首先通过拟合第二阶多项式, 然后从数据中减去数据。
	</ol> </li> <li> 在弹出窗口中选择一个单飞活动文件. 
	注意: 第一个图形是数据功率谱, 而不是熟悉的活动图。从绘制的功率谱中, 确定主周期 T 0 : 在生理峰值上单击鼠标左键, 或者在第二个谐波峰值 (在 T 0 /2) 上用鼠标右键. </li>
	<li> 在打开的数据图中, 检查上午和晚上的峰值是否可视化。如果没有, 则通过右键单击关系图上的任意位置来更改 bin_interval 值, 并在对话框中输入新的 bin_interval 值。该程序将使用间隔的新值重绘数据。若要接受 bin_interval 值, 请在关系图上的任意位置单击左键. </li>
	<li> 程序将重新绘制数据, 并显示前五天的活动。在此图中, 单击将在分析中使用的第一个 M 峰值 (有时需要跳过一或两天). 
	注意: 该程序将重新绘制的图形从采摘的早晨峰值开始。蓝色和红色线将根据步骤2.4 中确定的期间分别显示 E 峰值和第二天 M 峰值的大致位置. </li>
	<li> 在同一图形上, 为数据的初步拟合选择数据与模型: 单击以下点 (按此顺序; 请注意, 单击的位置将在底部显示一个红星): (i) M 峰值的顶部;(ii) M 峰值的结束;(iii) 电子山顶的开始;(iv) 山顶;(v) 电子山顶的终点;(vi) 第二天最高的 M 峰值. </li>
	<li> 请注意, 该程序现在显示了功率谱. 
	注意: x 轴现在是以频率给定的。
	<ol> <li> 在打开的窗口中使用功率谱, 选取将用于拟合模型功率谱的解析表达式的点。在步骤2.4 中检测到的期间标记为红色线。要选取拟合点, 首先粗略确定主周期, 类似于步骤2.4。然后使用滑块, 微调主周期值, 以便在移动滑块后显示的管接头点 (以红色圆圈显示) 将关闭到峰值值. </li>
	</ol> </li> <li> 在视觉峰值选择之后, 单击并 #34; 接受和 #34; 该程序将与分析表达式的选取点配合以计算模型参数. </li>
	<li> 请注意, 参数和频谱拟合错误保存到文件和 #34; model_fit_parameters. txt 和 #34;; 该程序将另外保存2数字与适合的运动数据和它的功率谱. </li>
</ol>

<ol><li>Pittendrigh, C. S. <a target="_blank" href="https://link.springer.com/chapter/10.1007%2F978-1-4615-6552-9_5">Circadian systems: general perspective.</a> Biological Rhythms. II, 57-80 (1981).</li>
<li>Zhang, E. E., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clocks+not+winding+down%3A+unravelling+circadian+networks%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Clocks not winding down: unravelling circadian networks.</a> Nat Rev Mol Cell Biol. 11 (11), 764-776 (2010).</li>
<li>Tataroglu, O., Emery, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+ticks+of+the+Drosophila+circadian+clock%5BTitle%5D)+AND+%22Curr+Opin+Insect+Sci%22%5BJournal%5D)">The molecular ticks of the Drosophila circadian clock.</a> Curr Opin Insect Sci. 7, 51-57 (2015).</li>
<li>Plautz, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+Drosophila+period+gene+transcription+in+living+animals%5BTitle%5D)+AND+%22J+Biol+Rhythms%22%5BJournal%5D)">Quantitative analysis of Drosophila period gene transcription in living animals.</a> J Biol Rhythms. 12 (3), 204-217 (1997).</li>
<li>Chiu, J. C., Low, K. H., Pike, D. H., Yildirim, E., Edery, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assaying+locomotor+activity+to+study+circadian+rhythms+and+sleep+parameters+in+Drosophila%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Assaying locomotor activity to study circadian rhythms and sleep parameters in Drosophila.</a> J Vis Exp. (43), e2157(2010).</li>
<li>Helfrich-Förster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+control+of+morning+and+evening+components+in+the+activity+rhythm+of+Drosophila+melanogaster--sex-specific+differences+suggest+a+different+quality+of+activity%5BTitle%5D)+AND+%22J+Biol+Rhythms%22%5BJournal%5D)">Differential control of morning and evening components in the activity rhythm of Drosophila melanogaster--sex-specific differences suggest a different quality of activity.</a> J Biol Rhythms. 15 (2), 135-154 (2000).</li>
<li>Dautzenberg, F. M., Neysari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Irreversible+binding+kinetics+of+neuropeptide+Y+ligands+to+Y2+but+not+to+Y1+and+Y5+receptors%5BTitle%5D)+AND+%22Pharmacology%22%5BJournal%5D)">Irreversible binding kinetics of neuropeptide Y ligands to Y2 but not to Y1 and Y5 receptors.</a> Pharmacology. 75 (1), 21-29 (2005).</li>
<li>Lazopulo, A., Syed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mathematical+model+provides+mechanistic+links+to+temporal+patterns+in+Drosophila+daily+activity%5BTitle%5D)+AND+%22BMC+Neuroscience%22%5BJournal%5D)">A mathematical model provides mechanistic links to temporal patterns in Drosophila daily activity.</a> BMC Neuroscience. 17 (1), 14(2016).</li>
<li>Donelson, N., Kim, E. Z., Slawson, J. B., Vecsey, C. G., Huber, R., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+positional+tracking+for+long-term+analysis+of+Drosophila+sleep+and+locomotion+using+the+%26%2334%3Btracker%26%2334%3B+program%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">High-resolution positional tracking for long-term analysis of Drosophila sleep and locomotion using the &#34;tracker&#34; program.</a> PloS ONE. 7 (5), e37250(2012).</li>
<li>Schlichting, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Neural+Network+Underlying+Circadian+Entrainment+and+Photoperiodic+Adjustment+of+Sleep+and+Activity+in+Drosophila%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">A Neural Network Underlying Circadian Entrainment and Photoperiodic Adjustment of Sleep and Activity in Drosophila.</a> J Neurosci. 36 (35), 9084-9096 (2016).</li>
<li>Guo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+neuron+feedback+controls+the+Drosophila+sleep-activity+profile%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Circadian neuron feedback controls the Drosophila sleep-activity profile.</a> Nature. 536 (7616), 292-297 (2016).</li>
</ol>

作者没有什么可透露的。

这项工作给出了使用计算工具的说明, 它提供了对飞行运动模式的定量描述。该工具适用于运动数据与一个数学模型, 由四指数术语, 共同描述的形状和大小的 M 和 E 峰。模型参数的最终值是从拟合数据的功率谱得到的, 在这里, 原始数据的使用可以避免数据分或过滤对参数值施加的 artefactual 影响。模型参数可用于进一步研究飞行运动和引起活动峰的机制。
该管接头的质量可以...

生物钟是一种内生的生化振荡器, 周期约24小时, 在动植物中几乎无处不在1,2。时钟有助于将生物体的内部过程和行为与外部光暗循环同步。自二十世纪六十年代使用果蝇、四腹果蝇以来, 昼夜节律时钟的遗传结构得到了广泛的研究。在这种昆虫中, 生物钟的核心包括四种蛋白质: 周期、永恒、时钟和周期。这些核心组分与其他分子一起形成一个反馈回路, 产生时钟基因的几乎正弦振荡3,4。在苍蝇的生物钟是广泛的研究使用多日运动记录, 飞行活动是检测到的一个单一的红外光束跨越中间的单个管5。一个典型的飞行记录有一个复杂的双峰模式与两个很好的可区分峰: 早晨峰值 (M), 开始在晚上的结束, 并有一个最大的灯打开;和傍晚峰值 (E), 它在一天结束时开始, 在指示灯关闭6时具有最大值。有趣的是, 这种行为记录的形状与在分子水平上观察到的简单的正弦振荡有很大的不同, 这暗示了额外的机制对观测到的时间模式的作用。为了更好地理解这些隐藏的机制, 我们开发了一个计算工具, 它提供了对时态模式的定量描述。
在我们的工作中, 运动节律的定义是一个波形, 模仿飞行活动模式。由于简单的正弦波不能用来对活动中观察到的节律变化进行建模, 我们测试了各种信号形状, 以选择最简单的方法来捕捉记录中所看到的所有显著特征。果蝇昼夜节律行为是由时钟神经元的活动控制的, 通常有指数模式的激活和失活7。数据的指数动力学和可视化分析促使我们建立一个指数条件的模型, 由四指数组成, 具有九独立参数, 与飞行活动模式8相似。除了运动数据外, 我们还分析了它的功率谱。典型的飞行活动谱显示多个峰值在谐波 t0/2, t0/3,等, 除了预期的基本峰值在生理周期 t0。根据傅立叶定理, 只有一个纯正弦波在功率谱中产生一个峰值, 而更复杂的波形在主周期的谐波中显示多个谱峰 (图 1)。因此, 考虑到飞行活动中的正弦时间模式8, 数据的峰功率谱在数学上是预期的, 并不一定意味着存在多个周期的振荡。重要的是, 所提出的模型波形的功率谱也显示了主周期所有谐波的峰值, 类似于飞行的运动记录, 从而强调了我们的模型描述的时间和频率上的飞行数据的高保真度。
在时间分辨率为几分钟或更少的情况下, 飞行活动数据出现噪音, 使得很难直接从原始数据中提取参数。分数据进入更长的时间间隔可以降低噪声水平, 但是, 可以改变数据的方式, 可能会影响模型参数的估计。因此, 我们从记录的功率谱中获取参数, 使用从模型函数的傅里叶变换中计算出的期望功率谱的解析表达式8 (参见附加文件1的引用8)。这种从功率谱获取参数的方法产生了精确的参数值, 而不需要对原始活动数据进行任何附加操作 (如分或过滤)。在参考8中描述了模型的数学细节以及对野生类型和突变数据的应用。这里介绍的协议集中于使用计算工具的 step-by 步骤指令。

<table><tbody><tr><td>Drosophila Activity Monitor</td><td>TriKinetics</td><td>DAM2, DAM5</td><td>Measures fly locootion using single infrared beam</td></tr><tr><td>MatLab</td><td>Mathworks</td><td></td><td>Computing environment and programming language, MatLab should include Optimization and Symbolic Math toolboxes</td></tr><tr><td>Drosophila melanogaster&amp;nbsp;</td><td></td><td>per[S], per[L], iso31(wild type)</td><td>Our analysis can be performed with fly mutants of any circadian period</td></tr></tbody></table>

a computational method to quantify fly circadian activity

在大多数动物和植物中, 昼夜节律时钟协调行为和分子过程, 并使它们与每日暗周期同步。这种时间控制基础的基本机制被广泛地研究使用果蝇, 作为一个模型生物体 .在苍蝇, 时钟通常是研究通过分析多日运动记录。这样的记录显示了一个复杂的双峰模式, 有两个活动的高峰: 黎明时分发生的峰值, 以及黄昏时分发生的傍晚高峰。这两个峰值一起形成一个波形, 它与时钟基因中的正弦振荡非常不同, 这表明除了时钟之外的机制对产生行为数据中的观察到的模式有深远的影响。在这里, 我们提供了使用最近开发的计算方法的说明, 数学描述了飞行活动中的时态模式。该方法符合活动数据的模型波形, 包括四指数术语和九独立的参数, 充分描述的形状和大小的上午和晚上的活动高峰。提取的参数可以帮助阐明基底的动力学机制的基础上普遍观察到的双峰活动模式的飞行节律。

这里提出的方法可以量化的主要特点, 在飞行的运动模式。量化是通过将活动数据与由四指数术语组成的模型拟合来实现的:
<img alt="Equation 1" src="/files/ftp_upload/55977/55977eq1.jpg">
该模型具有描述活动模式的九独立参数。参数 bmd、bMR、bED、和...

Watch this Scientific Journal Video about A Computational Method to Quantify Fly Circadian Activity at JoVE.com

A Computational Method to Quantify Fly Circadian Activity

量化飞行昼夜活动的一种计算方法

本文提出了一种量化飞行节律中主要时间特征的方法。通过拟合参数模型波形, 实现了飞行活动的量化。模型参数描述了每日活动的早晚峰值的形状和大小。

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

a-computational-method-to-quantify-fly-circadian-activity

Research

JoVE Journal

Behavior

5.9K 次观看.  University of Miami. 本文提出了一种量化飞行节律中主要时间特征的方法。通过拟合参数模型波形, 实现了飞行活动的量化。模型参数描述了每日活动的早晚峰值的形状和大小。

视频: A Computational Method to Quantify Fly Circadian Activity

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

含量自发活动，研究昼夜节律和睡眠参数果蝇</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

科研

神经科学

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

测量小鼠使用车轮运行活动的昼夜和急性对光反应

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

免疫与感染

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 ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

在分析癫痫样活动和运动的一种低成本方法<em&gt;果蝇</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal kingdom. As a consequence, sleep deprivation and sleep restriction are powerful tools that are commonly used to provide insight into sleep function. Nonetheless, sleep deprivation experiments are inherently problematic in that the deprivation stimulus itself may be the cause of observed changes in physiology and behavior. Accordingly, successful sleep deprivation techniques should keep animals awake and, ideally, result in a robust sleep rebound without also inducing a large number of unintended consequences. Here, we describe a sleep deprivation technique for Drosophila melanogaster. The Sleep Nullifying Apparatus (SNAP) administers a stimulus every 10s to induce negative geotaxis. Although the stimulus is predictable, the SNAP effectively prevents &#62;95% of nighttime sleep even in flies with high sleep drive. Importantly, the subsequent homeostatic response is very similar to that achieved using hand-deprivation. The timing and spacing of the stimuli can be modified to minimize sleep loss and thus examine non-specific effects of the stimulus on physiology and behavior. The SNAP can also be used for sleep restriction and to assess arousal thresholds. The SNAP is a powerful sleep disruption technique that can be used to better understand sleep function.

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Sleep deprivation is a powerful tool to investigate sleep function and regulation. We describe a protocol to sleep deprive Drosophila using the Sleep Nullifying Apparatus, and to determine the extent of rebound sleep induced by deprivation.

睡眠无效装置:剥夺嗜睡者睡眠的高效方法

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal ...

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 ...

生物学

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant environmental parameters that synchronize animal circadian clocks. In Drosophila circadian photo-responses are mediated by a solely blue light photoreceptor named CRYPTOCHROME (CRY). Upon photoreception, CRY changes its conformation and initiates the proteosomal dependent degradation of TIMELESS (TIM). TIM is an important pacemaker protein, thus degradation of TIM will reset the circadian clock. Under constant light conditions (LL), wild type flies quickly become arrhythmic because of the constant degradation of TIM, while flies bearing defects with circadian photo-responses will still be rhythmic. Thus LL triggered arrhythmicity has been used for screening of components in circadian light input pathways. A brief short light pulse in the night can also dramatically shift phases of circadian rhythms. As expected, this phase shift response is reduced in flied with defects in circadian photoresponse. Thus analyzing locomotion behavior rhythmicity under LL or phase changes after short light pulse in constant darkness (DD) are two major methods to study circadian photoresponse. Here we describe how to design and analyze LL and phase response experiments. LL arrhythmicity is suitable for screening light input pathways mutants, whereas phase response validates the results and provide further information for light sensitivity.

Drosophila locomotor activity is a robust and quantitative measurement of circadian photo-responses. We describe protocols for designing behavior experiments for circadian photo-responses and analyzing the data. Studying the circadian photo-responses is important for dissecting the neuronal and molecular mechanisms of light entrainment.

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant ...

Circadian Entrainment of Drosophila Melanogaster

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this rhythm and to understand the resulting outputs, entrainment of model organisms to defined circadian time-points is required. Here we detail a procedure to entrain many Drosophila to a defined circadian rhythm. Furthermore, we detail post-entrainment steps to prepare samples for immunofluorescence, nucleic acid, or protein extraction-based analysis.

Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology.

果蝇黑色素的循环训练

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this ...

量化飞行昼夜活动的一种计算方法

摘要

探索更多视频

含量自发活动，研究昼夜节律和睡眠参数果蝇

测量小鼠使用车轮运行活动的昼夜和急性对光反应

定量测定的免疫反应和睡眠模式

在自发活动的测定

在分析癫痫样活动和运动的一种低成本方法

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

睡眠无效装置:剥夺嗜睡者睡眠的高效方法

幼虫活动的测量

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

果蝇黑色素的循环训练