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Feb 2021

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Ethanol-induced Sedative Behavior: An Assay to Investigate Increased Dopamine Signaling in Caenorhabditis elegans
乙醇诱导的镇静行为:一项在秀丽隐杆线虫研究增加多巴胺信号   

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Abstract

Dopamine (DA) signaling affects locomotion, feeding, learning, and memory in C. elegans. Various assays have been developed to study the proteins involved in these behaviors; however, these assays show behavioral output only when there is a drastic change in DA levels. We designed an assay capable of observing behavioral output even with only slight alterations in DA levels. To achieve this, we designed a behavioral paradigm where we combined C. elegans movement with ethanol (EtOH) administration. The behavioral response to alcohol/EtOH and susceptibility to alcohol-use disorders (AUDs) have been linked to DA. Our assay correlates an increase in DA levels due to EtOH and movement obstruction due to a dry surface to a circular sedative behavior, which we designated as EtOH-induced sedative (EIS) behavior. We successfully utilized this assay to assign physiological and behavioral functions to a DA autoreceptor, DOP-2.

Keywords: EtOH (Ethanol) (乙醇), C. elegans (秀丽隐杆线虫), Dopamine (DA) (质多巴胺), Sedative behavior (镇静剂的行为)

Background

Alcohol is a widely abused drug with a plethora of associated diseases that can impact societal functioning. Multiple studies have focused on unravelling the mode of action and effect of this drug; however, the neuronal mechanisms underlying alcohol susceptibility and disinhibition are unclear. Studies across various species have demonstrated that alcohol intake increases the release of the neurotransmitter DA that induces the reward pathway (Imperato and Di Chiara, 1986; Weiss et al., 1996; Baik, 2013). Although C. elegans does not mimic all the complexities of the mammalian system, it has been successfully modeled for studying alcohol-dependent neuronal behaviors. Studies show that C. elegans display diverse aspects of alcohol responses (Davies et al., 2003 and 2004). Previously, investigations in C. elegans have revealed that there is a dose-dependent decline in the locomotor activity upon acute and chronic alcohol exposure at a concentration of 400-500 mM (Davies et al., 2003; Lee et al., 2009). The DA system in C. elegans is involved in feeding, movement, learning, and memory; and similar to that of mammals, signals through two receptor subfamilies D1-like and D2-like receptors. Mutants of the D2-like receptor, dop-2, exhibited no obvious phenotype when analyzed for DA-dependent behaviors, despite being expressed in all dopaminergic neurons and predicted to be an autoreceptor (Chase et al., 2004). We devised an assay utilizing the EIS behavior observed in mutants of dop-2 to investigate the neuronal circuitry involved in regulating locomotory behavior under the influence of EtOH (Pandey et al., 2021).


Materials and Reagents

  1. 60-mm Petri dishes (Tarsons, catalog number: 460061)

  2. Spreader (Tarsons, catalog number: 920081)

  3. 99.99% platinum wire (Sigma-Aldrich, catalog number: 267201)

  4. C. elegans: N2 (wild-type (WT) and dop-2 (vs105) adult animals with 3-4 eggs (University of Minnesota, Caenorhabditis Genetic Center)

  5. Escherichia coli OP50 (University of Minnesota, Caenorhabditis Genetic Center)

  6. Ethanol (EtOH) (Fisher chemical, catalog number: UN1170)

  7. Cholesterol (SRL Sisco Research Laboratories, catalog number: 54181)

  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 (HiMedia Laboratories, catalog number: GRM026)

  14. Bacto-peptone (BD, catalog number: 211677)

  15. 400 mM EtOH (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)

Equipment

  1. Pipettes (Eppendorf, model: Research® plus, catalog number: 2231000224)

  2. 2-L glass conical flask (DWK Life Sciences, DURAN, catalog number: 2121763)

  3. Autoclave (Equitron-7431 SLEFA)

  4. Microscope (ZEISS, model: Stemi 2000 C)

Software

  1. GraphPad Prism v6 (GraphPad Software)

  2. ImageJ (developed by the National Institutes of Health)

Procedure

  1. Prepare 60-mm NGM plates for the maintenance of C. elegans. Seed the plates with E. coli OP50 (serves as food for C. elegans) under sterile conditions in a bacterial hood and allow to grow overnight at 37°C.

  2. Synchronize the C. elegans strains to be analyzed by bleaching. Collect the animals from the NGM plates using 1 ml M9 solution in 2-ml microcentrifuge tubes (MCT). Add 400 µl freshly prepared bleach solution (1:1 ratio of 5 N sodium hydroxide and sodium hypochlorite) to the tubes and vortex at medium speed for 5 min. Spin the MCT at 6,097 × g (4000 rpm) for 60 s and discard the supernatant. Wash the pellet again with fresh M9 and plate on 60-mm NGM plates.

  3. To perform the assay, prepare fresh 60-mm (NGM) plates containing 8 ml media one day before the assay and store at 4°C until use.

  4. On the day of the assay, dry the plates in the laminar airflow for 3-4 h with the lids open.

  5. Spread 400 mM (196 µl) 100% EtOH on the dry 60-mm plates with 8 ml media using a glass spreader. Dry plates without EtOH (– EtOH) are used as control plates.

  6. Seal the EtOH assay plates with parafilm to avoid any loss of ethanol and place at 20°C for 2 h to allow the EtOH to equilibrate across the plates.

  7. The EtOH assay plates are now ready and can be used for the assay.

  8. Transfer 10 animals of the genotype being studied from a seeded plate (+ food) to an unseeded plate (– food) for 15-20 s (to get rid of the food). Next, transfer the animals to the assay plate or control plate and leave undisturbed for 2 h at 20°C.

  9. Observe the C. elegans after a 2-h time interval. Count the number of animals on the plates and make 1-min videos for each animal to observe and analyze the locomotory behavior by counting the number (anterior and posterior) and amplitude of body bends to characterize the behavior.

  10. The number of body bends are scored manually using ImageJ while maintaining identical parameters when analyzing anterior and posterior body bends (Figure 1A).

  11. Calculate the amplitude of the body bends manually using the NIH ImageJ software, quantitating the anterior and posterior body bends separately. To quantitate the amplitude of the body bends, measure the distance between the deepest angle of the body bend. Draw a tangential line from the tip of the head to the midsection of the body and measure the vertical distance from the line to the body as the amplitude of the anterior body bend (Figure 1B). Correspondingly, from the midsection to the tail of the animal, measurements are obtained for the posterior body bend amplitude (Figure 1B). Normalize the measurements to the length of the animal to derive the measured values in micrometers.



    Figure 1. Representative image used for the analysis of the amplitude of body bends. A. Image represents the evaluation of anterior and posterior body bends separately. B. Image indicates the amplitude of body bends; a double-sided arrow depicts the amplitude (Amp) in the anterior and posterior regions.


  12. Perform timeline-based analysis of behavior. Starting from EtOH exposure, different time points (30, 60, 120, 300 min), including overnight exposure (16 h), are analyzed (Figure 2A).



    Figure 2. Illustration of the timeline for EtOH treatment of C. elegans to study EIS behavior. A. The timepoints denote different phases of WT animals upon EtOH exposure, as indicated in the timeline. Experiments to test the number and amplitude of body bends are performed at ~2 h. B. Illustration of EIS behavior, where WT animals recover from EtOH-induced paralysis and show normal sinusoidal movement, while dop-2 mutant animals show EtOH-induced sedative behavior (Pandey et al., 2021).


  13. WT animals show normal behavior 2 h after EtOH paralysis (Video 1). While dopaminergic autoreceptor, dop-2, mutant animals show EtOH-induced sedative (EIS) behavior at the same time point (Figure 2B and Video 2).


    Video 1. A representative video of WT animals showing a single C. elegansthat has recovered from EtOH-induced paralysis. The animal can be seen moving in normal sinusoidal wave-like patterns, with no observable defects in locomotion (Pandey et al., 2021).


    Video 2. A representative video of the EIS behavior shown by dop-2 mutant C. elegans upon EtOH exposure at the 2-h time point. The animal shows defects in locomotion and moves by dragging its posterior region (Pandey et al., 2021).

  14. Recovery from the EIS behavior can be observed upon transferring the mutant animals to normal NGM plates with food after a 2-h exposure to EtOH. Mutants of dop-2 recover from the EIS phenotype 1 h after being transferred.

Data analysis

  1. Calculate the number of anterior and posterior body bends separately in a 1-min video at the 2-h time point.

  2. Software: GraphPad Prism v6.

  3. Transfer 10 animals to each assay plate and perform the experiment in triplicate for each strain.

  4. Statistical analysis: Use one way-ANOVA to determine statistical P-values.

Notes

  1. Drying the NGM plates perfectly is important, and the surface of the plates should be free from any dust or other surface aberrations for proper visualization of the tracks.

  2. Seal the plates with parafilm to avoid loss of ethanol and appropriate equilibration.

Recipes

  1. 5 mg/ml cholesterol

    Add 500 mg cholesterol to 100 ml 95% ethanol and mix by rotating at room temperature for a few hours to dissolve.

    Store at 4°C.

  2. 1 M CaCl2 stock solution

    Dissolve 14.7 g CaCl2·2H2O in 100 ml ddH2O and autoclave for 15 min at 121°C.

    Store at 4°C.

  3. 1 M MgSO4 stock solution

    Dissolve 12.04 g MgSO4 in 100 ml ddH2O and autoclave for 15 min at 121°C.

    Store at 4°C.

  4. 1 M KPO4, pH 6.0 stock solution

    1. Mix 108.3 g KH2PO4 and 35.6 g K2HPO4 in 500 ml ddH2O.

    2. Adjust the pH to 6.0 by adding NaOH; finally, make up the volume to 1 L.

    3. Aliquot the solution and autoclave for 15 min at 121°C.

    4. Store at 4°C.

  5. Nematode growth medium (NGM) agar plate

    1. Add 3 g NaCl, 16 g Bacto-agar, and 2.5 g Bacto-peptone to 975 ml ddH2O in a 2-L flask.

    2. Autoclave for 50 min at 121°C.

    3. Allow the NGM agar to cool to 55-60°C. Add 1 ml 5 mg/ml cholesterol, 1 ml 1 M CaCl2, 1 ml 1 M MgSO4, and 25 ml 1 M KPO4, pH 6.0.

  6. 400 mM ethanol

    Spread 196 ml ethanol on 8 ml media in a 60-mm plate.

    Seal the plate with parafilm and allow equilibration at 20°C.

Acknowledgments

This protocol was adapted from Pandey et al. (2021). AS thanks the Council of Scientific and Industrial Research (CSIR) – University Grants Commission (UGC) for a graduate fellowship. PP acknowledges support from a Department of Science and Technology (DST) – Women of Science (WOS-A) grant as well as past funding from the Department of Biotechnology (DBT) Bio-CARe, the Indian Institute of Science Education and Research (IISER) Mohali, and an IA grant awarded to KB. KB was an Intermediate Fellow of the India Alliance (IA) and is currently an IA Senior Fellow. KB thanks the Alliance for funding support. KB also thanks DBT, DST – Science and Engineering Research Board (SERB), and the Ministry of Human Resources Development (MHRD) – Scheme for Transformational and Advanced Research in Sciences (STARS) for funding support.


Funding

This work was supported by the DBT/Wellcome Trust India Alliance fellowships [grant numbers IA/S/19/2/504649 and IA/I/12/1/500516] awarded to KB and partially supported by DBT, MHRD–STARS, and DST–SERB grants [BT/PR24038/BRB/10/1693/2018, STARS/APR2019/BS/454/FS and SERB/F/7047] as well as a DBT-IISc partnership grant to KB. PP is supported by a DST WOS-A grant [SR/WOS-A/LS-285/2018] and was earlier supported by a DBT Bio-CARe grant [BioCARe/01/10167].

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Baik, J. H. (2013). Dopamine signaling in reward-related behaviors. Front Neural Circuits 7: 152.
  2. Chase, D. L., Pepper, J. S. and Koelle, M. R. (2004). Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans. Nat Neurosci 7(10): 1096-1103.
  3. Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele, T. R., Bonci, A., Bargmann, C. I. and McIntire, S. L. (2003). A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell 115(6): 655-666.
  4. Davies, A. G. and McIntire, S. L. (2004). Using C. elegans to screen for targets of ethanol and behavior-altering drugs. Biol Proced Online 6: 113-119.
  5. Imperato, A. and Di Chiara, G. (1986). Preferential stimulation of dopamine release in the nucleus accumbens of freely moving rats by ethanol. J Pharmacol Exp Ther 239(1): 219-228.
  6. Lee, J., Jee, C. and McIntire, S. L. (2009). Ethanol preference in C. elegans. Genes Brain Behav 8(6): 578-585.
  7. Pandey, P., Singh, A., Kaur, H., Ghosh-Roy, A. and Babu, K. (2021). Increased dopaminergic neurotransmission results in ethanol dependent sedative behaviors in Caenorhabditis elegans. PLoS Genet 17(2): e1009346.
  8. Weiss, F., Parsons, L. H., Schulteis, G., Hyytia, P., Lorang, M. T., Bloom, F. E. and Koob, G. F. (1996). Ethanol self-administration restores withdrawal-associated deficiencies in accumbal dopamine and 5-hydroxytryptamine release in dependent rats. J Neurosci 16(10): 3474-3485.

简介

[摘要]多巴胺(DA)信令影响运动,送料,学习,并在内存中线虫。已经开发了各种检测方法来研究参与这些行为的蛋白质;^ h H但是,这些实验表明行为只输出时,有一个在DA水平急剧变化。我们设计了一个实验能够OBSERV荷兰国际集团行为的输出,即使只有轻微的DA水平的改变。为实现这一目标,我们设计了一种行为范式,将秀丽隐杆线虫运动与乙醇 (EtOH)管理相结合。 的b ehavioral到醇/ EtOH中反应和易感性醇-使用障碍(AUD中)一直立Ñ的KED到DA 。我们的测定相关的由于DA水平由于EtOH中,运动障碍增加一个干燥的表面上的圆形镇静剂行为,其中我们指定为EtOH中-i EP3受体激动剂诱导小号edative(EIS)的行为。我们成功地利用该测定将生理和行为功能分配给 DA 自动受体DOP-2。

[背景]醇是一个广泛被滥用的药物与相关的疾病的过多是c的影响社会的运作。多项研究集中于解开这种药物的作用方式和效果;ħ H但是,神经元机制未derlying醇易感性和去抑制是未明确。对不同物种的研究表明,酒精摄入会增加神经递质 DA 的释放,从而诱导奖励途径(Imperato 和 Di Chiara,1986;Weiss等,199 6 ;Baik,2013 )。虽然线虫不模仿哺乳动物系统的所有复杂性,它已经成功地模拟了研究酒精-依赖的神经行为。研究表明,秀丽隐杆线虫表现出酒精反应的不同方面(Davies等,2003和2004)。以前,在调查线虫已经表明,有一个剂量-在后急性运动活性依赖性下降和在400的浓度慢性酒精暴露- 500毫米(戴维斯等人,2003;李等人,2009) . 秀丽隐杆线虫的 DA 系统参与进食、运动、学习和记忆;与哺乳动物类似,通过两个受体亚家族 D1 样和 D2 样受体发出信号。突变体的所述D2样受体,DOP-2分析时为DA,没有表现出明显的表型-依赖性的行为,尽管被在所有多巴胺能神经元中表达并预测为一个自身受体(大通等。人,2004)。我们设计了一种利用在dop-2突变体中观察到的 EIS 行为的分析,以研究在EtOH 影响下参与调节运动行为的神经元回路(Pandey等,2021)。

关键字:乙醇, 秀丽隐杆线虫, 质多巴胺, 镇静剂的行为


材料和试剂

1. 60 - mm 培养皿(Tarsons ,目录号:460061)     
2.吊具(Tarsons ,目录号:920081)     
3. 99.99% 铂丝(Sigma-Aldrich,目录号:267201)     
4.线虫:N2(野生型(WT)和做P-2(vs105)成年动物用3 - 4个鸡蛋(明尼苏达大学,秀丽隐杆线虫遗传中心)     
5.大肠杆菌OP50(明尼苏达大学,Caenorhabditis遗传中心)     
6.乙醇(EtOH)(Fisher 化学品,目录号:UN1170)     
7.胆固醇(SRL Sisco Research Laboratories,目录号:54181)     
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 PO 4 )(Sigma-Aldrich,目录号:P5379) 
11.磷酸二氢钾(K 2 HPO 4 )(Sigma-Aldrich,目录号:P8281) 
12.氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653) 
13. Bacto -agar(HiMedia Laboratories,目录号:GRM026) 
14. Bacto-蛋白胨(BD,目录号:211677) 
15. 400mM的乙醇(见配方小号) 
16. 5mg / ml的胆固醇(见配方小号) 
17. 1M的氯化钙2储液(见配方小号) 
18. 1M的用MgSO 4储备溶液(见配方小号) 
19. 1M的KPO 4 ,pH 6.0的储备溶液(见配方小号) 
20.线虫生长培养基(NGM)琼脂板(参见配方小号) 

设备

移液器仪(Eppendorf,型号:研究®加,目录号:2231000224)
2 - L玻璃锥形瓶(DWK Life Sciences,DURAN,目录号:2121763)
高压釜(Equitron-7431 SLEFA)
显微镜(蔡司,型号:Stemi 2000 C)

软件

GraphP ad Prism v6(GraphPad 软件)
ImageJ的(由开发该研究所Š健康)

程序

制备60 -毫米NGM高原ES为所述维护的秀丽隐杆线虫。在无菌条件下,在细菌罩中用大肠杆菌OP50(作为秀丽隐杆线虫的食物)接种平板,并使其在 37°C 下生长过夜。
通过漂白同步线虫菌株进行分析。收集的从用1ml M9溶液中的NGM板动物在2 -毫升微量离心管(MCT)。添加400 μ以中等速度向管和涡流5分钟:升新鲜制备的漂白剂溶液(1比5 N氢氧化钠和次氯酸钠的1)。在6 , 097旋转 MCT× g ( 40 00 rpm ) 60秒,弃上清。W¯¯灰60上的用新鲜的M9再次沉淀和板-毫米NGM板。
为了进行该测定,制备新鲜60 -在4℃下直到使用含有所述测定和存储的前一天8毫升媒体毫米(NGM)板。
上的天的测定中,干燥3在层流气流将板- 4小时用的盖子打开。
扩展400毫米(196 μ升)100%乙醇上的博士ÿ 60 -与使用玻璃吊具8毫升媒体毫米的板。博士ÿ (不含乙醇板- EtOH中)被用作对照板。
密封吨用parafilm他EtOH中测定板,以避免乙醇和任何损失的地方,在20℃进行2 ħ以允许EtOH中横跨板平衡。
EtOH 检测板现已准备就绪,可用于检测。
转移10只动物的基因型从正在研究接种的板(+食品),以非种子选手板(-食品)15 - 20秒(摆脱食物的)。接下来,将动物转移到测定板或对照板,并在 20°C 下静置2小时。
观察线虫2后- ħ时间间隔。计数平板上的动物的数量,并使1 -分钟的视频的每个动物观察和分析通过计算运动器官行为的身体弯曲的数目(前和后)和振幅来表征的行为。
身体弯曲的数量是使用 ImageJ 手动评分的,同时在分析前后身体弯曲时保持相同的参数(图 1A)。
计算所述的振幅的手动使用NIH ImageJ软件,身体弯曲孔定量达分别荷兰国际集团的前部和后部主体弯曲。到孔定量泰特的振幅的身体弯曲,米easure主体弯曲的最深角度之间的距离。画一条从头部尖端到身体中部的切线,并测量从该线到身体的垂直距离作为前身体弯曲的幅度(图 1B)。相应地,从中间部分到所述动物的尾部,是为后体弯曲振幅(图1B)获得的测量结果。归一化的测量到的动物在微米至导出测量值的长度。
 
图标描述已自动生成
图1 。形象代表ü的sed对于该分析的身体弯曲的幅度。A.图像分别代表前体弯曲和后体弯曲的评估。B.图像表示身体弯曲的幅度;双面箭头描绘š的中前部和后部区域振幅(AMP)。

执行基于时间轴的分析B的ehavior。从EtOH博览会开始URE ,不同的时间点(30,60,120,300分钟),包括曝光过夜(16小时)是analyz ED(图2A)。

图标描述已自动生成
图2 。EtOH 处理秀丽隐杆线虫以研究 EIS 行为的时间表插图。A.时间点表示 WT 动物在 EtOH 暴露后的不同阶段,如时间线所示。测试身体弯曲的数量和幅度的实验在约 2 小时进行。B.的EIS行为插图,其中WT动物从EtOH恢复-诱导的麻痹和显示正常的正弦运动,而DOP-2突变体动物显示出乙醇-诱导的镇静行为(潘迪等人,2021)。

WT动物在乙醇麻痹后2 小时表现出正常行为(视频 1)。尽管多巴胺自身受体,DOP -2 ,突变体动物显示的EtOH -i EP3受体激动剂诱导小号edative(EIS)在同一时间点(图2B和视频2)的行为。

图形用户界面描述已自动生成
视频 1 . 代表性的六DEO的WT动物表示单线虫已经从EtOH中回收-诱导的麻痹。所述动物可以看出,在正常的正弦波运动-状图案,与没有可观察到在运动缺陷(潘迪等人,2021)。

图形用户界面,应用程序描述已自动生成
视频2 。代表性的视频的由示出的EIS行为DOP-2突变体线虫在2在EtOH中曝光- h时相点。该动物表现出运动缺陷,并通过拖动其后部区域来移动(Pandey等人,2021 年)。

从EIS行为恢复可以观察到在后转移突变体动物与正常食物NGM板一个2 -小时暴露于EtOH中。dop-2突变体在转移后1小时从 EIS 表型中恢复。

数据分析

分别计算在1前部和后部的身体弯曲数-在2分钟视频- ħ时间点。
软件:GraphPad Prism v6。
转移10只动物每块测定板,并执行的一式三份实验每个菌株。
统计分析:使用单向方差分析来确定统计P 值。

笔记

干燥所述NGM板完全是即时通讯portant ,和冲浪的ACE的板应该是免费从任何灰尘或用于适当可视化的其他表面像差的轨道。
用封口膜密封板以避免乙醇损失和适当的平衡。

食谱

5 毫克/毫升胆固醇
加入500毫克的胆固醇至百毫升95%乙醇,并通过在室温下混合旋转为几个小时以溶解。
储存在 4°C。
1 M CaCl 2原液
将 14.7 g CaCl 2 · 2H 2 O溶解在 100 ml ddH 2 O 中,并在 121°C 下高压灭菌 15 分钟。
储存在 4°C。
1 M MgSO 4储备溶液
将 12.04 g MgSO 4溶解在 100 ml ddH 2 O 中,并在 121°C 下高压灭菌 15 分钟。
储存在 4°C。
1 M KPO 4 ,pH 6.0 储备溶液
将 108.3 g KH 2 PO 4和 35.6 g K 2 HPO 4 混合在 500 ml ddH 2 O 中。
加入 NaOH 调节 pH 至 6.0 ;最后补到1升。
分装溶液并在 121°C 下高压灭菌 15 分钟。
储存在 4 °C。
线虫生长培养基 (NGM) 琼脂平板
加入3克氯化钠16克细菌用-琼脂和2.5g细菌用-peptone到975毫升的DDH 2 ○在2 -大号烧瓶中。
在 121°C 下高压灭菌 50 分钟。
允许的NGM琼脂到冷却到55 - 60℃。添加 1 ml 5 mg/ml 胆固醇、1 ml 1 M CaCl 2 、1 ml 1 M MgSO 4和 25 ml 1 M KPO 4 ,pH 6.0。
400mM的È THANOL
传播196毫升乙醇8 ml培养基在60 -毫米板。
密封用石蜡膜板和允许equilibrat离子在20℃。

致谢

该协议改编自 (Pandey et al. , 2021)。AS感谢该协会科学和工业研究委员会(CSIR)的-大学教育资助委员会(教资会)的研究生奖学金。PP从科学与技术(DST)的一个部门承认的支持-口碑Ë n个科学(WOS-A)授予,以及从过去的资金与生物技术(DBT)生物系护理,在科学教育和研究的印度理工学院(IISER) Mohali ,以及授予 KB 的 IA 赠款。KB 是印度联盟 (IA) 的中级研究员,目前是 IA 高级研究员。KB 感谢联盟提供资金支持。KB也感谢DBT,DST -科学与工程研究理事会(SERB) ,以及人力资源开发部(MHRD)-方案转型和高级研究在科学(STARS)的资金支持。

资金
这个工作是由DBT /支持威康信托印度联盟研究金[资助号IA / S / 19 /504649分之2和IA / I / 12 /五十万○五百十六分之一]授予KB和部分地由DBT,MHRD-STARS支持,并DST-SERB 向 KB 授予 [BT/PR24038/BRB/10/1693/2018、STARS/APR2019/BS/454/FS 和 SERB/F/7047] 以及 DBT-IISc 合作伙伴关系资助。PP是由DST WOS-A基金的支持[SR / WOS-A / LS-二千〇一十八分之二百八十五]和由DBT BIOCARE较早支持准予[ BIOCARE / 01/10167]。

利益争夺

在一个uthors声明没有冲突小号的利益或竞争利益小号。

参考

白克,JH(2013 年)。奖励相关行为中的多巴胺信号。前神经电路7:152。
Chase, DL, Pepper, JS 和Koelle , MR (2004)。秀丽隐杆线虫突触外多巴胺信号传导机制。Nat Neurosci 7(10): 1096-1103。
Davies, AG, Pierce-Shimomura, JT, Kim, H., VanHoven , MK, Thiele, TR, Bonci , A., Bargmann , CI 和 McIntire, SL (2003)。BK 钾通道在秀丽隐杆线虫乙醇行为反应中的核心作用。单元格115(6):655-666。              
Davies, AG 和 McIntire, SL (2004)。使用秀丽隐杆线虫筛选乙醇和改变行为的药物的目标。Biol Proced Online 6:113-119。
Imperato , A. 和 Di Chiara, G. (1986)。乙醇优先刺激自由运动大鼠伏隔核中的多巴胺释放。J Pharmacol Exp Ther 239(1): 219-228。              
Lee, J., Jee , C. 和 McIntire, SL (2009)。C. elegans 中的乙醇偏好。基因脑Behav 8(6):578-585。
Pandey, P.、Singh, A.、Kaur, H.、Ghosh-Roy, A. 和 Babu, K.(2021 年)。增加的多巴胺能神经传递导致秀丽隐杆线虫的乙醇依赖性镇静行为。PLoS基因17(2):e1009346。
Weiss, F., Parsons, LH, Schulteis , G., Hyytia , P., Lorang , MT, Bloom, FE 和Koob , GF (1996)。乙醇自我给药可恢复依赖大鼠中累积多巴胺和 5-羟色胺释放的戒断相关缺陷。J Neurosci 16(10):3474-3485。
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引用:Singh, A., Babu, K. and Pandey, P. (2021). Ethanol-induced Sedative Behavior: An Assay to Investigate Increased Dopamine Signaling in Caenorhabditis elegans. Bio-protocol 11(13): e4083. DOI: 10.21769/BioProtoc.4083.
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