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Aldicarb-induced Paralysis Assay to Determine Defects in Synaptic Transmission in Caenorhabditis elegans
涕灭威诱导的麻痹分析法测定秀丽隐杆线虫突触传递缺陷   

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Abstract

Aldicarb treatment causes an accumulation of acetylcholine in the synaptic cleft of the neuromuscular junction, resulting in sustained muscle activation and eventually paralysis. Aldicarb-induced paralysis assay is an easy and fast method to determine whether synaptic transmission of a C. elegans mutant of interest is altered. This assay is based on the correlation of the rate of neurotransmitter release with the rate of paralysis. In this protocol, we describe a method for simultaneously assessing the aldicarb sensitivity of animals with different genotypes.

Keywords: Aldicarb(涕灭威), C. elegans(秀丽隐杆线虫), Acetylcholinesterase(乙酰胆碱酯酶), Synaptic transmission(突触传递)

Background

Synaptic transmission is initiated by arrival of action potential at presynaptic terminals, which in turn results in the release of neurotransmitters. The released neurotransmitters bind to and activate postsynaptic receptors (Sudhof, 2013). C. elegans locomotion is controlled by acetylcholine-releasing excitatory motor neurons and GABA (γ-aminobutyric acid)-releasing-inhibitory motor neurons (Richmond and Jorgensen, 1999; Zhen and Samuel, 2015). Acetylcholine released from cholinergic motor neurons activates acetylcholine receptors on the muscle membrane, leading to muscle excitation and contraction. Acetylcholinesterase breaks down acetylcholine in the synaptic cleft and thus terminates neurotransmission. Aldicarb is an acetylcholinesterase inhibitor. In the presence of aldicarb, acetylcholine continues to accumulate, causing persistent muscle contraction and eventual paralysis. Mutant animals with decreased levels of synaptic transmission are resistant to the paralyzing effect of aldicarb because acetylcholine more slowly accumulates in the synaptic cleft of these animals than that of wild type animals. Conversely, mutant animals with increased levels of synaptic transmission, and as a result, a faster accumulation of acetylcholine, are more sensitive to the paralyzing effect of aldicarb than wild-type animals (Rand, 2007). Thus, by comparing the time-course of aldicarb-induced paralysis, it is possible to infer the relative efficiency of synaptic transmission. However, it is necessary to note that this assay does not necessarily demonstrate that abnormal aldicarb sensitivity is a result of a presynaptic defect. For example, a defect in post-synaptic acetylcholine receptor function may also result in resistance to aldicarb (Loria et al., 2004). Thus, the aldicarb-induced paralysis assay should be considered as the first step in investigating the involvement of synaptic transmission, and further corroborated by other means, such as electrophysiological analysis (Richmond, 2006).

Materials and Reagents

  1. 60 mm Petri dish (Genesee Scienntific, catalog number: 32-105G )
  2. 90% platinum, 10% iridium wire (Tritech Research, catalog number: PT-9010 )
  3. Copper rings (PlumbMaster, catalog number: 17668 )
  4. E. coli OP50 (University of Minnesota, Caenorhabditis Genetic Center)
  5. Aldicarb (ChemService, catalog number: N-11044 )
  6. Ethanol (Ethyl alcohol, 190 proof, ACS-USP grade) (PHARMCO-AAPER, catalog number: 111000190 )
  7. Cholesterol (Sigma-Aldrich, catalog number: C3045 )
  8. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3306 )
  9. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  10. Potassium phosphate, monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379 )
  11. Potassium phosphate, dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P8281 )
  12. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  13. Bacto-agar (BD, BactoTM, catalog number: 214040 )
  14. Bacto-pepton (BD, BactoTM, catalog number: 211820 )
  15. 100 mM aldicarb stock solution (see Recipes)
  16. 5 mg/ml cholesterol (see Recipes)
  17. 1 M CaCl2 stock solution (see Recipes)
  18. 1 M MgSO4 stock solution (see Recipes)
  19. 1 M KPO4, pH 6.0 stock solution (see Recipes)
  20. Nematode growth medium (NGM) agar plates (see Recipes)
  21. Nematode growth medium (NGM) agar plate containing aldicarb (see Recipes)

Equipment

  1. Forceps
  2. Rotator
  3. Autoclave
  4. 2 L flask
  5. Magnetic stir bar
  6. Microscope (Leica Microsystems, model: Leica MZ6 ) with KL 200 LED light source (Leica Microsystems, model: KL 200 LED )

Software

  1. GraphPad Prism 6.0, RRID:SCR_002798

Procedure

  1. For the routine maintenance of worms, see Stiernagle, T. Maintenance of C. elegans (Stiernagle, 2006).
  2. Prepare NGM agar plates containing 1 mM aldicarb (see Recipes) at least one day before the assay and store at 4 °C until use. Regular NGM agar plates for the routine maintenance should be seeded with OP50 E. coli and be available before the assay.
  3. Pick 30-40 L4 stage wild-type N2 and mutants of interest onto separate NGM plates seeded with OP50 E. coli (but not containing aldicarb), and culture them at 20 °C for 20 to 24 h.
  4. Use forceps to pick up a copper ring and dip into 70% ethanol. Flame the copper ring for a few seconds and immediately place it on the aldicarb-containing NGM agar plate. The copper ring will be lightly embedded in the agar surface of the plate (Figure 1). This will corral worms inside the ring. Place 3 copper rings on each assay plate and add 10 μl of OP50 E. coli (OD600 = 1.2) at the center of the ring. Bacteria help keep the worms around the center of the ring. Let the plates dry for 30 min. Three genotypes can be assayed on the same plate.


    Figure 1. A representative image of a copper ring embedded agar plate. Three copper rings are placed on a 60 mm NGM agar plate, and 10 μl of OP50 is added at the center of the ring.

  5. Transfer 20-30 worms of each genotype from step 3 to the center of each ring. Record the number of worms placed in each ring. Stagger worm transfer every 2-3 min so that all 3 genotypes are examined within a 10-min interval.
  6. Observe the worms in a 10-min interval. If there are worms not moving, then gently prod their heads with a platinum wire until they move (Videos 1 and 2). If they fail to move their head in response to even harsh touch, then remove them from the plate and record. Continue until no worm is left. Make sure that all of the worms are accounted for throughout the assay.

    Video 1. A video recording of responses of worms with different genotypes in the presence of aldicarb. The video (120 frames) was recorded for 4 min after 50 min incubation on an aldicarb plate. * indicates the paralyzed worms. WT: wild-type animals, slo-1: slo-1(eg142lf), mutant: an uncharacterized mutant.

    Video 2. A video recording shows paralyzed worms that do not respond to harsh touch

Data analysis

  1. Software: Prism 6.0
  2. Use ‘Survival analysis’ function to plot the survival graph (Figure 2). At least 20 worms of each genotype are used per assay. Repeat the assay two more times on different days (total 3 trials) and report the summary of the statistics as shown in Tables 1A and 1B.


    Figure 2. Survival curve for aldicarb-induced paralysis assay. Wild-type, slo-1(ky399 gain-of-function) and slo-1(eg142 loss-of-function) mutant animals were simultaneously tested for aldicarb sensitivity in a copper ring-embedded assay plate (adapted from Oh et al., 2017).

    Table 1A. Median survival time of C. elegans exposed to aldicarb (Median survival unit is minutes. The number in parenthesis is sample number)


    Table 1B. P values of Log-rank test


  3. Statistical analysis: The log-rank test is used to analyze the data. Prism software returns overall curve comparison results. However, pair-wise comparisons can be performed to compare 2 specific groups. Statistically significant P-values need to be adjusted for multiple comparisons if more than 2 pair-wise comparisons are performed. A Bonferroni corrected threshold is used to determine statistically significant P-values (http://www.graphpad.com/guides/prism/6/statistics/index.htm?survival_curves.htm). In this example, 3 comparisons, N2 vs. slo-1(ky399), N2 vs. slo-1(eg142), and slo-1(ky399) vs. slo-1(eg142), are performed and P-values for each comparison is obtained. If threshold for statistical significance is set at a P-value of 0.05, then any P-value that is less than the corrected threshold, i.e., 0.05 divided by 3, is considered statistically significant in this example.

Notes

  1. As per standard scientific procedure, the experimenter should be blinded to the genotypes of the test worms. Ideally a colleague should place the worms (procedure step 5) and label the worms to prevent the experimenter from identifying the genotypes. For mutants with obvious phenotypes, an additional mutant with a similar phenotype, which is unrelated to the study of interest, may be added.
  2. The resolution of differential sensitivity can be changed by varying the concentration of aldicarb. For example, mutants that have higher rates of neurotransmission than wild type worms may be better distinguishable at a lower concentration of aldicarb (e.g., 0.5 mM), which increases the time for the worms to get paralyzed.

Recipes

  1. 100 mM aldicarb stock solution
    Dissolve 100 mg of aldicarb in 5.25 ml of 70% ethanol at room temperature
    Store at 4 °C
    Note: We have stored the stock up to 2 weeks and have not tried longer storage.
  2. 5 mg/ml cholesterol
    Add 250 mg of cholesterol in 50 ml of 95 % ethanol and mix by rotating on a rotator at room temperature. It takes a few hours to dissolve. Store at room temperature
  3. 1 M CaCl2 stock solution
    Dissolve 14.7 g of CaCl2·2H2O in 100 ml ddH2O and autoclave for 30 min at 121 °C. Store at room temperature
  4. 1 M MgSO4 stock solution
    Dissolve 12.04 g of MgSO4 in 100 ml ddH2O and autoclave for 30 min at 121 °C. Store at room temperature
  5. 1 M KPO4, pH 6.0 stock solution
    Make 500 ml of 1 M KH2PO4 (monobasic) and 250 ml of 1 M K2HPO4 (dibasic) solution. While measuring pH of 1 M KH2PO4 (pH is below 5), add and stir 1 M K2HPO4 slowly until pH reaches 6.0. It will take less than 250 ml of K2HPO4 to reach pH 6.0. Aliquot the solution and autoclave for 30 min at 121 °C. Store at room temperature
  6. Nematode growth medium (NGM) agar plate
    1. Add 3 g of NaCl, 16 g of Bacto-agar, and 2.5 g of Bacto-pepton in a 1 L of ddH2O in a 2 L flask with magnetic stir bar
    2. Autoclave for 30 min at 121 °C
    3. Let the NGM agar cool to 55 to 60 °C while stirring on a stirrer, Add 1 ml of 5 mg/ml cholesterol, 1 ml of 1 M CaCl2, 1 ml of 1 M MgSO4, and 25 ml of 1 M KPO4, pH 6.0 while stirring
    4. Dispense 10 ml per 60 mm Petri dish and let them solidify without perturbation
  7. Nematode growth medium (NGM) agar plate containing aldicarb
    1. For aldicarb-containing NGM plates, add aldicarb solution to a desired final concentration along with cholesterol, CaCl2, MgSO4, and 1 M KPO4, pH 6.0 when NGM agar medium is cooled to 55 to 60 °C
    2. For 1 mM aldicarb, add 1 ml of 100 mM aldicarb for 100 ml of NGM. Pour 10 ml per 60 mm Petri dish and let them dry for at least 1 day. Plates can be stored at 4 °C

      Note: We have used plates stored at 4 °C for up to 2 weeks without any issue.

Acknowledgments

The protocol has been adapted from Oh et al. (2017), eLife 6: e24733. This work was supported, in part, by a grant from the National Institute of Health.

References

  1. Loria, P. M., Hodgkin, J. and Hobert, O. (2004). A conserved postsynaptic transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans. J Neurosci 24(9): 2191-2201.
  2. Oh, K. H., Haney, J. J., Wang, X., Chuang, C. F., Richmond, J. E. and Kim, H. (2017). ERG-28 controls BK channel trafficking in the ER to regulate synaptic function and alcohol response in C. elegans. Elife 6.
  3. Rand, J. B. (2007). Acetylcholine. WormBook 30: 1-21.
  4. Richmond, J. E. (2006). Electrophysiological recordings from the neuromuscular junction of C. elegans. WormBook 6: 1-8.
  5. Richmond, J. E. and Jorgensen, E. M. (1999). One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat Neurosci 2(9): 791-797.
  6. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook 11: 1-11.
  7. Sudhof, T. C. (2013). Neurotransmitter release: the last millisecond in the life of a synaptic vesicle. Neuron 80(3): 675-690.
  8. Zhen, M. and Samuel, A. D. (2015). C. elegans locomotion: small circuits, complex functions. Curr Opin Neurobiol 33: 117-126.

简介

涕灭威的治疗导致神经肌肉接头突触裂缝中的乙酰胆碱积聚,导致持续的肌肉活化并最终导致麻痹。 涕灭威诱导的麻痹测定是确定是否突触传播的一种简单且快速的方法。 线虫兴趣突变体被改变。 该测定是基于神经递质释放速率与麻痹率的相关性。 在该方案中,我们描述了同时评估具有不同基因型的动物的涕灭威敏感性的方法。
【背景】突触传递通过动作电位到达突触前末端而开始,这又导致神经递质的释放。释放的神经递质结合并激活突触后受体(Sudhof,2013)。 ℃。线虫运动由乙酰胆碱释放兴奋性运动神经元和GABA(γ-氨基丁酸)释放抑制运动神经元控制(Richmond和Jorgensen,1999; Zhen和Samuel,2015)。从胆碱能运动神经元释放的乙酰胆碱激活肌肉膜上的乙酰胆碱受体,导致肌肉的激发和收缩。乙酰胆碱酯酶在突触裂缝中分解乙酰胆碱,从而终止神经传递。涕灭威是乙酰胆碱酯酶抑制剂。在涕灭威的存在下,乙酰胆碱继续积累,引起持续的肌肉收缩和最终的麻痹。具有降低突触传递水平的突变动物对涕灭威的麻痹作用具有抗性,因为乙酰胆碱在这些动物的突触裂隙中比慢性积累比野生型动物慢。相反,具有突触传递水平升高的突变体动物,并且结果是乙酰胆碱的更快积累对涕灭威的麻痹效应比野生型动物更敏感(Rand,2007)。因此,通过比较涕灭威诱导的麻痹的时间过程,可以推断突触传播的相对效率。然而,有必要指出,该测定法并不一定表明异常涕灭威的敏感性是突触前缺陷的结果。例如,突触后乙酰胆碱受体功能的缺陷也可能导致对涕灭威的抗性(Loria et al。,2004)。因此,涕灭威诱导的麻痹试验应被视为调查突触传播参与的第一步,并进一步通过其他手段证实,如电生理分析(Richmond,2006)。

关键字:涕灭威, 秀丽隐杆线虫, 乙酰胆碱酯酶, 突触传递

材料和试剂

  1. 60毫米培养皿(Genesee Scienntific,目录号:32-105G)
  2. 90%铂,10%铱丝(Tritech Research,目录号:PT-9010)
  3. 铜环(PlumbMaster,目录号:17668)
  4. 电子。大肠杆菌OP50(明尼苏达大学,秀丽菌属遗传中心)
  5. 涕灭威(ChemService,目录号:N-11044)
  6. 乙醇(乙醇,190证明,ACS-USP级)(PHARMCO-AAPER,目录号:111000190)
  7. 胆固醇(Sigma-Aldrich,目录号:C3045)
  8. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C3306)
  9. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  10. 磷酸二氢钾(KH 2 O 3 PO 4)(Sigma-Aldrich,目录号:P5379)
  11. 磷酸二钾(K 2 O 3 HPO 4)(Sigma-Aldrich,目录号:P8281)
  12. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  13. Bacto-agar(BD,Bacto TM ,目录号:214040)
  14. Bacto-pepton(BD,Bacto TM ,目录号:211820)
  15. 100mM涕灭威储备溶液(参见食谱)
  16. 5 mg / ml胆固醇(见食谱)
  17. 1M CaCl 2储备溶液(参见食谱)
  18. 1M MgSO 4储备溶液(参见食谱)
  19. 1 M KPO 4,pH 6.0储备溶液(参见食谱)
  20. 线虫生长培养基(NGM)琼脂平板(参见食谱)
  21. 含有涕灭威的线虫生长培养基(NGM)琼脂平板(参见食谱)

设备

  1. 镊子
  2. 旋转器
  3. 高压灭菌器
  4. 2升烧瓶
  5. 磁力搅拌棒
  6. 具有KL 200 LED光源(Leica Microsystems,型号:KL 200 LED)的显微镜(Leica Microsystems,型号:Leica MZ6)

软件

  1. GraphPad Prism 6.0,RRID:SCR_002798

程序

  1. 对于蠕虫的日常维护,请参阅Stiernagle,T.维护C. elegans (Stiernagle,2006)。
  2. 在测定前至少一天准备含有1mM涕灭威的NGM琼脂平板(见食谱),并在4℃下储存直至使用。常规维护的常规NGM琼脂平板应接种OP50大肠杆菌,并在测定前可用。
  3. 将30-40 L4级野生型N2和目标突变体选择在用OP50大肠杆菌(但不含涕灭威)接种的分离的NGM板上,并在20℃下培养20至24小时。
  4. 使用镊子拿起铜环并浸入70%乙醇中。将铜环阻燃几秒钟,立即将其放置在含有涕灭威的NGM琼脂平板上。铜环将轻轻地嵌入板的琼脂表面(图1)。这将是环内的珊瑚虫。在每个测定板上放置3个铜环,并加入10μlOP50E。大肠杆菌(OD 600)= 1.2)在环的中心。细菌有助于使蠕虫环绕环的中心。让板干燥30分钟。可以在同一板上测定三种基因型

    图1.铜环嵌入琼脂平板的代表性图像。 将三个铜环放置在60mm NGM琼脂板上,并在环的中心加入10μlOP50。

  5. 将每个基因型的20-30个蠕虫从步骤3转移到每个环的中心。记录每个环中放置的蠕虫数。每2-3分钟交错蠕虫传播,以便在10分钟的间隔内检查所有3种基因型。
  6. 以10分钟的间隔观察蠕虫。如果有蠕虫没有移动,那么用铂金丝轻轻地将其头部移动(视频1和2)。如果他们没有动摇他们的头,以反应甚至严酷的触摸,然后将其从盘子中删除并记录。继续,直到没有蠕虫。确保所有的蠕虫在整个测定中被考虑。

    Video 1. A video recording of responses of worms with different genotypes in the presence of aldicarb. The video (120 frames) was recorded for 4 min after 50 min incubation on an aldicarb plate. * indicates the paralyzed worms. WT: wild-type animals, slo-1: slo-1(eg142lf), mutant: an uncharacterized mutant.

    To play the video, you need to install a newer version of Adobe Flash Player.

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    Video 2. A video recording shows paralyzed worms that do not respond to harsh touch

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

翻译

Data analysis

  1. Software: Prism 6.0
  2. Use ‘Survival analysis’ function to plot the survival graph (Figure 2). At least 20 worms of each genotype are used per assay. Repeat the assay two more times on different days (total 3 trials) and report the summary of the statistics as shown in Tables 1A and 1B.


    Figure 2. Survival curve for aldicarb-induced paralysis assay. Wild-type, slo-1(ky399 gain-of-function) and slo-1(eg142 loss-of-function) mutant animals were simultaneously tested for aldicarb sensitivity in a copper ring-embedded assay plate (adapted from Oh et al., 2017).

    Table 1A. Median survival time of C. elegans exposed to aldicarb (Median survival unit is minutes. The number in parenthesis is sample number)


    Table 1B. P values of Log-rank test


  3. Statistical analysis: The log-rank test is used to analyze the data. Prism software returns overall curve comparison results. However, pair-wise comparisons can be performed to compare 2 specific groups. Statistically significant P-values need to be adjusted for multiple comparisons if more than 2 pair-wise comparisons are performed. A Bonferroni corrected threshold is used to determine statistically significant P-values (http://www.graphpad.com/guides/prism/6/statistics/index.htm?survival_curves.htm). In this example, 3 comparisons, N2 vs. slo-1(ky399), N2 vs. slo-1(eg142), and slo-1(ky399) vs. slo-1(eg142), are performed and P-values for each comparison is obtained. If threshold for statistical significance is set at a P-value of 0.05, then any P-value that is less than the corrected threshold, i.e., 0.05 divided by 3, is considered statistically significant in this example.

Notes

  1. As per standard scientific procedure, the experimenter should be blinded to the genotypes of the test worms. Ideally a colleague should place the worms (procedure step 5) and label the worms to prevent the experimenter from identifying the genotypes. For mutants with obvious phenotypes, an additional mutant with a similar phenotype, which is unrelated to the study of interest, may be added.
  2. The resolution of differential sensitivity can be changed by varying the concentration of aldicarb. For example, mutants that have higher rates of neurotransmission than wild type worms may be better distinguishable at a lower concentration of aldicarb (e.g., 0.5 mM), which increases the time for the worms to get paralyzed.
3274/5000

数据分析

  1. 软件:Prism 6.0
  2. 使用“生存分析”功能绘制生存图(图2)。每个测定使用每个基因型至少20个蠕虫。在不同的日子重复测定两次(共3次试验),并报告统计数据摘要,如表1A和1B所示。


    图2.涕灭威诱导麻痹测定的生存曲线。野生型,slo-1(ky399增益功能)和 slo-1
    表1A。暴露于涕灭威的线虫的中位生存时间(中位生存单位为分钟,括号中的数字为样本号)


    表1B。日志级别测试
    的P 值

  3. 统计分析:对数秩检验用于分析数据。 Prism软件返回总体曲线比较结果。然而,可以进行成对比较以比较2个特定组。如果进行多于2次的成对比较,则需要调整统计学上显着的P 值进行多次比较。 Bonferroni校正阈值用于确定统计学显着的P 值( http://www.graphpad.com/guides/prism/6/statistics/index.htm?survival_curves.htm )。在这个例子中,N2与 slo-1(ky399)的3个比较,N2与slo-1(eg142)和 slo-1(ky399) ) vs。执行 slo-1(eg142),并且获得每个比较的 P 值。如果将统计显着性的阈值设置为0.05的P值,则小于校正阈值的任何


    值,例如 ,0.05除以3,在本例中被认为具有统计学意义

笔记

  1. 按照标准的科学程序,实验者应该对测试蠕虫的基因型不了解。理想情况下,同事应该放置蠕虫(程序步骤5)并标记蠕虫,以防止实验者识别基因型。对于具有明显表型的突变体,可以添加与感兴趣研究无关的具有相似表型的另外的突变体。
  2. 差异灵敏度的分辨率可以通过改变涕灭威的浓度来改变。例如,具有比野生型蠕虫更高的神经传递速率的突变体可以在较低浓度的涕灭威(例如,0.5mM)下更好地区分,这增加了蠕虫瘫痪的时间。 br />

食谱

  1. 100mM涕灭威储备溶液
    在室温下将100毫克涕灭威溶解在5.25毫升70%乙醇中 储存于4°C
    注意:我们已经储存了两周的库存,并没有尝试更长的存储空间。
  2. 5 mg / ml胆固醇
    加入250毫克胆固醇在50ml的95%乙醇中,并在室温下旋转旋转混合。解散需要几个小时。在室温下存放
  3. 1M CaCl 2 储备溶液
    将14.7g CaCl 2·2H 2 O在100ml ddH 2 O中溶解,并在121℃下高压灭菌30分钟。在室温下存放
  4. 1 M MgSO 4储备溶液
    将12.04g MgSO 4在100ml ddH 2 O中溶解并在121℃下高压灭菌30分钟。在室温下存放
  5. 1 M KPO 4,pH 6.0储备溶液
    制备500毫升1M KH 2 PO 4(一元碱性)和250毫升1 MK 2 HPO 4 / (二元)溶液。在测量1M KH 2 PO 4的pH(pH低于5)的同时,加入并搅拌1KH 2 HPO 4, / sub>,直到pH达到6.0。将需要少于250毫升的K 2 HPO 4+达到pH6.0。将溶液等分并在121℃下高压灭菌30分钟。在室温下存放
  6. 线虫生长培养基(NGM)琼脂平板
    1. 在具有磁力搅拌棒的2L烧瓶中加入3g NaCl,16g Bacto-agar和2.5g Bacto-pepton在1L ddH 2 O中,
    2. 高压灭菌在121°C 30分钟
    3. 将NGM琼脂冷却至55至60℃,同时在搅拌器上搅拌,加入1ml 5mg / ml胆固醇,1ml 1M CaCl 2,1ml 1M MgSO 4 > 4 和25ml 1M KPO 4,pH 6.0,同时搅拌
    4. 分配10毫升每60毫米培养皿,让他们凝固而不扰动
  7. 含有涕灭威的线虫生长培养基(NGM)琼脂平板
    1. 对于含有涕灭威的NGM板,将涕灭威溶液与胆固醇,CaCl 2,MgSO 4和1M KPO 4 ,pH 6.0,当NGM琼脂培养基冷却至55至60℃时
    2. 对于1mM的涕灭威,加入1ml 100mM的涕灭威用于100ml的NGM。倒入10毫升每60毫米培养皿,让他们干燥至少1天。板材可以在4°C下储存

      注意:我们在4°C下使用的板材长达2周没有任何问题。

致谢

该协议已经从Oh等人改编(2017),eLife 6:e24733。这项工作部分得到国家卫生研究所的资助。

参考

  1. Loria,PM,Hodgkin,J.和Hobert,O。(2004)。在秀丽隐杆线虫中影响神经肌肉信号传导的保守的突触后跨膜蛋白。 J Neurosci 24(9):2191-2201。
  2. 哦,KH,Haney,JJ,Wang,X.,Chuang,CF,Richmond,JE and Kim,H。(2017)。 
  3. Rand,JB(2007)。  Acetylcholine。 WormBook 30:1-21。
  4. Richmond,JE(2006)。  神经肌肉的电生理记录 C. elegans 的连接。 WormBook 6:1-8。
  5. Richmond,JE和Jorgensen,EM(1999)。  一个GABA和两个乙酰胆碱受体在秀丽隐杆线虫神经肌肉接头处起作用。 Nat Neurosci 2(9):791-797。
  6. Stiernagle,T。(2006)。维护℃。电影。 WormBook 11:1-11。
  7. Sudhof,TC(2013)。 Neurotransmitter release:the last突触小泡生命中的毫秒数。神经元 80(3):675-690。
  8. Zhen,M. and Samuel,AD(2015)。  ℃。 elegans 运动:小电路,复杂功能。 Curr Opin Neurobiol 33:117-126。
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Copyright Oh and Kim. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Oh, K. H. and Kim, H. (2017). Aldicarb-induced Paralysis Assay to Determine Defects in Synaptic Transmission in Caenorhabditis elegans. Bio-protocol 7(14): e2400. DOI: 10.21769/BioProtoc.2400.
  2. Oh, K. H., Haney, J. J., Wang, X., Chuang, C. F., Richmond, J. E. and Kim, H. (2017). ERG-28 controls BK channel trafficking in the ER to regulate synaptic function and alcohol response in C. elegans. Elife 6.
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