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Behavioral Assays to Study Oxygen and Carbon Dioxide Sensing in Caenorhabditis elegans
研究秀丽隐杆线虫的氧气和二氧化碳传感的行为分析实验   

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本实验方案简略版
Scientific Reports
Aug 2017

Abstract

Animals use behavioral strategies to seek optimal environments. Population behavioral assays provide a robust means to determine the effect of genetic perturbations on the ability of animals to sense and respond to changes in the environment. Here, we describe a C. elegans population behavioral assay used to measure locomotory responses to changes in environmental oxygen (O2) and carbon dioxide (CO2) concentrations. These behavioral assays are high-throughput and enable examination of genetic, neuronal and circuit function.

Keywords: C. elegans (秀丽隐杆线虫), Behavior (行为), Oxygen (氧), Carbon dioxide (二氧化碳), Sensing (传感)

Background

Oxygen concentration provides C. elegans with information regarding environmental conditions. In laboratory conditions, when presented with an O2 gradient, C. elegans migrate towards intermediate concentrations (2%-12%) (Gray et al., 2004). Low levels of O2 may indicate the presence of bacteria (food) while high O2 levels may imply that the worms are close to the surface of its environmental substrate. Therefore, C. elegans responds in an exquisitely sensitive manner to changes in O2 concentration to enable navigation to optimal environments conducive to survival and propagation of offspring (Gray et al., 2004; Chang et al., 2006; Zimmer et al., 2009). Similarly, worms present a strong behavioral response to changes in CO2. Well-fed animals avoid CO2 while starved animals are attracted to CO2 (Hallem and Sternberg, 2008). This change in response may provide an evolutionary advantage to find food, as the bacterial food source releases CO2. Furthermore, pathogens generate CO2, which possibly indicates why well-fed worms avoid CO2. Specific neurons regulate gas sensing responses in C. elegans including the head neurons URXL/R, BAGL/R, AQR and the PQR neuron located in the tail (Hallem and Sternberg, 2008; Zimmer et al., 2009; Bretscher et al., 2011). The main regulators of O2 sensing are the URX and BAG neurons, which sense upshifts and downshifts of oxygen respectively. Regarding changes in CO2 levels, the BAG neurons are the principal sensors.

Materials and Reagents

  1. Worm pick made with platinum wire (Tritech Research, catalog number: PT-9901 )
  2. Filter paper (110 mm diameter) (GE Healthcare, Whatman, catalog number: 1001-110 )
  3. Petri dish 140 mm diameter (VWR, catalog number: 391-1500 )
  4. 90 mm Petri dishes (Techno Plas, catalog number: S9014UV20 )
  5. C. elegans strains
    The following protocol applies to strains derived from wild type animals (N2, Bristol strain). Suggested controls (available from the Caenorhabditis Genetics Center (CGC)):
    1. N2 (wild-type): positive control
    2. gcy-31(ok296): unable to respond to downshifts of O2 levels
    3. gcy-35(ok769): unable to respond to upshifts of O2 levels
    4. gcy-9(n4470): unable to respond to changes in CO2 concentration
    5. tax-4(p678): deficient in O2 and CO2 sensing
    Note: All strains should be grown and maintained under standard conditions (Brenner, 1974). All strains should be crosses a minimum of 4 times with wild type animals. Changes in temperature or starvation can affect the results of the assay. Therefore, strains should be maintained at a constant temperature (routinely 20 °C) and well-fed with OP50 Escherichia coli for at least two generations prior to the assay.
  6. Copper(II) chloride dihydrate (CuCl2·2H2O) (Sigma-Aldrich, catalog number: 221783-100G )
  7. NGM maintenance plates seeded with OP50 E. coli (see Recipes)
  8. KPO4 buffer (1 M, see Recipes)
    1. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662-500G )
    2. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: 3786-500G )
  9. Assay NGM plates (see Recipes)
    1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653-250G )
    2. Agar (SERVA high-gel strength agar) (SERVA Electrophoresis, catalog number: 11396.03 )
    3. Cholesterol (Sigma-Aldrich, catalog number: C8667-25G )
    4. Calcium chloride (CaCl2) (Merck, catalog number: 1.02382.0500 )
    5. KPO4 buffer (1 M)
  10. Starvation plates (see Recipes)

Equipment

  1. Computer compatible with the software
    Intel core i7, 32GB RAM, Windows 7, 64 bit. Needs one additional gigabit Ethernet card for connecting the GigE camera (Figure 1)
  2. Camera
    We use a 4-megapixel CCD camera (JAI, model: BM-500GE ), 35 mm objective: QIOPTIQ MEVIS 3516. Extension rings: PENT EXTENSION RING 1 (1 mm C-Mount) (Figure 1)


    Figure 1. Computer and camera setup

  3. Flow controllers
    Vögtlin Instruments, CO2: GSC-A9TA-BB21 50 mln/min, N2: GSC-A9TA-BB22 200 mln/min, O2: GSC-A9TA-BB21 50 mln/min (Figure 2)


    Figure 2. Gas and flow controller setup. A. Overview of mains gas supply connected to the flow controller. B. Close-up of the flow controller.

  4. LED illumination (CCS, catalog number: TH-211/200RD ), cable (CCS, catalog number: FCB-1 ), power supply (CCS, catalog number: PD2-5024 )
  5. Individual gas bottles for O2, CO2 and N2
    Note: Alternatively, premixed bottles may be used, depending on the flow controllers that are used. Nevertheless, we recommend individual gas bottles as they provide flexibility on the gas parameters that can be tested.
  6. Assay chamber: custom-made transparent Plexiglas device with a flow arena of 60 x 60 x 0.7 mm (Figure 3)


    Figure 3. Custom-designed assay chamber. A. Top diagram: view from the top. Bottom diagram: lateral view. Note that the diagram is not scaled. B. Photograph of the assay chamber taken from above.

  7. Autoclave
  8. Dryer (Binder, catalog number: 9010-0102 )
  9. 500 ml autoclaved bottle

Software

  1. Labview (to program and regulate the flow controllers)
  2. MATLAB (R13) (MathWorks) and Image Acquisition and Image Processing Toolbox (Figure 4)
  3. Parallel WormTracker (freely available: https://sourceforge.net/projects/wormtracker/)
  4. Streampix software (Norpix)


    Figure 4. Screenshots of WormTracker analysis. A. Tracks of individual worms (red lines) detected by the software. B. MATLAB analysis software screen showing tracking frames.

Procedure

  1. Growth and synchronization of C. elegans populations
    Recommendation: For each strain, pick 4 larval stage 4 (L4) hermaphrodites onto 4 maintenance plates every day (4 worms per plate), and use the following generation in the assay: For transgenic extrachromosomal strains, more than 4 transgenic L4s should be picked to ensure enough transgenic worms are available for the assay. In addition, for mutants with a lower brood size than wild type, a higher number of worms must be picked for synchronization.
  2. Pick approximately 150 L4/young adult worms to a starvation plate (not seeded with OP50 E. coli). So that bacteria are not transferred to the empty plate, do not pick using bacteria, instead use the worm pick as a spoon to transfer worms that have crawled off the bacterial lawn.
    Note: Worms should not be prepared by washing off a plate with M9 buffer, as behavioural assays are very sensitive to stress.
  3. Allow worms to starve for 1 h. Feeding status strongly affects behavioral responses to O2 and CO2, therefore, the starvation must be conducted for the same length of time for each strain tested. Furthermore, well-fed N2 worms do not respond to downshifts in O2 (Zimmer et al., 2009).
  4. Prepare the assay plate (Figure 5)
    1. Cut a 56 x 56 mm squared area (a hole puncher may be used) in the center of the Whatman filter paper.
    2. Place the filter paper on top of the behavioral assay plate (14 cm NGM assay plate).
    3. Soak the filter paper at the border of the square with 20 mM CuCl2 to corral worms within the assay area.


      Figure 5. Illustration of the assay plate. 14 cm NGM assay plate containing a 56 x 56 mm arena of Whatman filter paper soaked in 700 μl of 20 mM CuCl2. Hermaphrodites are placed in the arena and the assay chamber is carefully positioned on top of the plate.

  5. Spoon the worms from the starvation plate to the centre of the assay plate.
    Note: Be careful not to damage the agar as worms will burrow.
  6. Place the assay chamber on top of the assay plate. It is important that the 56 x 56 mm square within which the worms are located fits within the square of the chamber.
  7. Connect gas tubes to the chamber and start the gas flow with atmospheric concentrations (or alternatively with your desired starting conditions). Allow the gas to flow for at least 5 min prior commencing your experiments.
  8. Record videos while running the chosen gas concentration test.
    Note: To assay O2 and CO2 sensing, balance the concentrations with N2. For testing acute O2 sensing, we suggest the following timings for each condition: 6 min 21%O2/79%N2–6 min 10%O2/90%N2–6 min 21%O2/79%N2. For testing acute CO2 sensing, we suggest the following timings for each condition: 6 min 21%O2/79%N2–6 min 21%O2/78%N2/1%CO2–6 min 21%O2 79%N2.

Data analysis

The parallel WormTracker software package includes a detailed user manual that describes how to run it for extraction of necessary data from the videos (Chalasani et al., 2007; Ramot et al., 2008). It is important to state that the script can be updated to different operating systems and video formats. The original script was written for PC running Windows XP and uncompressed, grayscale (8-bit) movies in AVI format with a resolution of 640 x 480. The WormTracker identifies worms and tracks their position defined by the worm’s centre of mass. The tracking is performed after the video has been recorded. After the worms are tracked, the Wormanalyzer permits analysis of the tracks and detection of the speed of the worms and other parameters such as turning events. Those data can be extracted and further analysed with any standard statistical software such as GraphPad Prism.

Representative data
When wild type hermaphrodites experience changes in O2 levels, they reduce their speed and change direction. Using the population behavioral assay, we describe in this protocol how these changes of speed are measurable to enable evaluation of the gas-sensing capability of a population of worms. We have previously used these assays to determine the functional importance of multiple transcription factors required for the development of gas-sensing neurons in C. elegans (Brandt et al., 2012; Gramstrup Petersen et al., 2013; Rojo Romanos et al., 2015 and 2017). In Figure 6, we illustrate examples of results obtained: we show how wild type worms decrease their speed in response to a BAG neuron-mediated downshift (21%-10%) or a URX neuron-mediated upshift (10%-21%) in O2 concentration (Figure 6A). In contrast, lin-32(tm1446) mutant animals, which lack the LIN-32/Atoh1 transcription factor and have defects in URX development, fail to respond to an upshift in O2 concentration from 10% to 21% (Figure 6B) (Rojo Romanos et al., 2017). Finally, egl-13(ku194) mutant animals, in which the EGL-13/Sox transcription factor is deleted, fail to respond to upshifts and downshifts in O2 concentration due to a defect in specification of the BAG and URX neurons (Figure 6C) (Gramstrup Petersen et al., 2013).


Figure 6. C. elegans mutants that fail to respond to changes in O2 levels. Graphs showing the locomotion speed of wild-type (A), lin-32(tm1446) (B) and egl-13(ku194) (C) mutant animals when O2 levels are shifted every 6 min: 21%-10%-21%. The data represent averages of at least four independent assays (80-120 animals per assay). lin-32(tm1446) mutants fail to respond to O2 upshifts (URX-mediated) but show a similar response to wild type animals to O2 downshifts (BAG-mediated). In contrast, egl-13(ku194) mutant animals fail to respond to O2 upshifts and downshifts due to defects in BAG and URX specification. Red arrows indicate defective responses to changes in O2 levels.

Recipes

  1. Maintenance plates
    See He (2011)
  2. KPO4 (1 M)
    Prepare separate 1 M solutions of KH2PO4 (Sigma-Aldrich) and K2HPO4 (Sigma-Aldrich)
    Filter sterilize both solutions and measure 434 ml of 1 M KH2PO4 and 66 ml of 1 M K2HPO4 using an autoclaved measuring cylinder before transferring to a 500 ml autoclaved bottle
  3. Assay NGM plates (22 g/L agar)
    Use 14 cm plates (VWR, Petri dish 140 mm diameter)
    1. For 1 L NGM:
      3 g NaCl
      22 g agar (SERVA high-gel strength agar)
      1 ml cholesterol (5 mg/ml) (add after autoclaving)
      1 ml CaCl2 (1 M) (add after autoclaving)
      25 ml KPO4 (1 M) (add after autoclaving)
    2. Pour precisely 75 ml of NGM solution into each 14 cm plate and allow to solidify on a stable and perfectly flat surface
    3. When the agar is solid, remove residual water condensed on the lid of the plate with tissue paper
    4. Dry the plates in a dryer (Binder) overnight to 24 h at 50 °C with the lid facing down
    5. Take plates out of the dryer and remove residual water on the lid of the plate with tissue paper
    6. Leave plates at room temperature for one day
    7. Use these plates directly for behavioral experiments or store them in the cold room/fridge for up to 30 days
    Note: Do not seed these plates with bacteria. For one experiment, use plates poured from the same batch of NGM and allowed to dry for the same length of time.
  4. Starvation plates (17 g/L agar)
    Use 9 cm plates (90 mm Petri dishes, Techno Plas)
    For starvation plates use the same recipe as for assay plates but with 17 g agar per liter (instead of 22 g per liter)
    Note: Do not seed these plates with bacteria.

Acknowledgments

Some strains used in this study were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), and by Shohei Mitani at the National Bioresource Project (Japan). This work was supported by a grant from the European Research Council (ERC Starting Grant number 260807), Monash University Biomedicine Discovery Fellowship and veski innovation fellowship: VIF 23 to R.P. This protocol has been adapted from (Zimmer et al., 2009; Rojo Romanos et al., 2017).
The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC (grant agreement 281869) and the Research Institute of Molecular Pathology (IMP). The IMP is funded by Boehringer Ingelheim.
Conflict of interest statement: The authors declare no conflict of interest or competing interests.

References

  1. Brandt, J. P., Aziz-Zaman, S., Juozaityte, V., Martinez-Velazquez, L. A., Petersen, J. G., Pocock, R. and Ringstad, N. (2012). A single gene target of an ETS-family transcription factor determines neuronal CO2-chemosensitivity. PLoS One 7(3): e34014.
  2. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
  3. Bretscher, A. J., Kodama-Namba, E., Busch, K. E., Murphy, R. J., Soltesz, Z., Laurent, P. and de Bono, M. (2011). Temperature, oxygen, and salt-sensing neurons in C. elegans are carbon dioxide sensors that control avoidance behavior. Neuron 69(6): 1099-1113.
  4. Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B. and Bargmann, C. I. (2007). Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450(7166): 63-70.
  5. Chang, A. J., Chronis, N., Karow, D. S., Marletta, M. A. and Bargmann, C. I. (2006). A distributed chemosensory circuit for oxygen preference in C. elegans. PLoS Biol 4(9): e274.
  6. Gramstrup Petersen, J., Rojo Romanos, T., Juozaityte, V., Redo Riveiro, A., Hums, I., Traunmuller, L., Zimmer, M. and Pocock, R. (2013). EGL-13/SoxD specifies distinct O2 and CO2 sensory neuron fates in Caenorhabditis elegans. PLoS Genet 9(5): e1003511.
  7. Gray, J. M., Karow, D. S., Lu, H., Chang, A. J., Chang, J. S., Ellis, R. E., Marletta, M. A. and Bargmann, C. I. (2004). Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Nature 430(6997): 317-322.
  8. Hallem, E. A. and Sternberg, P. W. (2008). Acute carbon dioxide avoidance in Caenorhabditis elegans. Proc Natl Acad Sci U S A 105(23): 8038-8043.
  9. He, F. (2011). Common worm media and buffers. Bio Protoc Bio101: e55.
  10. Ramot, D., Johnson, B. E., Berry, T. L., Jr., Carnell, L. and Goodman, M. B. (2008). The Parallel Worm Tracker: a platform for measuring average speed and drug-induced paralysis in nematodes. PLoS One 3(5): e2208.
  11. Rojo Romanos, T., Petersen, J. G., Riveiro, A. R. and Pocock, R. (2015). A novel role for the zinc-finger transcription factor EGL-46 in the differentiation of gas-sensing neurons in Caenorhabditis elegans. Genetics 199(1): 157-163.
  12. Rojo Romanos, T., Pladevall-Morera, D., Langebeck-Jensen, K., Hansen, S., Ng, L. and Pocock, R. (2017). LIN-32/Atonal controls oxygen sensing neuron development in Caenorhabditis elegans. Sci Rep 7(1): 7294.
  13. Zimmer, M., Gray, J. M., Pokala, N., Chang, A. J., Karow, D. S., Marletta, M. A., Hudson, M. L., Morton, D. B., Chronis, N. and Bargmann, C. I. (2009). Neurons detect increases and decreases in oxygen levels using distinct guanylate cyclases. Neuron 61(6): 865-879.

简介

动物使用行为策略来寻求最佳的环境。 人口行为分析提供了一个强大的手段来确定遗传扰动对动物感知和响应环境变化的能力的影响。 在这里,我们描述一个 C。 elegans 人群行为测定用于测量对环境氧(O 2)和二氧化碳(CO 2)浓度变化的运动反应。 这些行为测定法是高通量的并且能够检查遗传,神经元和电路功能。

【背景】氧浓度提供了C。线虫与环境条件有关的信息。在实验室条件下,当呈现O 2梯度时,C。线虫向中等浓度(2%-12%)迁移(Gray等人,2004)。低水平的O 2可以指示细菌(食物)的存在,而高O 2水平可能意味着蠕虫靠近其环境基质的表面。因此,C。 elegans 以极其敏感的方式对O 2浓度的变化作出反应,使得能够导航到有利于后代存活和繁殖的最佳环境(Gray等人, 2004; Chang等人,2006; Zimmer等人,2009)。同样,蠕虫对CO 2的变化也有强烈的行为反应。喂食良好的动物避免CO 2而饥饿的动物被吸引到CO 2(Hallem和Sternberg,2008)。这种响应的变化可能提供寻找食物的进化优势,因为细菌食物来源释放CO 2。此外,病原体产生CO 2 2,这可能表明为什么喂食良好的蠕虫避免CO 2 2。特定的神经元调节气体感应反应。包括头部神经元URXL / R,BAGL / R,AQR和位于尾巴中的PQR神经元(Hallem和Sternberg,2008; Zimmer等人,2009; Bretscher et al。,2011)。 O 2感应的主要调节器是URX和BAG神经元,其分别感测氧的升档和降档。关于CO 2水平的变化,BAG神经元是主要的传感器。

关键字:秀丽隐杆线虫, 行为, 氧, 二氧化碳, 传感

材料和试剂

  1. 用铂丝制成的蜗杆(Tritech Research,目录号:PT-9901)
  2. 滤纸(110mm直径)(GE Healthcare,Whatman,目录号:1001-110)
  3. 直径140毫米的培养皿(VWR,目录号:391-1500)
  4. 90毫米培养皿(Techno Plas,目录号:S9014UV20)
  5. ℃。线虫株
    以下方案适用于源自野生型动物(N2,布里斯托尔菌株)的菌株。建议的对照(可从Caenorhabditis遗传中心(CGC)获得):
    1. N2(野生型):阳性对照
    2. gcy-31(ok296):无法响应O 2 级别的降档
    3. gcy-35(ok769):无法响应O 2 级别的升级
    4. gcy-9(n4470):无法回应CO <2>浓度的变化
    5. 税4(p678):O 2和CO 2感应不足
    注意:所有菌株都应在标准条件下培养和保存(Brenner,1974)。所有的菌株应该与野生型动物杂交至少4次。温度或饥饿的变化会影响测定结果。因此,菌株应该保持在恒定温度(通常20℃),并且在测定之前用OP50大肠杆菌充分喂养至少两代。
  6. 氯化铜(II)二水合物(CuCl 2•2H 2 O)(Sigma-Aldrich,目录号:221783-100G)
  7. 用OP50 E播种的NGM维护板。 (见食谱)
  8. KPO 4缓冲液(1M,参见食谱)
    1. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P0662-500G)
    2. 磷酸二氢钾(KH 2 HPO 4)(Sigma-Aldrich,目录号:3786-500G)
  9. 测定NGM板(参见食谱)
    1. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653-250G)
    2. 琼脂(SERVA高凝胶强度琼脂)(SERVA电泳,目录号:11396.03)
    3. 胆固醇(Sigma-Aldrich,目录号:C8667-25G)
    4. 氯化钙(CaCl 2)(Merck,目录号:1.02382.0500)
    5. KPO缓冲液(1M)
  10. 饥饿板(见食谱)

设备

  1. 电脑与软件兼容
    英特尔酷睿i7,32GB内存,Windows 7,64位。需要一个额外的千兆以太网卡来连接GigE摄像机(图1)
  2. 相机
    我们使用一个400万像素CCD相机(JAI,型号:BM-500GE),35毫米物镜:QIOPTIQ MEVIS 3516.延伸环:扩展环1(1毫米C型安装)(图1)


    图1.计算机和相机设置

  3. 流量控制器
    Vgtgt仪器,CO 2:GSC-A9TA-BB21 50mln / min,N 2:GSC-A9TA-BB22 200mln / min,O 2: GSC-A9TA-BB21 50 mln / min(图2)


    图2.气体和流量控制器设置:一种。连接到流量控制器的主电源总览。 B.流量控制器的特写。

  4. LED照明(CCS,目录号:TH-211 / 200RD),电缆(CCS,目录号:FCB-1),电源(CCS,目录号:PD2-5024)
  5. 个别气瓶为O 2,CO 2和N 2
    注意:也可以使用预混瓶,具体取决于所使用的流量控制器。尽管如此,我们还是建议使用单独的气瓶,因为它们可以对可测试的气体参数提供灵活性。
  6. 检测室:定制透明有机玻璃装置,流动空间为60 x 60 x 0.7 mm(图3)


    图3.定制设计的化验室。 :一种。顶部图:从顶部看。底图:侧视图。 注意图表没有被缩放。 B.从上面拍摄的化验室的照片。

  7. 高压灭菌器
  8. 干燥机(粘结剂,目录号:9010-0102)
  9. 500毫升高压灭菌瓶

软件

  1. Labview(编程和调节流量控制器)
  2. MATLAB(R13)(MathWorks)和图像采集与图像处理工具箱(图4)
  3. 并行WormTracker(免费提供: https://sourceforge.net/projects/wormtracker/ ) >
  4. Streampix软件(Norpix)


    图4. WormTracker分析的截图。 :一种。由软件检测到的单个蠕虫轨迹(红线)。 B. MATLAB分析软件屏幕显示跟踪帧。

程序

  1. C的增长和同步。线虫人群
    建议:对于每个品系,每天挑选4个幼虫阶段4(L4)雌雄同体到4个维护板上(每板4个蠕虫),并且在测定中使用下一代:对于转基因染色体外菌株,应该挑选超过4个转基因L4以确保足够的转基因蠕虫可用于测定。另外,对于小于野生型的突变体,必须选择更多的蠕虫进行同步。
  2. 选择大约150L4 /年轻的成虫到饥饿平板(不用OP50大肠杆菌接种)。因此,细菌不会转移到空盘子,不要选择使用细菌,而是使用蠕虫挑选作为一个勺子来传播爬出细菌草坪蠕虫。
    注意:蠕虫不应该用M9缓冲液冲洗掉,因为行为分析对压力非常敏感。
  3. 让蠕虫挨饿1小时。饲喂状态强烈地影响对O 2和CO 2的行为反应,因此,对于每个测试的菌株,饥饿必须进行相同的时间长度。此外,良好喂养的N2蠕虫对O 2(Zimmer等人,2009)中的降级无效。
  4. 准备测定板(图5)
    1. 在Whatman滤纸的中心切一个56×56平方毫米的面积(可以使用打孔机)。
    2. 将滤纸置于行为测定板(14cm NGM测定板)上。
    3. 将滤纸浸泡在20 mM CuCl 2 2的正方形边缘,以便在检测区域内检测到蠕虫。


      图5.分析板的图示。 14cm NGM测定板包含浸泡在700μl20mM CuCl 2中的Whatman滤纸的56×56mm的竞技场。
      。雌雄同体被放置在舞台上,化验室被仔细定位在板的顶部。

  5. 将饥饿板上的蠕虫勺到试验板的中心。
    注意:注意不要伤害琼脂,因为蠕虫会挖洞。
  6. 将分析室放在分析板的顶部。
    蠕虫所在的56×56平方毫米正方形内部很重要
  7. 将燃气管连接到燃烧室,启动大气浓度的气体流动(或者根据您所需的启动条件)。在开始实验之前让气体流动至少5分钟。
  8. 在运行选定的气体浓度测试时录制视频。
    2 和CO 2 > 感测,用N 2 平衡浓度。为了测试急性感觉,我们建议每种情况的下列时间:6分钟21%O 2 / 79%N 2 -6 min 10%O 2 / 90%N 2 -6分钟21%O <子> 2 / 79%N <子> 2 。为了测试急性感应,我们建议每种情况的下列时间:6分钟21%O 2 / 79%N 2 -6分钟21%O <子> 2 / 78%N <子> 2 / 1%CO

数据分析

平行的WormTracker软件包包括一个详细的用户手册,描述如何运行它从视频中提取必要的数据(Chalasani et al。,2007; Ramot et al。 ,2008)。说明脚本可以更新到不同的操作系统和视频格式是很重要的。最初的脚本是为运行Windows XP的PC和AVI格式的未压缩,灰度(8位)电影而编写的,分辨率为640 x 480.WormTracker识别蠕虫并跟踪蠕虫质心定义的位置。在录制视频之后进行跟踪。蠕虫跟踪后,Wormanalyzer允许分析轨道和检测蠕虫的速度和其他参数,如转弯事件。这些数据可以用任何标准的统计软件如GraphPad Prism来提取和进一步分析。

代表性数据
当野生型雌雄同体在O 2水平上发生变化时,它们会降低速度并改变方向。使用人口行为分析,我们在这个协议中描述了这些速度的变化是如何测量的,以便能够评估蠕虫种群的气体感应能力。我们以前使用这些测定来确定在气体感应神经元的发展所需的多种转录因子的功能重要性。 (Brandt等人,2012; Gramstrup Petersen等人,2013; Rojo Romanos 等人,2015)和2017年)。在图6中,我们举例说明了获得的结果:我们展示野生型蠕虫如何响应BAG神经元降档(21%-10%)或URX神经元介导的升高(10%-21%)而降低其速度在O 2浓度下(图6A)。相反,缺乏LIN-32 / Atoh1转录因子并且在URX发育中具有缺陷的突变动物lin-32( tm1446 )未能响应O 2浓度从10%升高至21%的升高(图6B)(Rojo Romanos等人,2017)。最后,其中EGL-13 / Sox转录因子被删除的突变体动物egl-13(ku194)突变体动物未能响应于O 2浓度的升高和降档到BAG和URX神经元规范的缺陷(图6C)(Gramstrup Petersen等人,2013)。


图6.线虫突变体不能响应O 2 水平的变化。 (B)和(egl-13)(ku194)的运动速度的图表显示野生型(A), (C)突变动物,当O 2水平每6分钟移动一次时:21%-10%-21%。数据表示至少四次独立测定的平均值(每个测定80-120个动物)。突变体对O 2升高(URX-但是对于野生型动物对O 2降档(BAG介导的)表现出类似的响应。相比之下,egl-13(ku194)突变体动物由于BAG和URX规格的缺陷而不能响应O 2升档和降档。红色箭头表示O 2水平变化的缺陷响应。

食谱

  1. 维护板
    见他(2011)
  2. KPO <4>(1M)
    制备KH 2 PO 4(Sigma-Aldrich)和K 2 HPO 4(分别为Sigma-Aldrich)的单独1M溶液西格玛奥德里奇)
    过滤灭菌两种溶液并测量434ml 1M KH 2 PO 4和66ml 1M K 2 HPO 4在转移到一个500毫升的高压灭菌瓶之前使用高压灭菌的量筒。
  3. 测定NGM板(22 g / L琼脂)
    使用14厘米板(VWR,培养皿140毫米直径)
    1. 对于1升NGM:
      3克NaCl
      22克琼脂(SERVA高凝胶琼脂)
      1毫升胆固醇(5毫克/毫升)(高压灭菌后添加)
      1毫升CaCl 2(1M)(高压灭菌后加入)
      25 ml KPO 4(1 M)(高压灭菌后添加)
    2. 将精确的75毫升NGM溶液倒入每个14厘米的平板中,使其固化在一个稳定且完美的平坦表面上
    3. 当琼脂是固体时,用薄纸去除凝结在盘子盖子上的残余水。

    4. 在干燥器(Binder)中将板干燥至50°C 24小时,盖子朝下
    5. 从烘干机中取出印版,用纸巾清除印版盖上的残留水分

    6. 在室温下放置一天
    7. 直接使用这些盘子进行行为实验,或将它们存放在冷藏室/冰箱中长达30天
    注意:不要用细菌对这些平板进行播种。对于一个实验,使用从同一批NGM中倒出的板,并允许干燥相同的时间。
  4. 饥饿板(17克/升琼脂)
    使用9厘米板(90毫米培养皿,Techno Plas)
    对于饥饿平板,使用与测定平板相同的配方,但每升含17克琼脂(而不是每升22克)。
    注意:不要用细菌对这些盘子进行播种。

致谢

本研究中使用的一些菌株由美国国立卫生研究院基础研究计划办公室(P40 OD010440)和国立日本生物资源项目(日本)的三井昌平提供资金的Caenorhabditis遗传中心提供。这项工作得到了欧洲研究理事会(ERC起始拨款号码260807),莫纳什大学生物医学发现奖学金和veski创新研究金的资助:VIF 23到RP。该协议已经改编自(Zimmer等人, 2009年; Rojo Romanos等人,2017年)。利益冲突声明:作者声明不存在利益冲突或利益冲突。

参考

  1. Brandt,J.P.,Aziz-Zaman,S.,Juozaityte,V.,Martinez-Velazquez,L.A.,Petersen,J.G.,Pocock,R。和Ringstad,N.(2012)。 ETS家族转录因子的单个基因靶标决定神经元CO <,2> 2
  2. Brenner,S。(1974)。 的遗传学 Caenorhabditis elegans Genetics 77(1):71-94。
  3. Bretscher,A.J.,Kodama-Namba,E.,Busch,K.E.,Murphy,R.J.,Soltesz,Z.,Laurent,P.and de Bono,M。(2011)。 温度,氧气和盐敏感神经元 C。 elegans 是控制回避行为的二氧化碳传感器。 Neuron 69(6):1099-1113。
  4. Chalasani,S.H.,Chronis,N.,Tsunozaki,M.,Gray,J.M。,Ramot,D.,Goodman,M.B。和Bargmann,C.I。(2007)。 秀丽隐杆线虫 450(7166):63-70。
  5. Chang,A.J.,Chronis,N.,Karow,D.S。,Marletta,M.A。和Bargmann,C.I。(2006)。 分布式化学感应电路,用于 C.elegans
  6. Gramstrup Petersen,J.,Rojo Romanos,T.,Juozaityte,V.,Redo Riveiro,A.,Hums,I.,Traunmuller,L.,Zimmer,M.和Pocock,R.(2013)。 EGL-13 / SoxD指定不同的O 2 and CO 2 感官神经元的命运 秀丽隐杆线虫
  7. Gray,J.M.,Karow,D.S.,Lu,H.,Chang,A.J.,Chang,J.S.,Ellis,R.E.,Marletta,M.A。和Bargmann,C.I。(2004)。 由一个介导的氧气感受和社会喂养 C。 elegans 鸟苷酸环化酶同系物。 Nature 430(6997):317-322。
  8. Hallem,E.A。和Sternberg,P.W。(2008)。 避免急性二氧化碳 秀丽隐杆线虫 。 Proc Natl Acad Sci USA 105(23):8038-8043。
  9. 他,楼(2011年)。 常见的蠕虫介质和缓冲区。 Bio Protoc Bio101:e55。
  10. Ramot,D.,Johnson,B.E.,Berry,T.L.,Jr.,Carnell,L。和Goodman,M.B。(2008)。并行蠕虫跟踪器:一种测量线虫平均速度和药物诱导的麻痹的平台。 PLoS One 3(5):e2208。
  11. Rojo Romanos,T.,Petersen,J.G.,Riveiro,A.R。和Pocock,R。(2015)。 锌指转录因子EGL-46在气体感觉神经元分化中的新作用在 秀丽隐杆线虫
  12. Rojo Romanos,T.,Pladevall-Morera,D.,Langebeck-Jensen,K.,Hansen,S.,Ng,L。和Pocock,R.(2017)。 LIN-32 / Atonal控制氧气感应神经元发育 秀丽隐杆线虫(Caenorhabditis elegans) 。 Sci Rep 7(1):7294。
  13. Zimmer,M.,Gray,J.M.,Pokala,N.,Chang,A.J.,Karow,D.S.,Marletta,M.A.,Hudson,M.L.,Morton,D.B.,Chronis,N.and Bargmann,C.I.(2009)。 神经元使用不同的鸟苷酸环化酶检测氧气水平的增加和减少 神经元 61(6):865-879。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rojo Romanos, T., Ng, L., Zimmer, M. and Pocock, R. (2018). Behavioral Assays to Study Oxygen and Carbon Dioxide Sensing in Caenorhabditis elegans. Bio-protocol 8(1): e2679. DOI: 10.21769/BioProtoc.2679.
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