JoVE Journal > Neuroscience
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Neuroscience

该平衡翼：管理对酒精苍蝇

Published: May 18, 2014
doi:
10.3791/50442
, , ,
1Department of Biological Science,Florida State University, 2Department of Biology and Biochemistry, Biology of Behavior Institute,University of Houston

Summary

果蝇已经成为解剖的行为反应，以酒精的细胞和分子基础的一个显著的模型系统。在这里，我们提出了一个协议，用于酒精的敏感性数据的，可以很容易地应用到其他的实验，是非常适合本科生科研昼夜上下文的集合。

Abstract

果蝇（ 果蝇 ）是一种成熟的模式为酒精的研究和昼夜生物。最近，我们发现，生物钟调节酒精的敏感性，但不能耐受的形成。在这里，我们详细描述了我们的协议。酒精是给予使用平衡翼的苍蝇。在这种设置中，饱和酒精蒸气与加湿空气混合比例设置，并给予苍蝇在同时四根管子。苍蝇是为了尽量减少重复之间的差异在标准条件下饲养。三日龄果蝇不同的基因型或治疗方法，所用的实验中，优选通过匹配两个不同的时间点（ 例如 ，CT 5和CT 17）苍蝇进行直接比较成为可能。在实验过程中，苍蝇暴露1小时，以酒精蒸气的预定百分比和苍蝇的数量表现出翻正反射消失（LORR）的损失或sed的振动性进行计数，每5分钟。中的数据可以使用三种不同的统计方法来分析。首先是要确定在哪个苍蝇的50％已失去翻正反射时，使用方差分析（ANOVA）来确定的时间点之间是否存在显著差异。第二个是确定的百分比苍蝇指定的分钟数，后跟一个单因素方差分析后显示翻正反射消失。最后一种方法是分析整个时间序列采用多元统计。该协议也可以用于基因型之间的非生理实验或比较。

Introduction

果蝇表现出双相的行为反应，以酒精1是类似于人类的反应，此药2,3。在初始接触低浓度的酒精，苍蝇展览增加自发活动，由于缺乏运动协调，姿势控制的损失和扶正反射（翻正反射消失的：翻正反射消失）取代，镇静（完全没有响应运动活动机械性刺激）作为暴露于酒精的进展4-9。内源性生物钟是一个强大酒精的敏感性和毒性的调制器在10,11小鼠，大鼠12观察，和人类13。在果蝇研究的最新进展表明，生物钟调节急性酒精的敏感性，但没有酒精的耐受1。通过突变体的研究和空间的转基因操作的强有力的遗传可在果蝇方法和时间上的基因表达提供了一个系统，允许在识别复杂行为的基本细胞和分子机制进展迅速。利用果蝇作为调查工具已经允许在了解酒精神经生物学实质性的进步，可以迅速转化为哺乳动物14-16。为了方便，通过该生物钟调节酒精的敏感性的分子机制的理解，并均匀地测量整个昼夜时间点的行为反应，适于在昏暗的红光条件下使用醇的给药方案是必需的。对于果蝇 ，酒精可通过食物补充对慢性接触或通过可靠的蒸汽急性暴露的形式，行政的酒精管理。在这里，我们描述了适用于失的扶正反射（翻正反射消失）1昼夜调制的评估，以及酒精管理协议镇静。

蝇夹带有12小时：12小时LD周期在恒定的温度和然后转移到控制光制度为2-5天取决于实验的问题。蝇中被称为平衡杆的装置暴露于乙醇蒸气。在该装置中，控制量的空气通过水和醇鼓泡;蒸气，然后混合并定向到小瓶外壳的苍蝇。每5苍蝇分钟被打进了失败，显示扶正反射或已成为镇静的数量。 LORR百分比为每个时间点计算并比较了昼夜时间点或苍蝇的菌株之间。酒精交付使用平衡翼酒精交付并结合行为分析选项的简便性和可靠性提供了黑暗的条件下进行昼夜实验一个显著的好处。

Protocol

在平衡翼1。大会基本原理和概况：该系统的设计管理酒精蒸气的控制百分比苍蝇。注意： 图1提供了一示意性的概述的平衡杆设置为三个阶段（空气流的组件，设置在醇和水的瓶子，并观察小瓶组件）下面描述。总之，一个稳定的气流被分成通过酒精和水，鼓泡分别混合，给药于4观察小瓶两个馏分。 气流大会连接一段简短的弹性硅胶管连接到无论?…

Representative Results

酒精的敏感性，使用的50％翻正反射消失为标志的昼夜调制。 一个典型的例子显示，白天昼夜调制在酒精的敏感性示于图2。LORR在进行DD在广州-S中的第 2天测量在六个时间点，50％翻正反射消失为每个时间点确定。分析表明，一天中的时间显著作用（ANOVA：F 5,45 = 7.39，P <0.001，N = 6-10每个时间点）。费雪LSD检验表明CT1之间的显著差异与CT5，C…

Discussion

酒精滥用和酒精中毒的社会成本是巨大的，无论是在人类29和经济成本方面30,31。 果蝇为模型提供了一个快速和灵活的系统可快速检查了大量个人的行为反应，因此已被广泛地用于这两种醇5,7,32-34和昼夜节律研究35-37。

在这里，我们描述了一个简单的协议，用于酒精蒸汽成蝇昼夜条件下的控制管理。

苍蝇在标准条件…

Disclosures

The authors have nothing to disclose.

Acknowledgements

资助这项研究是由一个程序从佛罗里达州立大学医学院和生物科学系于前苏联的支持提供了神经科学奖。额外的经费是由格兰特在急救从酒精饮料制造商的研究基金提供的。

Materials

Alcohol 190 proof Various
Name of Material Company Catalog Number Comments
Aerator Local pet store We use Whisper 60
Silicone tubing 1/8” VWR 408060-0030
120° Y Connector VWR 82017-256
Quick disconnects VWR 46600-048
Plastic tube clamps Bell-art products 132250000 Either this or next
Miniature Air Regulator McMaster-Carr 8727K11 Either this or previous
Miniature Air Regulator Mounting Bracket McMaster-Carr 9891K66
Gilmont size 12 flow meter VWR 29895-242
Tool clips McMaster-Carr 1722A43 To hold flow meters
Vial VWR 89092-722
Rubber stopper with two holes VWR 59585-186 Fits in vials
5 mm Pyrex Glass tubes Trikinetics PGT5x65 Fits best in previous stopper.
Teflon tape Hardware store To achieve snug fit in stoppers if necessary
Rubber stopper with two holes VWR 59582-122 Fits our bottles
Disposable glass pipets VWR 53283-768 Cut to length and bend by heating
Very fine nylon netting VWR Various
15 watt bulbs Hardware store Overhead red light
Photographic red safe light filters Overhead red light
Mini Flashlights with red filters Mag-light
DOWNLOAD MATERIALS LIST

References

  1. Linde, K., Lyons, L. C. Circadian modulation of acute alcohol sensitivity but not acute tolerance in Drosophila. Chronobiol. Int. 28, 397-406 (2011).
  2. Kaun, K. R., Azanchi, R., Maung, Z., Hirsh, J., Heberlein, U. A Drosophila model for alcohol reward. Nat Neurosci. 14, 612-619 (2011).
  3. Shohat-Ophir, G., Kaun, K. R., Azanchi, R., Mohammed, H., Heberlein, U. Sexual deprivation increases ethanol intake in Drosophila. Science. 335, 1351-1355 (2012).
  4. Bellen, H. J. The fruit fly: A model organism to study the genetics of alcohol abuse and addiction. Cell. 93, 909-912 (1998).
  5. Guarnieri, D. J., Heberlein, U. Drosophila melanogaster, a genetic model system for alcohol research. International Review of Neurobiology. 54, 203-232 (2003).
  6. Scholz, H. Intoxicated fly brains: Neurons mediating ethanol-induced behaviors. J. Neurogenet. 23, 111-119 (2009).
  7. Wolf, F. W., Rodan, A. R., Tsai, L. T. Y., Heberlein, U. High-resolution analysis of ethanol-induced locomotor stimulation in Drosophila. J. Neurosci. 22, 11035-11044 (2002).
  8. Schumann, G., Spanagel, R., Mann, K. Candidate genes for alcohol dependence: Animal studies. Alcoholism: Clinical and Experimental Research. 27, 880-888 (2003).
  9. Singh, C. M., Heberlein, U. Genetic control of acute ethanol-induced behaviors in Drosophila. Alcohol Clin Exp Res. 24, 1127-1136 (2000).
  10. Perreau-Lenz, S., Zghoul, T., de Fonseca, F. R., Spanagel, R., Bilbao, A. Circadian regulation of central ethanol sensitivity by the mPer2 gene. Addiction Biology. 14, 253-259 (2009).
  11. Brager, A. J., Prosser, R. A., Glass, J. D. Circadian and acamprosate modulation of elevated ethanol drinking in mPer2 clock gene mutant mice. Chronobiol. Int. 28, 664-672 (2011).
  12. Sinclair, J. D., Geller, I. Ethanol consumption by rats under different lighting conditions. Science. 175, 1143-1144 (1972).
  13. Danel, T., Jeanson, R., Touitou, Y. Temporal pattern in consumption of the first drink of the day in alcohol-dependent persons. Chronobiol. Int. 20, 1093-1102 (2003).
  14. Kapfhamer, D., et al. Taok2 controls behavioral response to ethanol in mice. Genes, brain, and behavior. 12 (1), 87-97 (2012).
  15. Lasek, A. W., et al. An evolutionary conserved role for anaplastic lymphoma kinase in behavioral responses to ethanol. PLoS One. 6, 226-236 (2011).
  16. Lasek, A. W., Giorgetti, F., Berger, K. H., Tayor, S., Heberlein, U. Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse. Alcohol Clin Exp Res. 35, 1600-1606 (2011).
  17. Lyons, L. C., Roman, G. Circadian modulation of short-term memory in Drosophila. Learning & Memory. 16, 19-27 (2009).
  18. Hamblen-Coyle, M. J., Wheeler, D. A., Rutila, J. E., Rosbash, M., Hall, J. C. Behavior of period-altered circadian-rhythm mutants of Drosophila in ligh-dark cycles (Diptera Drosophilidae). J. Insect Behav. 5, 417-446 (1992).
  19. Konopka, R. J., Pittendrigh, C., Orr, D. Reciprocal behavior associated with altered homeostasis and photosensitivity of Drosophila clock mutants. J. Neurogenet. 6, 1-10 (1989).
  20. Power, J. M., Ringo, J. M., Dowse, H. B. The effects of period mutations and light on the activity rhythms of Drosophila melanogaster. Journal of Biological Rhythms. 10, 267-280 (1995).
  21. Yoshii, T., et al. Temperature cycles drive Drosophila circadian oscillation in constant light that otherwise induces behavioural arrhythmicity. Eur. J. Neurosci. 22, 1176-1184 (2005).
  22. Berger, K. H., Heberlein, U., Moore, M. S. Rapid and chronic: two distinct forms of ethanol tolerance in Drosophila. Alcohol Clin Exp Res. 28, 1469-1480 (2004).
  23. Scholz, H., Ramond, J., Singh, C. M., Heberlein, U. Functional ethanol tolerance in Drosophila. Neuron. 28, 261-271 (2000).
  24. Kong, E. C., et al. Ethanol-regulated genes that contribute to ethanol sensitivity and rapid tolerance in Drosophila. Alcohol Clin Exp Res. 34, 302-316 (2010).
  25. Borycz, J., Borycz, J., Kubow, A., Lloyd, V., Meinertzhagen, I. Drosophila ABC transporter mutants white, brown and scarlet have altered contents and distribution of biogenic amines in the brain. J. Exp. Biol. 211, 3454-3466 (2008).
  26. Sitaraman, D., et al. Serotonin is necessary for place memory in Drosophila. Proceedings of the National Academy of Sciences. 105, 5579-5584 (2008).
  27. Bainton, R. J., et al. Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila. Current Biology. 10, 187-194 (2000).
  28. Kong, E. C., et al. A pair of dopamine neurons target the D1-like dopamine receptor DopR in the central complex to promote ethanol-stimulated locomotion in Drosophila. Plos One. 5, (2010).
  29. Xu, J., Kochanek, K. D., Murphy, S. L., Tejada-Vera, B. . Deaths: Final data for 2007. , (2010).
  30. . The National Center on Addiction and Substance Abuse. Shoveling up II: The impact of substance abuse on federal, state and local budgets. , (2009).
  31. NIAAA, Estimated economic costs of alcohol abuse in the United States. , (1992).
  32. Devineni, A. V., Heberlein, U. Preferential ethanol consumption in Drosophila models features of addiction. Current Biology. 19, 2126-2132 (2009).
  33. Devineni, A. V., Heberlein, U. Addiction-like behavior in Drosophila. Communicative & Integrative Biology. 3, 357-359 (2010).
  34. Rodan, A. R., Rothenfluh, A. The genetics of behavioral alcohol responses in Drosophila. International Review of Neurobiology. 91, 25-51 (2010).
  35. Boothroyd, C. E., Young, M. W., Pfaff, D. W., Kieffer, B. Molecular and Biophysical Mechanisms of Arousal, Alertness, and Attention. Annals of the New York Academy of Sciences. 1129, 350-357 (2008).
  36. Nitabach, M. N., Taghert, P. H. Organization of the Drosophila circadian control circuit. Current Biology. 18, 84-93 (2008).
  37. Sheeba, V. The Drosophila melanogaster circadian pacemaker circuit. J. Genet. 87, 485-493 (2008).
  38. Cohan, F. M., Graf, J. -. D. Latitudinal cline in Drosophila melanogaster for knockdown resistance to ethanol fumes and for rates of response to selection for further resistance. Evolution. , 278-293 (1985).
  39. Moore, M. S., et al. Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway. Cell. 93, 997-1007 (1998).
  40. Berger, K. H., et al. Ethanol sensitivity and tolerance in long-term memory mutants of Drosophila melanogaster. Alcohol Clin Exp Res. 32, 895-908 (2008).
  41. Pohl, J. B., et al. Circadian Genes Differentially Affect Tolerance to Ethanol. in Drosophila. Alcoholism: Clinical and Experimental Research. , (2013).
  42. Bhandari, P., Kendler, K. S., Bettinger, J. C., Davies, A. G., Grotewiel, M. An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance. Alcoholism: Clinical and Experimental Research. 33, 1794-1805 (2009).
  43. Rothenfluh, A., et al. Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms. Cell. 127, (1016).

Tags

Fruit FliesDrosophila MelanogasterAlcohol ResearchCircadian BiologyAlcohol SensitivityAlcohol ToleranceFlyBarAlcohol VaporLoss Of Righting ReflexSedationStatistical Analysis

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
van der Linde, K., Fumagalli, E., Roman, G., Lyons, L. C. The FlyBar: Administering Alcohol to Flies. J. Vis. Exp. (87), e50442, doi:10.3791/50442 (2014).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image