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Electroshock Induced Seizures in Adult C. elegans
成年秀丽隐杆线虫的电休克诱发性癫痫发作   

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Abstract

The nematode Caenorhabditis elegans is a useful model organism for dissecting molecular mechanisms of neurological diseases. While hermaphrodite C. elegans contain only 302 neurons, the conserved homologous neurotransmitters, simpler neuronal circuitry, and fully mapped connectome make it an appealing model system for neurological research. Here we developed an assay to induce an electroconvulsive seizure in C. elegans which can be used as a behavioral method of analyzing potential anti-epileptic therapeutics and novel genes involved in seizure susceptibility. In this assay, worms are suspended in an aqueous solution as current is passed through the liquid. At the onset of the shock, worms will briefly paralyze and twitch, and shortly after regain normal sinusoidal locomotion. The time to locomotor recovery is used as a metric of recovery from a seizure which can be reduced or extended by incorporating drugs that alter neuronal and muscular excitability.

Keywords: Epilepsy(癫痫), Seizure(发作), C. elegans(秀丽隐杆线虫), Electroshock(电休克), Electroconvulsion(电惊厥), Antiepileptic drugs(抗癫痫药), AEDs(AEDs)

Background

We were interested in using the powerful genetic model, Caenorhabditis elegans, to develop an electroconvulsive seizure assay that can be easily manipulated by pharmacology. Invertebrate models have been used in seizures research for decades (Lee and Wu, 2002) however there were no protocols specifically investigating electroconvulsive seizures in C. elegans. In the past, multiple groups have developed methods of analyzing paralysis in response to chemical proconvulsants such as the GABAA receptor blockers, pentylentetrazol (PTZ) and picrotoxin (PTX), as well as an acetylcholinesterase inhibitor, aldicarb (Williams et al., 2004; Vashlishan et al., 2008). While these methods typically analyze the time to paralysis, our method quantifies the time it takes to recover from an electric shock-induced seizure (Risley et al., 2016).

Materials and Reagents

  1. 60 x 15 mm Petri dishes (Excel Scientific, catalog number: D-901 )
  2. Pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 0094300120 )
  3. Vacuum filter (Corning, catalog number: 430320 )
  4. 2, 20 gauge insulated copper wire, cut to 8 cm segments (Del City, catalog number: 1120101 )
  5. Plastic tubing, cut into 9 mm segments (Emurdock, catalog number: AAQ04127 )
    Note: This product has been discontinued.
  6. 2 x Alligator clip wires (United Scientific Supplies, catalog number: WAG024-PK/6 )
  7. C. elegans wildtype strain N2 (obtained from Caenorhabditis Genetics Center)
  8. LB broth powder (Fisher Scientific, catalog number: BP1426-500 )
  9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  10. Agar (Sigma-Aldrich, catalog number: A7002 )
  11. Peptone (Fisher Scientific, catalog number: BP1420 )
  12. Calcium chloride solution (CaCl2) (Sigma-Aldrich, catalog number: 21115 )
  13. Magnesium sulfate solution (MgSO4) (Sigma-Aldrich, catalog number: M3409 )
  14. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  15. Ethanol (Fisher Scientific, catalog number: 04-355-226 )
  16. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  17. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  18. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 795410 )
  19. LB broth (see Recipes)
  20. Nematode growth medium (NGM) agar plates (see Recipes)
  21. KP buffer (see Recipes)
  22. E. coli strain OP50 (see Recipes)
  23. M9 solution (see Recipes)

Equipment

  1. 2 L flask
  2. Pipettes  
  3. Stir bar
  4. Dissection stereo microscope (Tritech Research, model: SMT1 )
  5. Dissecting stereoscope (AmScope, model: SM-1TSX )
  6. Timer output stimulator (Grass Instruments, model: S44 )
  7. Stimulator (Grass Instruments, model: SD9 )
  8. CCD color microscope camera (Hitachi, model: KP-D20BU )
  9. Television monitor (RCA, model: 19LA30RQ )
  10. HDD and DVD recorder (Magnavox, catalog number: MDR535H/F7 )
  11. DVD-Recordable discs (Verbatim, catalog number: 95032 )
  12. Digital oscilloscope (OWON Technology, catalog number: PDS5022T )
  13. Ethanol lamp (Carolina, catalog number: 706604 )
  14. Water bath (50 °C) (Corning, model: Corning® LSETM Digital Water Bath, catalog number:6783)
  15. Incubators (37 °C) (Thelco, model: 4 )
  16. Incubator (20 °C) (Cuisinart, catalog number: CWC 1200DZ )
  17. Timer (VWR, catalog number: 62344-641 )
  18. A metric ruler (Fisher Scientific, catalog number: 09-016 )
  19. Infrared Temperature Gun (J-1 Trading Wholesale, Nubee, catalog number: NUB8380 )

Software

  1. Open source image processing program, we use VLC media player (version 2.2.4)
  2. SigmaPlot 11.0 (Systat Software, Inc., San Jose, CA, USA)

Procedure

  1. Setup the microscope camera to the TV and digital video recorder as per the camera instructions (Figures 1 and 2A).


    Figure 1. The experimental setup. The experimental setup includes a stereoscope (A), oscilloscope (B), timer output stimulator (S44) (C), stimulator (SD9) (D), and the recording equipment (E).


    Figure 2. A simplified schematic of the experimental setup and analysis. A. The stereoscope (center), the recording equipment (left), and a stimulator (right) represent a simplified schematic of a basic experimental setup. To control duration, our setup includes an additional stimulator (S44); however, timing can be controlled by many different methods. B. A close up schematic of the experimental tube plugged on either end with copper wires; C. This schematic represents the normal, sinusoidal body position (arrow) of a worm before the shock. During and after the shock, the worms generally exhibit convulsions and paralysis, and then typically resume normal sinusoidal locomotion. This figure was adapted from Risley et al., 2016.

  2. Connect the output on the S44 stimulator via a cable with a banana plug splitter and connect it to the ground and mod banana plug inputs on the SD9 stimulator. The goal here is to connect the S44 to the SD9 stimulator so that the S44 can be used to modulate duration.
    Note: Be sure the polarity of the banana plugs is consistent.
  3. Set the S44 to at least 5 V and desired stimulus duration; we use 3 sec.
  4. Clip one of the alligator clips onto the positive (+) output on the SD9 and the other onto the negative (-) output. These alligator clip wires will connect the SD9 stimulator to the experimental tube (Figures 3 and 2B).


    Figure 3. Close-up image of the experimental tube. The tube is plugged on either end with the insulated copper wires that are attached to the stimulator. The distance between the copper wire should be exactly 10 mm.

  5. Next clip each of the copper wires to the open end of each alligator clip. The open end of each copper wire will later be plugged directly into experimental tube when the experiment is ready to begin. Setup the microscope camera to the TV as per the camera instructions (Figures 1 and 2A).
    Note: It is very important to setup the experimental tubes consistently to obtain a consistent.
  6. 1-day old adult Caenorhabditis elegans are used for the experiments. 24-h before experimentation, approximately 30 L4 stage worms per genotype are transferred to fresh NGM plates seeded with OP50 E. coli. The worms are placed in a 20 °C incubator overnight; and will mature into 1-day old adults in approximately 18 h. During experimentation the following day, the worm plates are left on the lab bench at room temperature (23 °C).
  7. Typically, the drug solutions are prepared on the morning of experimentation. The drugs are dissolved directly into a vial of M9 solution with a total volume of 3-5 ml, depending on the number of experimental trials. Sham controls are also prepared at this time. The prepared reagents can be stored, at 4 °C or based on the specifications of the drug, for experiments performed on consecutive days.
    Note: Water-soluble drugs were dissolved directly into M9 (Risley et al., 2016); however, a DMSO concentration curve was conducted in Risley et al. (2016) and it was reported that DMSO concentrations up to 0.5% had no significant effect on wild-type locomotor recovery after electric shock.  
  8. Next, load a blank DVD+R into the HDD/DVD recorder and turn on the TV screen. Also, verify that the settings are correct on the timer output stimulator and stimulator using the oscilloscope. We typically set the electroshock parameters to 47 V, 3 sec duration. This voltage was selected based off a voltage-response curve. The maximum voltage in which wild-type worms recovered was about 60 V and they recovered, on average, in 85 sec. 47 V was selected as worms recover in approximately one half the time of maximum limit for recovery (Figure 4).
    Note: The timer output stimulator (S44) is used only as a trigger for the SD9 stimulator, which sends current to the worms. The duration, typically 3 sec, is set on the S44 and used as a trigger to regulate the duration. There are many other ways to control for the duration including using a digital trigger via a computer or an Arduino.


    Figure 4. A voltage response curve demonstrates locomotor recovery times in wild-type worms. Wild-type worms did not recover at voltages ≥ 70 V. The maximum tested limit for recovery was 60 V where worms recovered in approximately 80 seconds. 47 V was the voltage in which recovery time was 50% of the maximum limit. This figure was adapted from Risley et al., 2016.

  9. Load approximately 20 µl of control solution or drug solution. Immediately following, load 6-8 worms into the tube using a platinum pick and set a timer for 30 min. 30 sec before the timer expires, gently place the tube under the microscope/camera setup with the light base turned on, and plug each copper wire into the two ends of the tube; the wires should be 10 mm apart (Figures 3 and 2B). Verify the distance between the copper wires is precise (a ruler will suffice) and initiate the electroshock. Allow the experiment to record for 10 min. After the 10 min recording, the tube and its contents are discarded.
    Note: Generally, a tube is loaded every 10 min. Each tube is shocked and recorded for 10 min before it is discarded and another tube is immediately prepared. Also, OP50 transferred from the platinum pick to the tube should be minimized as it could impede the view of the worms.

Data analysis

  1. The analysis is manually scored using a video player. Each worm is scored individually in each tube. An example screenshot of a raw video file can be seen in Figure 5 and a schematic representation in Figure 2C. The time of the shock onset (depicted by the appearance of bubbles) is recorded in a notebook and subsequent recovery times of each worm are recorded.


    Figure 5. Example screenshot of recorded raw data. The image is a screenshot from a raw data video file of an experiment. There are five worms centered inside the middle of the tube. The tube is plugged by the copper wire on either end. This is an example of the video file that is analyzed by the researcher.

  2. Recovery is defined as a worm regaining its ability to move in a sinusoidal movement, similar to the locomotion it exhibited prior to the shock. Partial recovery of only head or tail is not considered recovery. (Video 1)
  3. Recovery time of each worm was considered n = 1 and the total number of experimental tubes was N = 1. For all experiments, a full data set is considered n ≥ 10 and N ≥ 6.
  4. Data is analyzed in SigmaPlot using One-Way ANOVA followed by a post-hoc Multiple Comparison’s test or a Student’s t-test; however, appropriate statistical analysis should be determined by the researcher. Data was typically represented by a bar graph ± SEM. An example of the data bar graphs can be seen in the voltage-response curve in Figure 4.

    Video 1. Example video of the experimental setup and analysis. This video shows another visual of the experimental setup, followed by a raw data video file. The video is annotated with notes describing when each worm is considered recovered. This figure was adapted from Risley et al., 2016.

Notes

  1. Due to electrolysis during the stimulus, bubbles will form on the edge of the tube. Worms that are covered by the bubbles are excluded from analysis. We have used an infrared temperature gun to record temperature fluctuations over 3 sec. The temperature fluctuations are acute and minimal, 1 ± 0.5 °C, however temperature should be recorded to be sure increased temperature will not compound the effect of the electroshock. If the shock is administered for more than 3 sec, care should be taken that the temperature does not increase substantially as this could confound the results.
  2. The speed of the sinusoidal locomotion was not taken into consideration during analysis.
  3. Worms that did not recover were excluded from analysis.

Recipes

  1. LB broth
    Add 25 g LB broth powder and 1 L dH2O to a 2 L flask
    Autoclave for 30 min
  2. Nematode growth medium (NGM) agar plates
    1. Add 3 g NaCl, 17 g agar, 2.5 g peptone, and 975 ml dH2O to a 2 L flask
    2. Autoclave for 30 min
    3. Let cool to 50 °C in the water bath
    4. Add 1 ml 1 M CaCl2, 1 ml 1 M MgSO4, 5 mg cholesterol dissolved in 1 ml 95% ethanol, 25 ml KP buffer to the agar solution, mixing well after each addition
    5. Pipette 10 ml into each 60 x 15 mm Petri dish
    6. Cover and store at 4 °C
    7. When needed, seed plates with 50 µl of OP50 E. coli and incubate overnight at 37 °C
  3. KP buffer
    Add 2.46 g KH2PO4 and 1.205 g K2HPO4, to 25 ml diH2O with a stir bar
    Adjust pH to 6.0 and vacuum filter
  4. OP50 E. coli
    Suspend 1 colony of OP50 E.coli from a stock plate in LB broth
    Incubate overnight at 37 °C with the lid loosely on
    Store at 4 °C
  5. M9 solution
    Add 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4 to a 2 L flask and fill with diH2O to 1 L
    Autoclave for 15 min and store at room temperature

Acknowledgments

We have published this protocol in (Risley et al., 2016; Opperman et al., 2017). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

References

  1. Lee, J. and Wu, C. F. (2002). Electroconvulsive seizure behavior in Drosophila: analysis of the physiological repertoire underlying a stereotyped action pattern in bang-sensitive mutants. J Neurosci 22(24): 11065-11079.
  2. Opperman, K. J., Mulcahy, B., Giles, A. C., Risley, M. G., Birnbaum, R. L., Tulgren, E. D., Dawson-Scully, K., Zhen, M. and Grill, B. (2017). The HECT family ubiquitin ligase EEL-1 regulates neuronal function and development. Cell Rep 19(4): 822-835.
  3. Risley, M. G., Kelly, S. P., Jia, K., Grill, B. and Dawson-Scully, K. (2016). Modulating behavior in C. elegans using electroshock and antiepileptic drugs. PLoS One 11(9): e0163786.
  4. Vashlishan, A. B., Madison, J. M., Dybbs, M., Bai, J., Sieburth, D., Ch'ng, Q., Tavazoie, M. and Kaplan, J. M. (2008). An RNAi screen identifies genes that regulate GABA synapses. Neuron 58(3): 346-361.
  5. Williams, S. N., Locke, C. J., Braden, A. L., Caldwell, K. A. and Caldwell, G. A. (2004). Epileptic-like convulsions associated with LIS-1 in the cytoskeletal control of neurotransmitter signaling in Caenorhabditis elegans. Hum Mol Genet 13(18): 2043-2059.

简介

线虫秀丽隐杆线虫是解剖神经系统疾病分子机制的有用的模型生物。而雌雄同体线虫仅包含302个神经元,保守的同源神经递质,更简单的神经元电路和完全映射的连接体使其成为神经学研究的吸引人的模型系统。在这里,我们开发了一种诱发电刺激性癫痫发作的检测方法。线虫可用作分析潜在的抗癫痫药物和涉及癫痫发作敏感性的新基因的行为方法。在该测定中,当电流通过液体时,将蠕虫悬浮在水溶液中。在休克开始时,蠕虫会短暂瘫痪和抽搐,并在恢复正常的正弦运动后不久。运动恢复的时间被用作从癫痫发作恢复的指标,可以通过掺入改变神经元和肌肉兴奋性的药物来减少或延长。

背景 我们有兴趣使用强大的遗传模型,即秀丽隐杆线虫,开发可以容易地被药理学操作的电惊厥发作测定。无脊椎动物模型已被用于癫痫发作研究几十年(Lee和Wu,2002),但是没有专门研究电刺激性癫痫发作的方案。线虫。过去,多组已经开发出响应于化学前同质醇如GABA A受体阻断剂戊戊四唑(PTZ)和微毒素(PTX)以及乙酰胆碱酯酶抑制剂涕灭威分析麻痹的方法(Williams等人,2004; Vashlishan等人,2008)。虽然这些方法通常分析麻痹的时间,我们的方法量化了从电击诱发的癫痫发作恢复所需的时间(Risley等,2016)。

关键字:癫痫, 发作, 秀丽隐杆线虫, 电休克, 电惊厥, 抗癫痫药, AEDs

材料和试剂

  1. 60 x 15 mm培养皿(Excel Scientific,目录号:D-901)
  2. 移液器吸头(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:0094300120)
  3. 真空过滤器(Corning,目录号:430320)
  4. 2,20规格绝缘铜线,切割至8厘米段(德尔市,目录号:1120101)
  5. 塑料管,切成9毫米段(Emurdock,目录号:AAQ04127)
    注意:本产品已停产。
  6. 2 x鳄鱼夹线(United Scientific Supplies,目录号:WAG024-PK/6)
  7. ℃。线虫野生型菌株N2(从Caenorhabditis获得遗传学中心)
  8. LB肉汤粉(Fisher Scientific,目录号:BP1426-500)
  9. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  10. 琼脂(Sigma-Aldrich,目录号:A7002)
  11. 蛋白胨(Fisher Scientific,目录号:BP1420)
  12. 氯化钙溶液(CaCl 2)(Sigma-Aldrich,目录号:21115)
  13. 硫酸镁溶液(MgSO 4)(Sigma-Aldrich,目录号:M3409)
  14. 胆固醇(Sigma-Aldrich,目录编号:C8667)
  15. 乙醇(Fisher Scientific,目录号:04-355-226)
  16. 磷酸氢二钾(K 2/2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  17. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  18. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:795410)
  19. LB肉汤(见食谱)
  20. 线虫生长培养基(NGM)琼脂平板(参见食谱)
  21. KP缓冲(见配方)
  22. 电子。大肠杆菌菌株OP50(参见食谱)
  23. M9解决方案(请参阅食谱)

设备

  1. 2升烧瓶
  2. 移液器
  3. 搅拌棒
  4. 解剖立体显微镜(Tritech Research,型号:SMT1)
  5. 解剖立体镜(AmScope,型号:SM-1TSX)
  6. 定时器输出刺激器(Grass Instruments,型号:S44)
  7. 刺激器(Grass Instruments,型号:SD9)
  8. CCD彩色显微镜相机(日立,型号:KP-D20BU)
  9. 电视监视器(RCA,型号:19LA30RQ)
  10. HDD和DVD刻录机(Magnavox,目录号:MDR535H/F7)
  11. DVD可刻录光盘(Verbatim,目录号:95032)
  12. 数字示波器(OWON Technology,目录号:PDS5022T)
  13. 乙醇灯(Carolina,目录号:706604)
  14. 水浴(50°C)(Corning,型号:Corning ® LSE TM数字水浴,目录号:6783)
  15. 孵化器(37°C)(Thelco,型号:4)
  16. 培养箱(20℃)(Cuisinart,目录号:CWC 1200DZ)
  17. 定时器(VWR,目录号:62344-641)
  18. 公制尺(Fisher Scientific,目录号:09-016)
  19. 红外温度枪(J-1贸易批发,Nubee,目录号:NUB8380)

软件

  1. 开源图像处理程序,我们使用VLC媒体播放器(版本2.2.4)
  2. SigmaPlot 11.0(Systat Software,Inc.,San Jose,CA,USA)

程序

  1. 根据相机说明将显微镜相机设置到电视机和数字录像机(图1和2A)。


    实验装置包括立体镜(A),示波器(B),定时器输出激励器(S44)(C),刺激器(SD9)(D)以及记录设备(E)。


    图2.实验设置和分析的简化示意图A.立体镜(中心),记录设备(左)和刺激器(右)表示基本实验设置的简化示意图。为了控制持续时间,我们的设置包括一个额外的刺激器(S44);然而,时间可以通过许多不同的方法来控制。 B.用铜线插入任一端的实验管的近似示意图; C.该示意图表示震动前蠕虫的正常正弦体位置(箭头)。在休克期间和之后,蠕虫通常会出现抽搐和麻痹,然后通常恢复正常的正弦运动。这个数字改编自Risley等人,2016年。

  2. 通过带有香蕉插头分离器的电缆将S44刺激器上的输出连接到SD9刺激器上的地面和模拟香蕉插头输入。这里的目标是将S44连接到SD9刺激器,以便S44可用于调整持续时间。
    注意:确保香蕉插头的极性一致。

  3. 将S44设置为至少5 V和所需的刺激持续时间;我们使用3秒。
  4. 将其中一个鳄鱼夹夹在SD9的正(+)输出上,另一根到另外的( - )输出。这些鳄鱼夹线将SD9刺激器连接到实验管(图3和图2B)

    图3.实验管的特写镜像。将管连接到与刺激器连接的绝缘铜线的两端。铜线之间的距离应为10毫米。

  5. 接下来将每根铜线夹到每个鳄鱼夹的开口端。当实验准备开始时,每根铜线的开口端将被直接插入实验管中。根据相机说明将显微镜相机设置到电视机(图1和图2A)。
    注意:一直设置实验管以获得一致性是非常重要的。
  6. 1天龄的成年人秀丽隐杆线虫用于实验。在实验前24小时,每基因型大约30个L4级蠕虫转移到用OP50E接种的新鲜NGM板上。大肠杆菌。将蠕虫置于20℃培养箱中过夜;并将在约18小时内成熟为1日龄的成年人。在第二天的实验中,将蜗杆放在室温(23℃)的实验台上
  7. 通常,药物溶液在实验的早晨制备。根据实验试验的数量,将药物直接溶解到总量为3-5ml的M9溶液瓶中。此时也准备了假手术控制。所制备的试剂可以在4℃下或基于药物的规格储存,用于连续进行的实验。
    注意:水溶性药物直接溶解到M9(Risley等,2016);然而,在Risley等人中进行DMSO浓度曲线。 (2016),据报道,DMSO浓度高达0.5%,对电击后的野生型运动恢复无明显影响。
  8. 接下来,将空白DVD + R加载到HDD/DVD刻录机中,然后打开电视屏幕。此外,请使用示波器验证定时器输出激励器和激励器上的设置是否正确。我们通常将电击参数设置为47 V,3秒持续时间。该电压根据电压 - 响应曲线选择。野生型蠕虫恢复的最大电压约为60 V,平均恢复时间为85秒。选择47 V作为蠕虫恢复大约恢复最大限制时间的一半(图4)。
    注意:定时器输出激励器(S44)仅用作SD9刺激器的触发器,SD9刺激器向蠕虫发送电流。持续时间(通常为3秒)在S44上设置,并用作调节持续时间的触发器。还有许多其他方式来控制持续时间,包括通过计算机或Arduino使用数字触发。


    图4.电压响应曲线表明野生型蠕虫的运动恢复时间。野生型蠕虫在电压≥70V时没有恢复。恢复的最大测试极限为60 V,其中蠕虫恢复约80秒。 47 V是恢复时间为最大限制的50%的电压。这个数字改编自Risley等人,2016年。

  9. 加载约20μl对照溶液或药物溶液。紧随其后,使用铂金挑选装入6-8个蠕虫管,并设置一个计时器30分钟。定时器超时前30秒,将灯管轻轻放置在显微镜/摄像机设置下,灯座开启,并将每根铜线插入管的两端;电线应相隔10 mm(图3和2B)。验证铜线之间的距离是否精确(标尺就足够了),并启动电击。让实验记录10分钟。记录10分钟后,管子及其内容物被丢弃。
    注意:通常,每10分钟加载一个管。每个管被震动并记录10分钟,然后丢弃,并立即准备另一个管。此外,应将铂选择转移到管的OP50应尽量减少,因为它可能会阻碍蠕虫的观察。

数据分析

  1. 使用视频播放器手动分析分析。每个蠕虫在每个管中分别得分。图5中可以看到原始视频文件的示例屏幕截图和图2C中的示意图。记录休克时间(由气泡的外观所示)记录在笔记本中,并记录每个蠕虫的随后恢复时间。


    图5.记录的原始数据的截图示例。图像是实验的原始数据视频文件的屏幕截图。管中间有五个蠕虫。管子由两端的铜线堵塞。这是由研究人员分析的视频文件的示例。

  2. 恢复被定义为一种蠕虫恢复其在正弦运动中移动的能力,类似于在休克之前展示的运动。只有头部或尾部的部分恢复不被认为是恢复。 (视频1)
  3. 每个蠕虫的恢复时间被认为是n = 1,实验管的总数为N = 1。对于所有实验,全数据集被认为是n≥10且N≥6。
  4. 数据在SigmaPlot中使用单向ANOVA分析,随后进行事后多重比较测试或学生测试;然而,适当的统计分析应由研究人员确定。数据通常由条形图±SEM表示。数据条形图的一个例子可以在图4的电压 - 响应曲线中看到。

笔记

  1. 由于在刺激期间的电解,气泡将形成在管的边缘上。被气泡覆盖的蠕虫被排除在分析之外。我们使用红外温度枪记录3秒以上的温度波动。温度波动是急剧和极小的,1±0.5℃,但应记录温度以确保升高的温度不会增加电击的影响。如果冲击时间超过3秒,应注意温度不会大幅度增加,这可能会导致结果的混乱。
  2. 分析过程中没有考虑正弦运动的速度
  3. 没有恢复的蠕虫被排除在分析之外。

食谱

  1. LB肉汤
    将25g LB肉汤粉末和1L dH 2 O加入到2L烧瓶中 高压灭菌30分钟
  2. 线虫生长培养基(NGM)琼脂平板
    1. 将3g NaCl,17g琼脂,2.5g蛋白胨和975ml dH 2 O加入到2L烧瓶中。
    2. 高压灭菌30分钟
    3. 在水浴中冷却至50°C
    4. 加入1ml 1M CaCl 2,1ml 1M MgSO 4,溶于1ml 95%乙醇中的5mg胆固醇,25ml KP缓冲液加到琼脂溶液中,混合每次添加之后,
    5. 吸取10毫升到每个60 x 15毫米培养皿
    6. 盖住并在4°C下存放
    7. 当需要时,种子板用50μlOP50E。大肠杆菌,并在37℃下孵育过夜
  3. KP缓冲区
    将2.46g KH 2 PO 4和1.205g K 2 HPO 4加入到25ml二氯甲烷中, 2 O与搅拌棒
    调节pH至6.0和真空过滤器
  4. OP50 E。大肠杆菌
    从LB肉汤中的板材上悬挂1个OP50大肠杆菌菌落 在37℃下孵育过夜,盖子松动地放在
    上 储存于4°C
  5. M9解决方案
    加入3g KH 2 PO 4,6g Na 2 HPO 4,5g NaCl,1ml 1将M MgSO 4向2L烧瓶中加入并加入二氢氯化铵至1L的二氯甲烷/ 高压灭菌15分钟,并在室温下储存

致谢

我们已经在(Risley等人,2016)发布了这个协议。一些菌株由CGC提供,由国家卫生研究基础设施项目办公室(P40 OD010440)资助。

参考

  1. Lee,J. and Wu,CF(2002)。  在果蝇中的电惊厥发作行为:分析敏感突变体中定型动作模式的生理谱系。 Neurosci 22(24):11065-11079 。
  2. Risley,MG,Kelly,SP,Jia,K.,Grill,B.and Dawson-Scully,K.(2016)。  调制C中的行为。 elegans 使用电击和抗癫痫药物。 PLoS One 11(9):e0163786。
  3. Vashlishan,AB,Madison,JM,Dybbs,M.,Bai,J.,Sieburth,D.,Ch'ng,Q.,Tavazoie,M.and Kaplan,JM(2008)。 RNAi屏幕识别调节GABA突触的基因。神经元< em> 58(3):346-361。
  4. Williams,SN,Locke,CJ,Braden,AL,Caldwell,KA和Caldwell,GA(2004)。  在秀丽隐杆线虫中的神经递质信号的细胞骨架控制中与LIS-1相关的癫痫样抽搐 Hum Mol Genet 13(18):2043-2059。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 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. Risley, M. G., Kelly, S. P. and Dawson-Scully, K. (2017). Electroshock Induced Seizures in Adult C. elegans. Bio-protocol 7(9): e2270. DOI: 10.21769/BioProtoc.2270.
  2. Risley, M. G., Kelly, S. P., Jia, K., Grill, B. and Dawson-Scully, K. (2016). Modulating behavior in C. elegans using electroshock and antiepileptic drugs. PLoS One 11(9): e0163786.
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