一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受<em>果蝇</em
Summary
Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.
Abstract
酒精使用障碍（AUD）是一个严重的健康挑战。尽管大量的遗传成分澳元，一些基因已被明确地牵连的病因。果蝇， 果蝇 ，是一个功能强大的模式探索潜在的酒精有关的行为的分子遗传机制，因此保持着识别和理解基因影响澳元的作用巨大潜力。利用果蝇模型对这些类型的研究依赖于测定中，能可靠地测量的行为反应，以乙醇的可用性。这份报告介绍了适用于评估乙醇的灵敏度和快速耐受在果蝇的实验。在该测定中测得的乙醇灵敏度由体积和乙醇浓度使用，各种先前报道遗传操作，并且时间还长的影响苍蝇收容没有食物之前立即测试。与此相反，乙醇sensitivity在此测定法测量不受苍蝇处理，苍蝇的性别，并补充以抗生素或活酵母生长培养基的活力。三种不同的方法来定量乙醇灵敏度描述，所有领先的基本上没有区别乙醇的敏感性结果。该测定的可伸缩的性质，结合其总体简单性设置和相对低的费用，使其适合于乙醇的灵敏度和快速耐受在果蝇的小和大规模基因分析。
Introduction
酒精使用障碍（AUD）是一个巨大的健康问题的全球（1回顾）。虽然驱动澳元的发展机制是复杂的，这些疾病有重要的遗传成分（ 例如，2）。元和保守的行为反应的大遗传跨越许多物种（在3,4审查）已经产生在利用遗传模式生物，调查在乙醇相关的行为的特定基因的朝向更好地理解的分子基础的介入的强烈兴趣乙醇澳元。果蝇， 果蝇 ，已成为一个主要模式生物研究乙醇相关行为的分子遗传机制（3,4审查）。研究苍蝇突出角色的几个信号通路的行为反应，以乙醇（5审查）。有趣的是，某些基因和通路的影响行为respons下载到乙醇中蝇也被牵连在啮齿类乙醇相关的行为和/或人AUD（ 例如，6-14）。机制的保护驾驶乙醇相关的行为跨物种，加上在果蝇模型系统中可用的遗传工具套件，强调果蝇模型的效用调查的行为反应的遗传乙醇。
灵敏度15,16和公差（在17中综述）在人中乙醇被链接到元的发展。这两种行为反应的乙醇可在果蝇经由各种实验室测定法（在3,4综述）进行建模。所有已知的作者飞测定法是基于从乙醇镇静要么依赖于时间的乙醇诱导镇静/共济失调或依赖于时间的恢复。
从我们组对乙醇的灵敏度和r的遗传学以前的文章在果蝇 APID宽容的基础上，苍蝇乙醇蒸气引起镇静一个行为实验检测18。试验在该试验中被转印活成人发起蝇无需麻醉清空食品小瓶，诱捕苍蝇与醋酸纤维素插塞的小瓶中，加入乙醇到乙酸纤维素插塞的顶部（ 即，非飞边），并且密封含有苍蝇，醋酸纤维素插塞和乙醇用硅酮塞子的小瓶（参见示意图图S3中 ，参考18）。代表不同组的苍蝇的多个小瓶评估平行，提高吞吐量该测定的。小瓶给一个匿名代码和实验者被蒙蔽治疗组，以防止意外的偏见镇静的评估。在标准实验中，苍蝇在小瓶轻轻敲打以6分钟的间隔，并在30秒后恢复，镇静剂苍蝇在每个小瓶中的数目进行计数并转换器具d可％活性苍蝇。苍蝇吸收从醋酸纤维素插塞乙醇蒸汽以时间依赖的方式，导致逐步增加内部乙醇18和镇静（比照参考18和图1A和1B在本报告中）。镇静在此测定法操作上定义为果蝇（ⅰ）站立在没有步行或（ii）具有或不具有拍打翅膀躺在背上的。这里，这乙醇镇静测定进行详细说明，有关使用它进一步操作优化设置，并测定用于寻址的食物补充剂的选项上飞镇静灵敏度的贡献。
Protocol
Representative Results
Discussion
简单的实验是重复定量有意义的表型是对行为的分析具有重要价值。这里所描述的工作涉及测量乙醇镇静敏感和快速耐受果蝇的实验的几个实际问题。虽然这项工作不是一个焦点，行为分析是通过维持对环境和遗传背景常数为测试对象的研究中变得容易。此外，比较通常应苍蝇饲养和测试并排侧的组之间进行比较。为此，从个人实验之内的所有的苍蝇在这项工作中有相同的遗传背景和均匀?…
Declarações
The authors have nothing to disclose.
Acknowledgements
These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse to M.G. (P20AA017828, R01AA020634, P50 AA022537). The authors thank Jill Bettinger for helpful discussions and Jacqueline DeLoyht for technical assistance.
Materials
| food vials | VWR | 89092-772 | narrow |
| Flugs | Genesee/flystuff.com | 49-102 | narrow |
| silicone stopper | Fisher Scientific | 09-704-1l | #4 |
| ethanol | Pharmaco-Aaper | 111000200 | 200 proof |
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