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Flight and Climbing Assay for Assessing Motor Functions in Drosophila
评估果蝇运动功能的飞行和攀爬分析   

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参见作者原研究论文

本实验方案简略版
The Journal of Neuroscience
Oct 2015

Abstract

Motor control requires the central nervous system to integrate different sensory inputs and convey this information to the relevant central pattern generator for execution of motor function through motor neurons and muscles. Proper motor control is essential for any mobile organism to survive and interact with the external environment. For flying insects, motor control is required for flying, walking, feeding and mating apart from other more advanced behaviours such as grooming and aggression. Any perturbation to the sensory input or malfunctioning of neural connections to the motor output can result in motor defects. Here, we describe simple protocols for assessing flight and climbing ability of fruit flies, which can be used as two general tests to assess their motor function.

Keywords: Tethers (系绳), Air-puff (空气脉冲), Cold anaesthesia (冷麻醉), Cylinder (圆筒)

Background

Coordinated motor functions are important for every mobile organism for survival as the major needs of finding food, shelter, mates and escaping from predators involve motor activity. Here we describe protocols to assess the flight and climbing ability of both individual and groups of Drosophila melanogaster. Both these protocols have been used extensively in earlier studies (Agrawal and Hasan, 2015; Pathak et al., 2015; Richhariya et al., 2017).


Part I: Flight protocol

Materials and Reagents

  1. Plastic tray of approximately 22 x 18 x 5 cm
  2. Petri dish of ~9 cm diameter (Fisher Scientific, catalog number: 12033333; Manufacturer: Pyrex, catalog number: 1480/08D ) with its outer side covered with Whatman filter paper (GE Healthcare, catalog number: 1001-918 ) (Figure 1A)
  3. Tethers made of stainless steel rod (diameter 0.2 cm) attached to stainless steel wire (diameter 0.01 cm) (Figure1B)
  4. Polystyrene foam of approximately 10 x 6 x 5 cm
  5. One glass slide (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3011-002 )
  6. Round synthetic hair brush, size 4 (Camlin, series 66, size 4)
  7. Fly strains to be tested of either sex, aged 3-4 days
  8. Ice
  9. Transparent nail polish

Equipment

  1. Wide field microscope (Olympus, model: SZX9 )
  2. Timer/stop watch (Fisher Scientific, catalog number: S02272 )
  3. Empty glass vials for cold anaesthesia
  4. Behaviour room with controlled temperature (~25 °C) and humidity (~60%)
  5. JVC colour video camera–ModelTK-C1481BEG (JVCKENWOOD, model: TK-C1481BEG )

Software

  1. Origin 8.0 software (MicroCal, Origin Lab, Northampton, MA, USA)
  2. Streampix digital video recording software

Procedure

  1. Place the Petri dish in a plastic tray filled with ice. Make sure that the filter paper edges are not touching the ice (Figure 1F). Keep the Petri dish on ice for 10 min before anaesthetising the flies.
  2. Add 5-6 flies of either sex into an empty vial. Flies are transferred to the empty vial by tapping the vial with flies on a pounding pad so that flies are located away from the cotton plug and are at the bottom of the vial. Following the tapping, the plug is removed and the vial is inverted rapidly onto a fresh empty vial, tapped hard so as to transfer all the flies into the empty vial which is closed immediately with another cotton plug to prevent flies from escaping (Figure 1C). Anaesthetise the flies in the empty vial by keeping the vial in ice for 5 min (Figure 1D).
  3. Gently tap the empty vial with flies on ice after 1 min and make sure all flies are at the bottom of the vial. Once anaesthetized, all flies will be immobile and will remain at the bottom of the vial (Figure 1E).
  4. Once the flies are fully anaesthetised (within 5 min; it is important that flies are not kept on ice longer than 5 min), gently place the flies with a fine brush with their dorsal side up on the cold Petri dish (Figure 1G).
  5. Add a tiny drop of nail polish (~10 µl) onto a glass slide at room temperature, as the nail polish dries up faster on a pre-cooled glass slide. Take a very small drop of it (~1-2 μl) on the tip of the tether by gently dipping the tip of the tether (0.7-1 mm) on the drop of nail polish.
  6. Using a very fine brush in your left hand, gently hold the fly above its wings on the abdomen and hold the tether in your right hand. Gently place the tether on the neck in such a way that the drop of nail polish touches the posterior of the head segment and anterior of the thorax. This position of the tether is important to ensure that the body and head of the fly are not tilted. If tethered correctly a normal individual will fly between 5-15 min. It is important that the nail polish drop is of the optimum size, as bigger drops might cover the eyes or the sensory bristles on the thorax and thus affect flight durations. Tether only a maximum of 5-10 flies as a batch (Video 1). Once every fly is tethered, fix the tether to a polystyrene foam (Figures 1H and 1I).

    Video 1. Tethering a single fly for measuring flight durations

  7. Once all flies recover from anaesthesia (~5 min), touch the legs gently using a brush to stop them flying spontaneously. Keep a stop watch ready, give a gentle mouth blown or automated air puff to all the flies in a batch and immediately turn on the stop watch. Observe flight duration for all the flies in a batch (Figure 1J and Video 2).

    Video 2. Recording flight durations after an air-puff

  8. Note down the time at which each fly stops flying. This is the flight duration of a single fly. The flight duration can be monitored either manually or by recording a video using any smart phone camera. We use JVC colour video camera–Model TK-C1481BEG and StreamPix digital video recording software to record flight videos. In the latter case flight durations of each fly can be noted down later from the video.
  9. A minimum of 30 flies should be tested per genotype in batches of 5-10.

Notes:

  1. In this protocol, both short and prolonged flight durations can be recorded by varying the duration of recording. For measuring short flight durations, flight time is recorded for 30 sec whereas to measure prolonged flight durations, the recording time is extended to 15 min/900 sec or longer as required. A majority of wild type (CS) flies fly for more than 5 min. In instances where the flies (experimental genotypes) are severely flight defective, they will fly for only a few seconds. In such instances flight times can be measured for 30 sec.
  2. The flies used for flight assays should not be exposed to CO2 for anaesthesia once they eclose as adults from pupal cases.


    Figure 1. Tethering. A. Petri dish covered with filter paper; B. Tether with dimensions; C. Flies transferred to empty vial for cold anaesthesia; D. Empty vial with flies in ice; E. Anaesthetised flies in an empty vial; F. Tray filled with ice to keep the Petri dish cold for tethering; G. Orientation of anaesthetised flies for tethering on the Petri dish; H. Tethered flies fixed to polystyrene foam before assessing the flight durations; I. Tethered fly before air puff stimulated flight; J. Tethered fly after an air puff.   

Data analysis

  1. Mean, median and SEM are calculated for a minimum of 30 flies per genotype. We did not find any significant difference between the flight and climbing ability of males and females. Flight durations are depicted as box plots with mean, median and the flight duration of individual flies are overlaid on the box using Origin 8.0 software (MicroCal, Origin Lab, Northampton, MA, USA)
  2. Mann Whitney U-test is applied to evaluate differences between samples as the data do not follow a normal distribution.

Flight durations for 30 Canton S (wild type) flies monitored for 15 min (900 sec) are given in Table 1 and a representative box plot of these data is depicted in Figure 2.

Table 1. Flight durations of 30 Canton S–4 days old (wild type) flies of either sex



Figure 2. Flight durations of Canton S (wild type) flies depicted as a box plot. The filled square represents mean of the distribution, the box represents 25-75% of the distribution and open diamonds represent flight durations of individual flies.

Part II: Climbing protocol

Materials and Reagents

  1. Glass cylinder of diameter 2.5 cm (borosilicate glass measuring cylinder 50 ml with a diameter of 2.5 cm) with a mark at the height of 8 cm from the bottom
  2. Flies to be tested of appropriate genotypes and either sex aged 3-4 days

Equipment

  1. Timer/stop watch (Fisher Scientific, catalog number: S02272 )
  2. Behaviour room with controlled temperature (~25 °C) and humidity (~60%)

Procedure

  1. Freshly eclosed flies of either sex should be collected in vials with standard corn meal media in batches of 10 and aged for 3-4 days.
  2. Transfer a batch of 10 flies (non anaesthetised) to the glass cylinder by tapping the vial with the flies on the pounding pad, immediately removing the cotton plug, inverting the flies into the glass cylinder, tapping hard and closing the cylinder with a fresh cotton plug.
  3. Allow the flies to get accustomed to the new environment for 3-4 min.
  4. Keep the stop watch ready, tap the measuring cylinder with 10 flies 3 times gently making sure all the flies reach the bottom of the cylinder and immediately turn on the stop watch (Video 3).

    Video 3. Climbing assay with a batch of 10 flies

  5. Count the number of flies that cross the 8 cm mark within 12 sec.
  6. Repeat the experiment twice more with the same set of flies with an interval of 3-4 min in between trials and calculate the average climbing ability for the single batch of flies. Wipe the cylinder with dry tissue paper once the assay is done for one genotype. Wash the cylinder with water once the entire set of experiments are done for a day and let it dry. Make sure not to use soap, any other detergent or ethanol for cleaning.
  7. A minimum of 3 batches of 10 flies should be tested per genotype, each batch tested 3 times in total.

Data analysis

  1. The average number of flies that crossed the 8 cm mark in 12 sec of time can be plotted as a bar graph with SEM calculated from a minimum of 3 independent batches of 10 flies using Origin 8.0 software (MicroCal, Origin Lab, Northampton, MA, USA). A majority of wild type/control flies reach the 8 cm mark within 12 sec. Bar graphs allow for easier visualization and comparison between various genotypes. Box plots with individual batch values overlaid on the box can also be used for representing these data.
  2. Statistical significance between different genotypes was calculated using One-way analysis of variance (ANOVA) for P < 0.05 using Origin 8.0 software (MicroCal, Origin Lab, Northampton, MA, USA).
  3. Climbing data for Canton S flies are given in Table 2 and the same data are depicted as a bar graph in Figure 3.

    Table 2. Data for climbing assay of wild type (Canton S) flies



    Figure 3. Climbing ability of flies (Canton S) depicted as percentage climbers. Bar graph represents the percentage of flies that crossed the 8 cm mark within 12 sec with error bar representing SEM and open diamonds on the bar representing the average of percentage climbers per batch.

Notes

The diameter of the cylinder used for climbing assay should not be more than 2.5 cm as higher diameter columns also allow the flies to fly and jump during the assay.

Acknowledgments

We would like to thank Santanu Banerjee, Sufia Sadaf and Tarjani Agrawal for setting up and improvising the flight protocol to the current format. We would also like to thank NCBS, TIFR for funding. This is a modified version of protocol explained in Agrawal and Hasan, 2015, Pathak et al., 2015 and Richhariya et al., 2017. The authors declare no competing financial interest.

References

  1. Agrawal, T. and Hasan, G. (2015). Maturation of a central brain flight circuit in Drosophila requires Fz2/Ca2+ signaling. Elife 4.
  2. Pathak, T., Agrawal, T., Richhariya, S., Sadaf, S. and Hasan, G. (2015). Store-operated Calcium entry through orai is required for transcriptional maturation of the flight circuit in Drosophila. J Neurosci 35(40): 13784-13799.
  3. Richhariya, S., Jayakumar, S., Abruzzi, K., Rosbash, M. and Hasan, G. (2017). A pupal transcriptomic screen identifies Ral as a target of store-operated calcium entry in Drosophila neurons. Sci Rep 7: 42586.

简介

电机控制要求中枢神经系统集成不同的感觉输入,并将该信息传达给相关的中枢模式发生器,以通过运动神经元和肌肉执行运动功能。 适当的电机控制对于任何移动生物体的生存和与外部环境的相互作用都至关重要。 对于飞行昆虫来说,飞行,行走,喂食和交配需要运动控制,而不是其他更高级的行为,例如梳理和攻击。 对感觉输入的任何干扰或与电机输出的神经连接的故障都可能导致运动缺陷。 在这里,我们描述了用于评估果蝇飞行和爬行能力的简单协议,其可以用作两种通用测试来评估它们的运动功能。

【背景】由于寻找食物,住所,配偶和逃离掠食者的主要需求涉及运动行为,协调运动功能对于每个移动生物体的生存都很重要。 在这里,我们描述协议来评估个体和果蝇组的飞行和爬升能力。 这两个方案已经在早期的研究中广泛使用(Agrawal和Hasan,2015; Pathak等人,2015; Richhariya等人,2017)。

关键字:系绳, 空气脉冲, 冷麻醉, 圆筒

第一部分:Flight protocol

材料和试剂

  1. 大约22 x 18 x 5厘米的塑料托盘
  2. 其外侧用Whatman滤纸(GE Healthcare,目录号:1001-918)覆盖约9cm直径的培养皿(Fisher Scientific,目录号:12033333;制造商:Pyrex,目录号:1480 / 08D) )
  3. 系绳由直径0.2厘米的不锈钢棒制成(直径0.01厘米)(图1B)

  4. 约10×6×5厘米的聚苯乙烯泡沫
  5. 一个载玻片(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3011-002)
  6. 圆形合成毛刷,4号(Camlin,66系列,4号)


  7. 飞行株需要进行任何性别测试,年龄3-4天

  8. 透明指甲油

设备

  1. 宽视场显微镜(奥林巴斯,型号:SZX9)
  2. 定时器/秒表(Fisher Scientific,产品目录号:S02272)
  3. 用于冷麻醉的空玻璃瓶
  4. 温度控制(〜25°C)和湿度(〜60%)的行为室
  5. JVC彩色摄像机 - 型号TK-C1481BEG(JVCKENWOOD,型号:TK-C1481BEG)

软件

  1. Origin 8.0软件(MicroCal,Origin Lab,Northampton,MA,USA)
  2. Streampix数字录像软件

程序

  1. 将培养皿放入盛有冰块的塑料托盘中。确保滤纸边缘不接触冰块(图1F)。

    在将蝇蛆进行麻醉之前,将培养皿放在冰上10分钟
  2. 将5-6只任何性别的苍蝇加入空瓶中。通过在敲击垫上用苍蝇拍打小瓶将苍蝇转移到空小瓶中,使得苍蝇远离棉塞并位于小瓶的底部。攻丝后,取下塞子,将瓶子迅速倒入新鲜空瓶中,用力敲击以便将所有苍蝇转移到空瓶中,立即用另一个棉塞塞住,以防止苍蝇逸出(图1C) 。通过将小瓶保持在冰中5分钟来使空瓶中的苍蝇麻醉(图1D)。
  3. 1分钟后用苍蝇轻轻敲打空的小瓶,并确保所有苍蝇都在小瓶的底部。一旦麻醉,所有的苍蝇都将不动,并将保留在小瓶的底部(图1E)。
  4. 一旦苍蝇被完全麻醉(5分钟内;重要的是苍蝇没有保存在冰上超过5分钟),轻轻地放置苍蝇与他们的背侧向上冷培养皿上的细刷(图1G)。
  5. 在室温下将一滴指甲油(约10μl)加到载玻片上,因为指甲油在预冷的载玻片上干得更快。
    轻轻地将系绳(0.7-1毫米)的尖端浸在指甲油滴上,在系绳的尖端取一小滴(〜1-2μl)。
  6. 用左手的细刷,轻轻地将苍蝇放在腹部的翅膀上方,并将系绳保持在右手中。轻轻地将系绳放在脖子上,使指甲油滴落在头部的后部和胸部的前部。系绳的这个位置对于确保苍蝇的身体和头部不会倾斜很重要。如果正确连接,正常人将在5-15分钟之间飞行。重要的是,指甲油滴是最佳的尺寸,因为更大的水滴可能会覆盖胸部上的眼睛或感觉刷毛,从而影响飞行持续时间。作为批次最多只有5-10只苍蝇(视频1)。一旦每只小苍蝇被系住,将系绳固定在聚苯乙烯泡沫上(图1H和1I)。

    视频1

  7. 一旦所有苍蝇从麻醉状态恢复(〜5分钟),用刷子轻轻地触碰腿部,以防止它们自发地飞行。准备好秒表,对所有苍蝇进行温和的吹嘴或自动吹气,然后立即打开秒表。观察批次中所有苍蝇的飞行持续时间(图1J和视频2)。

    视频2

  8. 记下每次飞行停止飞行的时间。这是一次飞行的飞行时间。飞行时间可以通过手动或使用任何智能手机摄像头录制视频进行监控。我们使用JVC彩色摄像机 - 型号TK-C1481BEG和StreamPix数字视频录制软件来记录飞行视频。在后一种情况下,每次飞行的飞行时间可以从视频中记下。

  9. 每个基因型应至少测试30只苍蝇,每批5-10只。

备注:

  1. 在这个协议中,可以通过改变记录的持续时间来记录短暂的和长时间的飞行持续时间。为了测量短飞行时间,记录飞行时间30秒,而为了测量长时间飞行时间,根据需要将记录时间延长到15分钟/ 900秒或更长。大多数野生型(CS)苍蝇飞行超过5分钟。在苍蝇(实验基因型)严重飞行缺陷的情况下,它们将仅飞行几秒钟。在这种情况下,飞行时间可以测量30秒。
  2. 用于飞行测定的苍蝇在成年后不应暴露于CO麻醉下,


    图1.共享。 :一种。用滤纸覆盖的培养皿; B.具有尺寸的系绳; C.苍蝇转移到空瓶中进行冷麻醉; D.用冰块中的苍蝇将小瓶倒空; E.在空瓶中麻醉苍蝇; F.装满冰块的托盘保持陪替氏培养皿冷藏的安全性; G.麻醉苍蝇定位在陪替氏培养皿上; H.在评估飞行持续时间之前固定在聚苯乙烯泡沫上的拴系苍蝇; I.喷气之前受束缚的飞行刺激飞行; J.抽气后拴住苍蝇。&nbsp;&nbsp;&nbsp;


数据分析

  1. 平均值,中位数和SEM计算每个基因型最少30只苍蝇。我们没有发现男性和女性的飞行能力和攀登能力之间存在显着差异。飞行持续时间被描绘为箱形图,其中使用Origin8.0软件(MicroCal,Origin Lab,Northampton,MA,USA)将单个苍蝇的平均值,中值和飞行持续时间覆盖在箱子上。
  2. 由于数据不符合正态分布,因此应用Mann Whitney U检验来评估样本之间的差异。

在表1中给出了30天广州S(野生型)苍蝇监测15分钟(900秒)的飞行持续时间,并且这些数据的代表性框图在图2中示出。

表1. 30天S-4天龄(野生型)任一性别苍蝇的飞行时间



图2. Canton S (野生型)苍蝇的飞行时间描绘为一个箱形图。 实心方块表示分布的均值,方框表示分布的25-75%,空心菱形表示单个苍蝇的飞行持续时间。

第二部分:攀登协议

材料和试剂

  1. 直径2.5厘米的玻璃圆筒(硼硅酸盐玻璃量筒50毫升,直径2.5厘米),在离底部8厘米的高度有一个标记
  2. 苍蝇被测试适当的基因型和任何性别年龄3-4天

设备

  1. 定时器/秒表(Fischer Scientific目录号:S02272)
  2. 温度控制(〜25°C)和湿度(〜60%)的行为室

程序

  1. 应将新鲜封闭的任一性别的苍蝇收集在装有标准玉米粉培养基的小瓶中,每批10只,老化3-4天。
  2. 将一批10只苍蝇(非麻醉)转移到玻璃瓶中,方法是用撞击垫上的苍蝇拍打小瓶,立即取下棉塞,将苍蝇倒入玻璃瓶中,轻轻拍打,用新鲜棉布关闭瓶子插头。
  3. 让苍蝇适应3-4分钟的新环境。
  4. 准备好秒表,轻拍10次苍蝇量筒,轻轻确保所有苍蝇都到达圆筒底部,并立即开启秒表(视频3)。

    视频3

  5. 计算在12秒内穿过8厘米标记的苍蝇数量。
  6. 在试验之间用相同的一组苍蝇重复实验两次,间隔为3-4分钟,并计算单批苍蝇的平均攀爬能力。一旦测定完成一种基因型,用干棉纸擦拭圆筒。一整套实验完成一天并让其干燥后,用水洗缸。确保不要使用肥皂,任何其他洗涤剂或乙醇进行清洁。
  7. 每个基因型至少应测试3批10只果蝇,每批测试3次。

数据分析

  1. 在12秒时间内跨过8cm标记的苍蝇的平均数可以作为条形图绘制,其中使用Origin 8.0软件(MicroCal,Origin Lab,Northampton,MA,USA)从最少3个独立批次的10只苍蝇计算SEM,美国)。大多数野生型/对照苍蝇在12秒内达到8厘米标记。条形图允许在各种基因型之间更容易可视化和比较。包装箱上重叠的单个批次值的箱子图也可用于表示这些数据。
  2. 使用单因素方差分析(ANOVA)计算不同基因型之间的统计学显着性(P <0.05) 0.05使用Origin 8.0软件(MicroCal,Origin Lab,Northampton,MA,USA)。
  3. 表2给出了 Canton S 苍蝇的攀登数据,同样的数据在图3中显示为条形图。

    表2.野生型( Canton S )苍蝇攀爬测定的数据



    图3.苍蝇攀爬能力( S )描绘为登山百分比。 条形图表示在12秒内穿过8厘米标记的苍蝇百分比,误差线代表SEM,条上的空心菱形代表每批攀登者的平均百分比。

笔记

用于攀爬测定的圆柱体直径不应超过2.5厘米,因为更高直径的柱子也允许苍蝇在测定期间飞行和跳跃。

致谢

我们要感谢Santanu Banerjee,Sufia Sadaf和Tarjani Agrawal设立和即兴将飞行协议改为当前格式。我们还要感谢NCBS,TIFR的资助。这是2015年Agrawal和Hasan,Pathak等人,2015年和Richhariya等人于2017年发布的协议的一个修改版本。作者宣称没有竞争的经济利益。

参考

  1. Agrawal,T。和Hasan,G。(2015)。 果蝇中央大脑飞行电路的成熟需要Fz2 / Ca < sup> 2 + 信号。 Elife 4。
  2. Pathak,T.,Agrawal,T.,Richhariya,S.,Sadaf,S.and Hasan,G。(2015)。 通过orai进行商店营业的钙进入是为了在果蝇的飞行线路转录成熟所必需的。 J Neurosci 35(40):13784-13799。
  3. Richhariya,S.,Jayakumar,S.,Abruzzi,K.,Rosbash,M.和Hasan,G。(2017)。 蛹转录组筛选将Ral识别为商店操作的钙进入目标果蝇 neurons。 Sci Rep 7:42586.
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Manjila, S. B. and Hasan, G. (2018). Flight and Climbing Assay for Assessing Motor Functions in Drosophila. Bio-protocol 8(5): e2742. DOI: 10.21769/BioProtoc.2742.
  2. Pathak, T., Agrawal, T., Richhariya, S., Sadaf, S. and Hasan, G. (2015). Store-operated Calcium entry through orai is required for transcriptional maturation of the flight circuit in Drosophila. J Neurosci 35(40): 13784-13799.
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