在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲
Summary
通过染色食品进行定量的食品摄入量测定, 为评价饲用动机提供了一种强有力的、高吞吐量的方法。将食品消费检测与 thermogenetic 和 optogenetic 筛结合起来, 是研究成人果蝇的食欲的神经回路的有力方法.
Abstract
食品消费受到大脑的严密控制, 它整合了食物的生理状态、适口性和营养成分, 并发出启动或停止进食的命令。破译及时和适度喂养决策的过程, 对我们理解与喂养控制有关的生理和心理障碍有重大影响。在实验操作后, 需要简单、定量和健壮的方法来测量动物的食物摄取量, 例如强行增加某些靶神经元的活性。在这里, 我们介绍了基于染料标记的饲料化验, 以促进遗传性研究的饲养控制在成年果蝇。我们回顾了现有的喂养方法, 然后从设置到分析, 逐步描述我们的方式, 这结合 thermogenetic 和 optogenetic 操作的神经元控制喂养动机与染料标签食品摄入量测定。我们还讨论了我们的方法的优势和局限性, 与其他喂养化验相比, 以帮助读者选择适当的化验。
Introduction
量化摄入的食物量对于评估大脑在响应内部需求 (如饥饿状态) 和外部因素 (如食品质量和适口性) 方面的多种喂养控制方面非常重要1,2,3,4,5,6,7,8,9. 近年来, 在果蝇中破译喂养控制的神经基质的努力导致了多种化验的发展, 直接量化摄取的食物量或作为喂养动机的指标。10,11,12,13,14,15,16。
毛细管进纸器 (咖啡馆) 检测12,13被开发用来测量玻璃 microcapillary 中液态食物的消耗量。咖啡馆化验是高度敏感和可重复的17 , 并简化了食品消耗量的测量, 特别是用于定量长期喂养18。然而, 这种化验要求苍蝇爬上 microcapillary 的尖端和饲料倒置, 这是不适合所有菌株。此外, 由于使用咖啡馆化验试验的苍蝇必须饲养在液态食品上, 这些饲养条件对新陈代谢状况或潜在营养不良的影响仍有待确定。
喙扩展响应 (每) 检测11,14计数的喙延伸反应的频率, 以温和的接触食物滴。每种化验结果证明, 作为评估个人飞行和驴子喂养动机的一个很好的方法食物适口性和食品含量的影响18,19。然而, 它不是直接定量的摄入量。
最近, 研制了一种半自动方法–人工喂养法 (MAFE)15。在 MAFE, 一只固定的苍蝇用含有食物的 microcapillary 人工喂养。鉴于可同时监测喙的延伸反应和食物消耗量, MAFE 适合于评估营养价值和药理操作的效果。然而, 固定一只苍蝇可能会对其行为表现产生负面影响, 包括进食。
此外, 还开发了飞行喙和活动检测器 (FlyPAD)10 , 以自动量化喂养行为。使用机器视觉方法, FlyPAD 记录苍蝇和食物之间的物理相互作用, 以量化喙延长的频率和持续时间, 作为喂养动机的指示器。FlyPAD 提供了一种高通量的方法来监视自由移动苍蝇的喂养行为, 尽管该系统的灵敏度和鲁棒性仍有待更多研究12进一步确认。
标签策略经常被用来估计苍蝇的食物摄取量。用化学示踪剂标记食物是常见的, 在进食后, 测量摄取的示踪物量来计算食物摄取量。放射性示踪剂16,17,20,21,22,23,24,25允许通过角质层检测不均匀的苍蝇。该方法提供了显著的低变异性和高灵敏度的18, 对食品的长期摄入量的研究是可行的。然而, 在使用这种化验时, 应考虑到可用的放射性同位素和不同的吸光度和排泄率。
标签和追踪食物摄取与无毒的食物颜色是一个更安全和更简单的选择2,3,26,27,28。在食用含有可溶性和不可吸收染料的食物后, 苍蝇是均匀的, 摄取的染料的用量后来用分光光度计3,24,28,29.标签策略易于执行, 并提供了高效率, 但有一个警告。从摄取染料中估计的食物摄入量比实际体积小, 因为在苍蝇开始喂养17之后, 排泄开始早于15分钟。此外, 该检测通常在60分钟内评估食物摄取量, 仅适用于短期喂养行为的调查24,28。此外, 多个内部和外部因素,如基因型17、性别17、交配状态17、饲养密度30、昼夜节律3132和食品质量3,8,16, 影响食物摄取量。因此, 喂养持续时间可能需要根据特定的实验条件进行调整。除了促进食品摄取量的量化, 食品颜色还用于评估食物选择2,19,27, 并可视化的半月板在咖啡馆化验12microcapillary。
在这里, 我们介绍了一个协议的结合操作的神经元活动与染料标记方法。这一策略已被证明是有益的, 在我们的遗传性研究的饲养控制在成年果蝇24。视觉评分法允许快速估计食物消耗量;因此, 它是有用的筛选通过大量的菌株及时的方式。然后用比色法对屏幕上的候选者进行详细分析, 以提供客观和精确的量化。
除了喂食化验, 我们还描述了 thermogenetic27,33,34,35和 optogenetic36在果蝇中强行激活目标神经元的方法。通过 thermogenetic 操作激活神经元是简单和方便的果蝇瞬态受体电位锚蛋白 1 (dTRPA1), 这是一个温度和电压门控阳离子通道, 增加神经元的兴奋性时, 环境温度升高23摄氏度以上33,37;然而, 在高温下测试动物可能会对行为产生不良影响。激活果蝇中神经元的另一种有效方法是使用光遗传学与 CsChrimson 36, 这是 channelrhodopsin 的一个红色移位变体, 它增加了当暴露于光时神经元的兴奋性.光遗传学提供更高的时间分辨率和较小的干扰行为比 thermogenetics。将食物摄取量与神经元活性的调控相结合, 是研究喂养神经机制的有效途径。
我们详细描述了喂养室的准备和要测试的苍蝇。使用Taotie-Gal4作为模型24, 我们描述了通过 thermogenetics 和光遗传学激活神经元。该议定书还介绍了两种用染料标记食品定量测定食品消耗量的试验。
Protocol
Representative Results
Discussion
本报告的重点是在 thermogenetic 和 optogenetic 激活的背景下, 对食品消费进行染料标记喂养测定的技术过程, 以操纵神经元控制喂养。这一简单而可靠的协议将有助于阐明候选神经元在喂养控制中的作用, 测量苍蝇的食物偏好, 并通过喂食的遗传屏幕24识别喂养控制回路中的新玩家。
染料标记策略是研究短期喂养行为的可行方法。染料, erioglaucine 钠, 溶入和摄取的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分是由中国国家基础研究计划 (2012CB825504)、中国国家自然科学基金 (91232720 和 9163210042)、中国科学院 (GJHZ201302 和 QYZDY-SSW-SMC015)、比尔和梅琳达盖茨支持的。基金会 (OPP1119434) 和 100-中国科学院人才培养计划。
Materials
| UAS-CsChrimson | Bloomintoon | 55135 | |
| UAS-dTrpA1 | Bloomintoon | 26263 | |
| TDC1-GAL4 | Bloomintoon | 9312 | |
| TDC2-GAL4 | Bloomintoon | 9313 | |
| sNPF-GAL4 | Provided by Z. Zhao | ||
| NPF-GAL4 | Provided by Y. Rao | ||
| TH-GAL4 | Provided by Y. Rao | ||
| 5-HT-GAL4 | Provided by Y. Rao | ||
| AKH-GAL4 | Provided by Y. Rao | ||
| dip2-GAL4 | Provided by Y. Rao | ||
| Taotie-GAL4 | Provided by J. Carlson | ||
| Agarose | Biowest | G-10 | |
| Sucrose | Sigma | S7903 | |
| Erioglaucine disodium salt | Sigma | 861146 | |
| all-trans-retinal | Sigma | R2500 | stored in darkness |
| Triton X-100 | Amresco | 9002-93-01 | |
| Fly food | 1 L food contains: 77.7 g corn meal, 32.19 g yeast, 5 g agar, 0.726 g CaCl2, 31.62 g sucrose, 63.2 g glucose, 2 g potassium sorbate, pH | ||
| 1x PBS buffer | 1 L 1X PBS contains: 8 g Nacl, 0.2 g Kcl, 1.44 g Na2HPO4, 0.24 g KH2PO4, pH 7.4 | ||
| PBST buffer | 1X PBS with 1% Triton X-100 | ||
| Grinding mill | Shang Hai Jing Xin | Tissuelyser-24 | |
| Incubator | Ning Bo Jiang Nan | HWS-80 | |
| Magnetic stirrer with a heat plate | Chang Zhou Bo Yuan | CJJ 78-1 | |
| Spectrometer | Thorlabs | CCS200/M | |
| Microplate Spectrophotometer | Thermo Scientific | Multiskan GO Type: 1510, REF 51119200 | |
| Fluorescence stereo microscope | Leica | M205FA | |
| Stereo microscope | Leica | S6E | |
| Outside container | Jiang Su Hai Men | glass vial with a diameter of 31.8 mm and a height of 80 mm (inside dimension) | |
| Inside container | Beijing Yi Ran machinery factory | plastic dish with a diameter of 13.6 mm and a height of 7.5 mm (inside dimension) | |
| 1.5 mL Eppendorf tubes | Hai Men Ning Mong | ||
| 96 well plate | Corning Incorporated | Costar 3599 | |
| LEDs | Xin Xing Yuan Guangdian | 607 nm, 3W | https://item.taobao.com/item.htm?id=20158878058 |
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