该平衡翼：管理对酒精苍蝇
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 |
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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).