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In situ Chemotaxis Assay in Caenorhabditis elegans (for the Study of Circadian Rhythms)
秀丽隐杆线虫原位趋化性实验(用于生命周期节律研究)   

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Abstract

Olfaction is a well-studied sensory mechanism in Caenorhabditis elegans (C. elegans). The nematodes respond to a wide range of chemicals by either attraction, repulsion or a mixture thereof (Bargmann et al., 1993). We have used olfaction to characterize behavioural and molecular circadian rhythms in C. elegans. The circadian clock is a biological oscillator that provides an endogenous temporal structure that approximately matches the 24-hour periodicity in the environment (due to the rotational movement of the Earth). Circadian rhythms are present in most organisms from cyanobacteria to humans and they typically regulate sensory functions among many other processes. Olfaction is under circadian control in many animals (Granados-Fuentes et al., 2006; Granados-Fuentes et al., 2011; Tanoue et al., 2008; Krishnan et al., 1999). This protocol was designed to allow the assessment of olfaction for a population of worms within a short time interval, in the same plate where the worms grew (to avoid washing steps that may disturb the rhythms), and in the presence of food.

Keywords: Circandian(昼夜), Olfaction(嗅觉), C. elegans(线虫), Behaviour(行为), Rhythms(节奏)

Materials and Reagents

  1. Egg preparation
    100 eggs are used for each petri dish. Assays are performed in triplicate.
  2. LB broth
  3. Cholesterol
  4. Ethanol
  5. CaCl2
  6. MgSO4
  7. KPO4
  8. 3.7% (v/v) 1-octanol (Merck KGaA, catalog number: 820931 ) in Ethanol
    This concentration was established after a titration assay to find the lowest concentration needed for a fast response (within the minutes range) (Olmedo et al., 2012)
  9. NGM plates (see Recipes)
    Note: For one experiment, always use plates that were poured from the same batch and were allowed to dry for the same amount of time.
  10. Escherichia coli (E. coli) OP50 culture at 100 g wet bacterial weight/liter (see Recipes)

Equipment

  1. Template for labeling plate (Figure 1)


    Figure 1. Template for plate labeling. On a piece of paper or plastic, draw a 5 cm-diameter circle in which to place the petri dish and a concentric 1 cm-diameter circle to mark the spot for the E. coli drop. Mark the center of this circle (A) with a dot. Draw another dot (B) at 0.8 cm from the center of the circle.

  2. 5 cm Petri plates
  3. Stereomicroscope equipped for picture acquisition (we use a Stereo Discovery V8 from Zeiss at a 10x magnification.)
  4. Centrifuge

Procedure

  1. Add 15 µl of the 100 g wet weight/L E. coli OP50 culture to the center of the NGM plate (inside A).
    Note: You may need to optimize the size of the drop for your stereomicroscope so that the pictures taken show the entire drop. A 15 µl drop of E. coli will form a circle of approximately 1 cm in diameter.
  2. Let the drop dry and allow the bacteria to grow O/N at room temperature.
  3. Add about 100 eggs on the E. coli drop. Grow the worms under the desired conditions until the developmental stage for which you want to score olfaction.
    Note: For circadian experiments, we grow the worms under temperature cycles of 16 h at 13 °C and 8 h at 16 °C. This 24-hour cycle is repeated 5 times until the worms become young adults. Then, the chemotaxis assay is performed every hour for 24 h to look for circadian rhythms in the response to 1-octanol.
  4. Chemotaxis assay: Place the NGM plate over the template, and center the E. coli drop with the circle of the template.
  5. Open the lid and add 1 µl of 1-octanol at dot B. Take care that the 1-octanol does not touch with the E. coli drop. If it does, discard the plate (since the worms that are in direct contact with the 1-octanol/ethanol solution will die).
  6. Close the lid of the plate and incubate for 15 min at the desired temperature.
    Note: For circadian experiments the assay can be performed over 24 h in constant conditions or under the same cycle used to entrain the worms. In both cases, the plates are incubated for 15 min at the same temperature at which they were before adding the 1-octanol.
  7. Take a picture of the final position of the worms. Always keep the dot of 1-octanol at the same position of the picture (Figure 2).


    Figure 2. Final picture of the E. coli drop of a plate showing high response to 1-octanol

  8. On the picture, draw a vertical line dividing the 1 cm circle in two equal parts (Figure 3). Count the number of worms in the proximal (p) and distal (d) sides of the circle. Worms that intersect the middle line are not counted.
  9. Calculate the chemotaxis index (CI=d-p/p+d).


    Figure 3. Calculation of chemotaxis index. The chemotaxis index is calculated by counting the worms in the proximal (p) and distal (p) sides of the circle and applying the formula CI = d - p/p + d.

Recipes

  1. NGM plates
    Prepare NGM media:
    Note: It is very important to prepare all the plates for the experiment in the same batch and allow them to dry for the same time. Variability in the level of humidity of the plates will introduce variability in the assay.
    1. NGM media: Mix 3 g NaCl, 17 g agar, and 2.5 g peptone. Add 975 ml dH2O and stir to mix. Pour half content into a second flask and autoclave. Cool media in a 55 °C water bath or incubator for at least 30 min.
    2. Using sterile conditions, add 1 ml 1 M CaCl2, 1 ml 5 mg/ml cholesterol in ethanol, 1 ml 1 M MgSO4 and 25 ml 1 M KPO4 buffer and mix well.
      Dispense 10 ml of NGM solution into 5 cm Petri plates using a pump. Leave plates at room temperature for 2 days before use to allow excess moisture to evaporate.
  2. 100 g wet weight/L E. coli OP50 culture
    Pick one single colony of OP50 bacteria and inoculate in 200 ml of LB broth. Grow O/N at 37 °C. Centrifuge the culture 10 min at 5,000 x g. Remove the supernatant, calculate the wet weight of bacteria and adjust concentration using LB broth. 

Acknowledgments

This method is based on work that was originally published in Olmedo et al. (2013). This work was supported by the Dutch Science Foundation (NWO; VICI Program).

References

  1. Bargmann, C. I., Hartwieg, E. and Horvitz, H. R. (1993). Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74(3): 515-527.
  2. Granados-Fuentes, D., Tseng, A. and Herzog, E. D. (2006). A circadian clock in the olfactory bulb controls olfactory responsivity. J Neurosci 26(47): 12219-12225.
  3. Granados-Fuentes, D., Ben-Josef, G., Perry, G., Wilson, D. A., Sullivan-Wilson, A. and Herzog, E. D. (2011). Daily rhythms in olfactory discrimination depend on clock genes but not the suprachiasmatic nucleus. J Biol Rhythms 26(6): 552-560.
  4. Krishnan, B., Dryer, S. E. and Hardin, P. E. (1999). Circadian rhythms in olfactory responses of Drosophila melanogaster. Nature 400(6742): 375-378. 
  5. Olmedo, M., O'Neill, J. S., Edgar, R. S., Valekunja, U. K., Reddy, A. B. and Merrow, M. (2012). Circadian regulation of olfaction and an evolutionarily conserved, nontranscriptional marker in Caenorhabditis elegans. Proc Natl Acad Sci U S A 109(50): 20479-20484. 
  6. Tanoue, S., Krishnan, P., Chatterjee, A. and Hardin, P. E. (2008). G protein-coupled receptor kinase 2 is required for rhythmic olfactory responses in Drosophila. Curr Biol 18(11): 787-794.

简介

嗅觉是Caenorhabditis elegans中的一个充分研究的感觉机制( C. elegans )。线虫通过吸引,排斥或其混合物对大范围的化学物质响应(Bargmann等人,1993)。我们使用嗅觉来表征行为和分子昼夜节律。 elegans 。生物钟是生物振荡器,其提供近似匹配环境中的24小时周期性(由于地球的旋转运动)的内生时间结构。昼夜节律存在于从蓝细菌到人的大多数生物体中,并且它们通常在许多其他过程中调节感觉功能。在许多动物中嗅觉处于昼夜节律控制之下(Granados-Fuentes等人,2006; Granados-Fuentes等人,2011; Tanoue等人, 2008; Krishnan等人,1999)。该协议被设计为允许在短时间间隔内,在蠕虫生长的同一板中(以避免可能干扰节律的洗涤步骤)以及在食物存在下,评估一群蠕虫的嗅觉。

关键字:昼夜, 嗅觉, 线虫, 行为, 节奏

材料和试剂

  1. 蛋准备
    每个培养皿使用100个鸡蛋。 测定一式三份。
  2. LB肉汤
  3. 胆固醇
  4. 乙醇
  5. CaCl <2>
  6. MgSO 4 4 /
  7. KPO 4
  8. 在乙醇中的3.7%(v/v)1-辛醇(Merck KGaA,目录号:820931) 在滴定测定后建立该浓度,以找到快速响应所需的最低浓度(在分钟范围内)(Olmedo等人,2012)
  9. NGM板(参见配方)
    注意:对于一个实验,总是使用从相同批次倾倒并允许干燥相同时间量的板。
  10. 大肠杆菌(大肠杆菌)OP50培养物以100g细菌湿重/升

设备

  1. 标签板模板(图1)


    图1.板标记模板。在一张纸或塑料上画一个5厘米直径的圆,在其中放置培养皿和一个同心的直径为1厘米的圆圈,以标记该点 e。 大肠杆菌下降。 用圆点标记此圆(A)的中心。 在离圆心中心0.8cm处绘制一个点(B)。

  2. 5厘米培养板
  3. 用于图像采集的立体显微镜(我们使用Zeiss的Stereo Discovery V8放大10倍)。
  4. 离心机

程序

  1. 加入15μl的100g湿重/L em。大肠杆菌 OP50培养物至NGM板的中心(在A内)。
    注意:您可能需要优化您的立体显微镜下降的大小,以便拍摄的照片显示整个下降。 15μl大肠杆菌将形成直径约1cm的圆形。
  2. 让滴干,并允许细菌在室温下生长O/N。
  3. 在 E上添加约100个鸡蛋。大肠杆菌下降。在所需的条件下生长蠕虫,直到你想要嗅觉的发育阶段。
    注意:对于昼夜节律实验,我们在13℃的16小时和16℃的8小时的温度循环下生长蠕虫。这个24小时的周期重复5次,直到蠕虫成为年轻的成年人。然后,每小时进行趋化性测定24小时,以寻找对1-辛醇的响应中的昼夜节律。
  4. 趋化性测定:将NGM板放置在模板上,并使EM中心。大肠杆菌随模板的圆圈。
  5. 打开盖子,在点B加入1μl的1-辛醇。注意1-辛醇不接触 E。大肠杆菌下降。如果是,丢弃板(因为与1-辛醇/乙醇溶液直接接触的蠕虫会死亡)。
  6. 关闭板的盖子,并在所需的温度下孵育15分钟。
    注意:对于昼夜节律实验,该测定可以在恒定条件下或在用于夹带蠕虫的相同循环下进行24小时。在两种情况下,将板在加入1-辛醇前相同的温度下温育15分钟。
  7. 拍摄蠕虫的最终位置的图片。始终将1-辛醇的点保持在图片的相同位置(图2)。


    图2. E的最终图片。大肠杆菌 下降对1-辛醇反应较强的平板

  8. 在图片上,绘制一条垂直线,将1厘米圆分成两等份(图3)。 计算在圆的近端(p)和远端(d)中的蠕虫数量。 与中间线相交的蠕虫不计入。
  9. 计算趋化指数(CI = d-p/p + d)。


    图3.趋化指数的计算。 通过计数圆的近端(p)和远端(p)中的蠕虫并应用公式CI = d-p/p + d来计算趋化性指数。

食谱

  1. NGM板
    准备NGM媒体:
    注意:在同一批次中准备所有实验板并允许它们同时干燥是非常重要的。 板的湿度水平的变化将在测定中引入变异性
    1. NGM培养基:混合3g NaCl,17g琼脂和2.5g蛋白胨。加入975ml dH 2 O并搅拌混合。将一半内容物倒入第二个烧瓶中并高压灭菌。在55°C水浴或培养箱中冷却培养基至少30分钟。
    2. 使用无菌条件,加入1ml 1M CaCl 2,1ml 5mg/ml胆固醇的乙醇溶液,1ml 1M MgSO 4和25ml 1M KPO 4 缓冲液并混匀。
      使用泵将10ml的NGM溶液分配到5cm培养皿中。使板在室温下放置2天,然后使用,以使过量的水分蒸发
  2. 100g湿重/L E。大肠杆菌 OP50培养物
    挑取一个单一菌落的OP50细菌并接种在200ml LB肉汤中。在37℃生长O/N。在5,000xg离心培养物10分钟。取出上清液,计算细菌的湿重,并使用LB肉汤调整浓度。

致谢

该方法基于最初在Olmedo等人(2013)中发表的工作。这项工作得到了荷兰科学基金会(NWO; VICI计划)的支持。

参考文献

  1. Bargmann,C.I.,Hartwieg,E。和Horvitz,H.R。(1993)。 气味选择性基因和神经元介导C中的嗅觉。 elegans 。 74(3):515-527。
  2. Granados-Fuentes,D.,Tseng,A.and Herzog,E.D。(2006)。 嗅觉灯泡中的生物钟控制嗅觉反应性 J Neurosci 26(47):12219-12225。
  3. Granados-Fuentes,D.,Ben-Josef,G.,Perry,G.,Wilson,D.A。,Sullivan-Wilson,A.and Herzog,E.D。 嗅觉歧视中的每日节律依赖于时钟基因,但不是视交叉上核。 J Biol Rhythms 26(6):552-560。
  4. Krishnan,B.,Dryer,SE和Hardin,PE(1999)。嗅觉反应中的昼夜节律 400 400(6742):375-378。 
  5. Olmedo,M.,O'Neill,J.S.,Edgar,R.S.,Valekunja,U.K.,Reddy,A.B.and Merrow,M。(2012)。 嗅觉的环境调节和在Caenorhabditis elegans中的进化保守,非转录标记。 Proc Natl Acad Sci US A 109(50):20479-20484。 
  6. Tanoue,S.,Krishnan,P.,Chatterjee,A.and Hardin,P.E。(2008)。 G蛋白偶联受体激酶2是果蝇中的节律性嗅觉反应所必需的。 Curr Biol 18(11):787-794。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Merrow, M. and Olmedo, M. (2014). In situ Chemotaxis Assay in Caenorhabditis elegans (for the Study of Circadian Rhythms). Bio-protocol 4(3): e1040. DOI: 10.21769/BioProtoc.1040.
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