毛细管馈线分析测量的摄食<em>果蝇</em
Summary
The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.
Abstract
For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.
Introduction
饮食是必不可少的;然而，导致进食障碍如食欲过盛，厌食或暴饮暴食强加的一般倾向的食物摄取的失调对个人和社会1，2，3，收费。目前研究的目的是揭开食物摄入量的监管机制，并提供规避障碍形成的战略。使用哺乳动物模型生物体许多研究在进食障碍4，5，6中提供的电路和信号系统的作用的新的见解。然而，我们的这些疾病背后的神经细胞和分子基础知识没有完成仍远。在最近几年，果蝇果蝇已成为解开基本机械分析上市metabolis的调节中有价值的模型系统M 7，8，9。对果蝇毛细管进纸器（CAFE）法成立于西摩·本泽在2007年的绿头苍蝇10,11灵感来自早期的工作由Dethier实验室。网吧分析使我们能够直接测量果蝇食物的摄入量。在这种行为测试系统，苍蝇喂上放在瓶内分级玻璃毛细管提供流质食物。毛细管半月板的衰落表明通过蒸发和食品消费的食品解决方案的损失。确定由小瓶蒸发率没有苍蝇允许摄入食物的准确定量。
网吧测定是用来测量果蝇喂养几个行为范式之一，研究人员选择最合适他们的具体题。使用特定试验应考虑以下几点决定：提供食物的性质;饲养条件;摄取或营养物的摄取和调查食物消耗或响应于食物的测量。
本报告中所描述的CAFE法是理想的直立饲养条件下以下的液体食物来源的食物的摄入量。可替代地，食物摄入量可以为苍蝇组来测量在小瓶或在平板上的着色的食物来源。蝇通常杀死或麻醉后喂养和摄取的染料的量由法或染色腹部的目测来确定。蝇开始排泄摄取食物摄入后仅30分钟，因此这种方法很难用于连续长馈送的分析行为12,13。
相比之下蝇保持完整时吸收染料s的放射性示踪剂的使用和它们的放射性同位素的消费在闪烁计数器14，15被评分。由飞消化系统的放射性标记的吸收，使长期摄取食物测量可能的，但可能会导致因非吸收而排出体外示踪分子的消费被低估。另一种方法来测量响应果蝇的食物是黑的长鼻扩展响应（PER），它通常出现在食物中摄取16。这间优雅的方法措施，食物刺激的初步反应，但不记录的摄入量。食物摄入量是使用用于馈送17，18的规临界几个后消化反馈信号馈送期间动态调整。多次尝试在最近几年已经进行了半自动化数据集合中的每个试验<s补课=“外部参照”> 19，20。该PER由电垫或电极的组合检测到的，并通过计算机计算。结合PER检测放射性同位素的摄取透露，这个实验是由低灵敏度限于检测量喂养差异18。手动进测定（MAFE）21，在其中一个苍蝇用玻璃毛细管手动馈送，最近开发用于测量在单一固定的飞食品摄取。该MAFE法消除觅食和进食引发的干扰，有几秒钟的时间分辨率，PER的启动和食品消费可以独立在试验进行监控。然而，其中一路飞的固定化的影响摄食行为的某些方面（ 如运动，动机）仍然需要进行调查。对于不同的检测方法用于测量果蝇我食品消费出色的横向评测lanogaster并帮助研究人员找到最合适的，看到德什潘德和马克思报告 13，22。
网吧法避免了一些上述其它测定法的缺点，并结合简单易用的食物的摄入量能够可靠地计量。这里，提供了CAFE测定的详细说明，我们示出了简单的设置的修改，以减少蒸发。代表性的成果包括两个食物选择实验（短期和长期）和苍蝇的蔗糖的吸收是证明。在讨论中，我们比较我们用其他的方法来执行的CAFE法，并突出显示潜在的局限性所描述的方法。
Protocol
Representative Results
Discussion
该报告描述了一步一步的时尚CAFE法，着眼于技术设置并在实验室的成功表现。由于其简单，该测定也可以教育上用作校实验。该实施例表明该测定允许食物感测，在短和更长的时间周期（数小时至数天）的偏好和消费在果蝇的调查。网吧法已在该领域广泛使用的调查对象包括食品和药品消费，吸毒，能量平衡和喂养16神经元控制<sup cl…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank the past and present members of the Scholz lab for discussion and Helga Döring for excellent technical support. We especially thank the members of the Biocenter workshop of the University of Cologne for their support and creativity. The work is supported by SFB 1340, SysMedAlc, and DAAD-Siemens.
Materials
| Vials (breeding) | Greiner Bio-One | 960177 | www.greinerbioone.com |
| Vials (CAFE assay) | Greiner Bio-One | 217101 | www.greinerbioone.com |
| Lid-CAFE assay | Workshop | – | – |
| Plastic box, low wall | Plastime | 353 | www.plastime.it |
| Cover for the plastic box | Workshop | – | – |
| Capillaries | BLAUBRAND | REF 7087 07 | www.brand.de |
| Pipette tips | Greiner Bio-One | 771290 | www.greinerbioone.com |
| Filter paper circles | Whatman | 10 311 804 | www.sigmaaldrich.com |
| D(+)-Sucrose | AppliChem | 57-50-1 | www.applichem.com |
| Ethanol absolute | VWR Chemicals | 20,821,330 | www.vwr.com |
| Food color (red, E124) | Backfun | 10027 | www.backfun.de |
| Food color (blue, E133) | Backfun | 10030 | www.backfun.de |
| Soap solution (CVK 8) | CVH | 103220 | www.cvh.de |
| Digital caliper | GARANT | 412,616 | www.hoffmann-group.com |
| Vials (breeding) | Height 9.8 cm, diameter 4.8 cm | ||
| Vials (CAFE assay) | Height 8 cm, diameter 3.3 cm | ||
| Lid-CAFE assay | Produced in university workshop, technical drawing supplied | ||
| Plastic box, low wall | A plastic grid inlay was custom-made for 8 x 10 vial positions | ||
| Cover for the plastic box | Dimensions (37 x29 x18 cm) | ||
| Capillaries | DIN ISO 7550 norm, IVD-guideline 98/79 EG, ends polished | ||
| Pipette tips | Pipettes for the outer circle are cut according to the lid | ||
| Filter paper circles | 45 mm diameter works nicely if folded for the vials used | ||
| D(+)-Sucrose | Not harmful | ||
| Ethanol absolute | Highly flammable liquid and vapor | ||
| Food color (red, E124) | Not stated | ||
| Food color (blue, E133) | Not stated | ||
| Soap solution (CVK 8) | Odor neutral soap | ||
| Digital caliper | |||
| Standard fly food | (for 20 L) | ||
| Agar | 160 g | ||
| Brewer`s Yeast | 299.33 g | ||
| Cornmeal | 1200g | ||
| Molasses | 1.6 L | ||
| Propionic acid | 57.3 mL | ||
| Nipagin 30% | 160 mL |
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