我们要感谢古尔本基安·德辛西亚研究所（IGC）为我们提供了获得本议定书中描述的部分实验设备的机会。这项工作得到了葡萄牙科学技术基金会（FCT）的支持，LISBOA-01-0145-FEDER-007660， PTDC/NEU-NMC/2459/2014、IF/00697/2014 和 La Caixa HR17-00595 到 PMD，以及澳大利亚研究理事会未来研究金 （FT170100259） 向 CKM 提供。

1. 准备蔗糖酵母 （SY） 饮食
<ol><li>称量所有干成分（阿加，酵母，蔗糖）的宏量营养素平衡和L3饲养饮食。准备1升食物所需的每种成分的克数在 图1B中注明。 注意:考虑到需要大约 13 mL 的食物来填充 60 毫米的培养皿。</li>
	<li>将所有成分溶解在无菌蒸馏水中（使用准备食物所需的总水量的约 50%），搅拌介质 5-10 分钟。</li>
	<li>自动滑轮50分钟。</li>
	<li>允许介质冷却后，在饮食中加入尼帕金和丙酸溶液，最终浓度（v/v）分别为3%和0.3%。在宏量营养素平衡饮食中，将蓝色食物染料添加到 1% 的最终浓度 （v/v） 中。用蒸馏水完成总体积。</li>
	<li>小心地将食物饮食倒入 60 毫米的培养皿中，使每个盘子中浇注的食物数量大致相同。用饮食的 P:C 比率标记盘子。 注意:在喂养检测当天准备宏量营养素平衡饮食。如果不可能，将准备好的饮食储存在4°C，密封容器中，最长持续3天。更长的储存期使饮食过于干燥和困难，幼虫无法钻进媒介。</li>
</ol>2. 父母线的遗传交叉
注:使用Gal4/UAS系统21 来设置遗传交叉。在此协议中，为了激活特定神经元群体的神经元功能，使用 UAS dTRPA1 线17 的女处女，并从 Janelia Gal4 线（图 2A）交叉到男性。使用的基因控制是 dTRPA1 线和"空 GAL4"线之间的交叉的后代，该线在用于生成 Rubin Gal4 集合的向量中携带 Gal4，但没有存在调节片段 （attP2）22。为了促进神经元抑制，可以使用 UAS 线编码 ShibireTS20， 而不是 dTRPA1。
<ol><li>设置60毫米胚胎收集笼与L3饲养饮食板，辅以一些活性酵母膏。</li>
	<li>将成年 UAS dTRPA1 女处女和 Janelia Gal4 雄性（5-8 天）转移到胚胎采集笼中，让交配在 25 °C、60% 湿度和 12:12 光暗周期（图 2A）下进行 24-48小时。对于60毫米胚胎采集笼，每个十字架使用约100个处女雌性和30个雄性。</li>
	<li>在交配期结束时，取出并丢弃遗传十字架中使用的L3饲养饮食板。用它们代替新鲜的L3饲养饮食板，以执行卵子产卵和幼虫分期。</li>
</ol>3. 准备三星幼虫 （L3）
<ol><li>将交美成人苍蝇转移到新鲜的L3饲养饮食板，并允许产卵发生3-4小时，在25°C（图2B）。确保所有的盘子都标有基因型、饮食的P:C比例和产卵日期。 注意:为了节省时间，将产卵直接放入 L3 饲养饮食中，从而避免对鸡蛋进行额外的处理。在小规模基因筛选的情况下，使用苹果汁阿加板可以优化产卵。</li>
	<li>在产卵期结束时，从笼子里取出盘子，用塑料盖盖住它们。如果酵母提取物用于补充L3饲养板，请务必在产卵结束时去除所有残留酵母。这对于避免幼虫生长过程中的不均匀喂养非常重要。 注:交美成人可以转移到新鲜的L3饲养饮食板，所以更多的蛋下进行，并可以获得更多的实验幼虫。在整个工作周内，可以与同一成年人一起连续产卵。</li>
	<li>估计每盘卵子的数量，并将幼虫密度保持在每盘最多200个胚胎。这种估计可以通过计算四分之一的盘子中的胚胎数量来完成。 注意:过度拥挤的盘子会延缓幼虫发育，影响幼虫喂养行为。</li>
	<li>在 18 °C（允许温度）、60% 湿度和 12:12 光暗循环下孵育 L3 饲养板，使幼虫生长 9 天（图 2B）。</li>
	<li>在产卵（AEL）后的第九天，从每个基因型（和复制品）中收集三组10L3进行测试。此外，收集 10 L3 组用于"零染料食品"控制。确保幼虫的收集是在一天中用于产卵的同等时间段内完成的（例如，如果卵子产卵发生在上午 10 点到下午 2 点之间，在 AEL 的同一时间段内收集幼虫），并且通过使用钳子#5或羽毛重量钳尽可能轻轻地进行。按照下一步（3.6）所示直接转移幼虫。 注:"零染料食品"控制动物是幼虫，在喂养检测中给予食物没有蓝色染料。这种控制是必要的，以消除后吸收的幼虫提取物。</li>
	<li>将收集的实验幼虫转移到含有1mL水的塑料盘重量船上。请按照 图 3中给出的指示，确保收集 L3 而不是 L2。 注:收集L3到含有水或1倍磷酸盐缓冲盐水（PBS）的塑料船，对于在喂养检测开始前保持幼虫良好的水分非常重要。如果同时收集来自不同基因型的几个实验 L3 组，这一点尤其重要。跟踪每个组的收集顺序，因此将每个组食物匮乏时间的差异降至最低。在这一步中使用塑料船有利于可选的步骤4.3，因为它使幼虫直接漂浮到水浴。</li>
</ol>4. 恒温遗传活化和无选择喂养检测
注意:建议在一天中大致相同的时间执行喂养检测，以尽量减少与昼夜节律相关的可能变化。此外，始终运行控制实验（"空Gal4"线的后代交叉到UAS dTRPA1和"零染料食物"幼虫），与兴趣的基因型平行。
<ol><li>设置一个孵化器到30°C（不允许的温度），并保持高湿度（至少65%）避免在检测过程中幼虫脱水。</li>
	<li>在开始进食检测之前，通过在 30 °C 加热板 30 分钟来平衡检测板的温度。</li>
	<li>（可选）在37°C的水浴中加热实验幼虫2分钟。执行这一步骤与动物在塑料重量船包含一些水。 注:此步骤的目的是通过促进自喂养检测开始以来神经元的启动来加强神经元激活。</li>
	<li>将多个定时器设置为1小时。使用的定时器数量取决于正在测试的实验组的数量和实验者处理幼虫的熟练程度。 注意:使用多个定时器对于保持所有基因型的检测持续时间一致至关重要。</li>
	<li>小心地从塑料船上排出水，用湿润的软刷，轻轻地将 L3 组从船上转移到检测板的中心。将板盖放回原处，启动每个板（或一组板）的限时器，以保持准确的 1 小时进餐会话。</li>
	<li>允许幼虫在黑暗中以30°C（图2C）为食1小时。 注意:在黑暗中检测的性能对于控制不同饮食中视觉暗示的差异非常重要，因为饮食的色调会有所不同，即使它们含有相同的染料浓度。</li>
	<li>通过将盘子转移到冰浴中来停止喂食检测。尽可能压下冰层，为板块提供稳定的表面。 注意:寒冷的温度会通过抑制挖洞和挖掘行为来促进喂养的结束。大多数幼虫会在几分钟后浮出水面，促进它们在以下步骤中的恢复。</li>
</ol>5. 食品染料提取
<ol><li>为每组 10 个 L3 测试准备 2 mL 微管，其中含有大约相同数量的 0.5 mm 玻璃珠（足以填充微管底部）和 300 μL 的冰冷甲醇。使用长凳冷却器将微管保持在寒冷中。 注意:甲醇具有高度易燃性和毒性。遵循所有安全程序建议处理这种试剂，包括在通风良好的区域工作，戴氮化物手套。 注意:使用甲醇对于修复幼虫样本和避免基质层中的黑色素化反应非常重要。</li>
	<li>使用#5或羽毛重量力，小心地从进食检测板中回收 10 L3 组，并将它们转移到含有一些水的检测板的盖子上。冲洗幼虫，以清除他们身上的任何食物碎片，同时轻轻地处理幼虫，以避免任何伤害。记录每个复制品的幼虫数量，以便量化每种幼虫的平均食物摄入量。 注意:受伤的幼虫应该丢弃，因为它们会有黑色素化角质层，不适合着色定量。</li>
	<li>将 L3 组转移到 5.1 中准备的 2 mL 微管。</li>
	<li>使用在第 5.1 步添加的组织裂解器和玻璃珠，用机械裂解方法将幼虫组织酶从肠道中提取食物染料。（如果组织解酒器不可用，请使用同质化的害虫）。优先执行此步骤在 4 °C （图 2D）。 注:此步骤的持续时间将取决于所使用的设备。使用传统的组织解压器，1分钟的提取就足够了。如果时间限制，协议可以在本步骤结束时暂停，然后继续。将样品储存在-20°C。</li>
	<li>通过将 2 mL 微管直接倒置到新的 1.5 mL 微管上，将提取物转移到清洁 1.5 mL 微管中。如果轻轻执行，大部分玻璃珠将停留在2毫升微管的底部。</li>
	<li>通过离心提取物（最高速度为 10 分钟）在 4 °C 下清除细胞碎片。</li>
	<li>收集超自然人清洁1.5 mL微管。如果细胞碎片在超自然体中仍然可见，则重复步骤 5.6 和 5.7。</li>
</ol>6. 食品消费的彩色定量
<ol><li>准备标准解决方案，通过在开始的蓝色染料溶液的甲醇中执行串行 1:2 稀释，生成校准曲线。作为空白，仅使用甲醇。标准的浓度取决于动物的食物摄入水平。 注意:在此处显示的试验屏幕中，由于幼虫提取物获得的染料浓度在 0.02 至 1.93 μL/mL 之间，因此使用通过测量甲醇中 2 μL/mL 蓝色染料溶液 8 个连续稀释的吸收量而获得的标准曲线。如有必要，根据实验样品的染料浓度增加或降低这些溶液的浓度。</li>
	<li>将实验样品的 100 μL（第 5.7 步获得）、标准和空白（步骤 6.1）转移到 96 井微板的井中，并使用板阅读器（图 2E）测量 600 nm 的吸收量。要去除背景吸收，测量从幼虫饲料中获得的提取物的吸收量，而不使用蓝色染料作为幼虫提取物的"零"（"零染料食物控制"）。</li>
	<li>生成标准曲线，并将每个实验幼虫组样品的吸收值与食物摄入量（mL 中的体积）相关联。通过考虑步骤 5.2 中每组采集的幼虫数量，查找每只幼虫的平均食物消耗量</li>
</ol>

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<li>Raubenheimer, D., Simpson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+models+of+nutrient+balancing%3A+application+to+insects+and+vertebrates%5BTitle%5D)+AND+%22Nutrition+Research+Reviews%22%5BJournal%5D)">Integrative models of nutrient balancing: application to insects and vertebrates.</a> Nutrition Research Reviews. 10 (1), 151-179 (1997).</li>
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</ol>

作者没有什么可透露的。

通过此协议，当接触不同P:C成分的饮食时，可以测试幼虫在特定神经元种群的恒温激活下调节蛋白质和碳水化合物（两种主要宏量营养素）的摄入水平的能力。这种方法是在幼虫初步筛选的背景下测试的，目的是确定与控制不同宏量营养素质量的饮食中的食物摄入量相关的神经元人群。这项工作还有助于证明 ，果蝇 幼虫是研究与营养平衡相关的喂养行为的神经元基础的有价值的动物模型?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61323/quantification-macronutrients-intake-thermogenetic-neuronal-screen">Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae</a>. A figure was updated.
Figure 1&#160;was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61323/61323fig1.jpg" /> 
Figure 1: The sucrose-yeast (SY) diets used in our protocol. (A) The blue dots represent the isocaloric (248 calories/L) macronutrient balancing diets used in the feeding assay, which differ in the protein to carbohydrate (P:C) ratios: 1:1, 1:4 and 1:16. The beige dot represents the diet used to rear the experimental third-instar larvae (L3), which contained a P:C ratio of 1:2 and a caloric density of 495 calories/L. (B) Detailed composition and nutritional information of the sucrose-yeast (SY) based diets. The components are the same for all the diets: agar, sucrose and yeast. The amount in grams of the components needed to prepare 1 L of diet is shown. Note that 1% (v/v) of blue dye must be added to the macronutrient balancing diets and to the L3 rearing diet nipagin and propionic acid solutions must be added to a final concentration (v/v) of 3% and 0.3%, respectively. <a href="https://www.jove.com/files/ftp_upload/61323/61323fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61323/61323fig1v2.jpg" /> 
Figure 1: The sucrose-yeast (SY) diets used in our protocol. (A) The blue dots represent the isocaloric (248 calories/L) macronutrient balancing diets used in the feeding assay, which differ in the protein to carbohydrate (P:C) ratios: 1:1, 1:4 and 1:16. The beige dot represents the diet used to rear the experimental third-instar larvae (L3), which contained a P:C ratio of 1:2 and a caloric density of 495 calories/L. (B) Detailed composition and nutritional information of the sucrose-yeast (SY) based diets. The components are the same for all the diets: agar, sucrose and yeast. The amount in grams of the components needed to prepare 1 L of diet is shown. Note that 1% (v/v) of blue dye must be added to the macronutrient balancing diets and to the L3 rearing diet nipagin and propionic acid solutions must be added to a final concentration (v/v) of 3% and 0.3%, respectively. <a href="https://www.jove.com/files/ftp_upload/61323/61323fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61323/quantification-macronutrients-intake-thermogenetic-neuronal-screen">Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae</a>. A figure was updated.

Erratum: Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

所有动物都依靠均衡的饮食来获得生存、生长和繁殖所需的营养物质。选择吃什么和吃多少受许多相互作用的因素的影响，相关的动物的内部状态，如饱腹程度和环境条件，如食物质量2，3，4，5。蛋白质和碳水化合物是两种主要的宏量营养素，其均衡摄入对维持动物的生理过程至关重要。因此，对控制喂养行为和维持这些宏量营养素均衡摄入的神经机制的理解是极其相关的。这是因为生命史特征，如寿命，胎儿和代谢健康直接影响蛋白质摄入量水平6，7，8，9，10。
使用更简单、更易操作的生物体，表现出与复杂动物（包括哺乳动物）一起进化保存的喂养习惯，对于这类研究至关重要。重要的是，这些更简单的动物模型提供了一个很好的机会，在昂贵，道德和技术更有效的环境中剖析复杂的生物问题。在过去的几十年里 ，Drosophila以其强大的遗传工具包、复杂而陈规定型的行为以及保存了哺乳动物的外围和营养感应机制结构，成为行为神经生物学家11的富有成效的模型。归根结底，希望通过了解这种动物的食物摄入是如何调节的，有了更简单的神经系统，我们就可以开始解开人类饮食失调背后的神经元故障。
对喂养行为的神经元基材的研究在很大程度上取决于能否同时测量动物的食物摄入量，同时操纵动物的神经元活动。由于摄入的食物数量最少，量化苍蝇吃的食物量极具挑战性，目前所有可用的方法都存在重大限制。因此，金本位制是采用互补方法12的组合。从历史上看，成年苍蝇一直被看好为遗传和行为模型。然而，果蝇幼虫，也提供了机会，以研究神经元基质编码喂养行为。幼虫中枢神经系统（CNS）约有12，000个神经元，比包含约150，000个神经元的成人要复杂得多。这种较低的复杂性不仅在数字上，而且在功能上都是如此，因为幼虫行为依赖于更简单的机车功能和感官系统。尽管幼虫的神经系统明显简单，但幼虫仍然表现出完整的喂养行为，一些方法可以量化果蝇幼虫的食物摄入量。通过与神经元活动的操纵配对，果蝇幼虫可以构成一个高度可牵引的模型，用于理解食物摄入的神经调节。
提供这里是一个详细的协议，量化食物摄入量在幼虫暴露在不同宏量营养素品质的饮食。饮食，所谓的宏量营养素平衡饮食，在蛋白质和碳水化合物含量上有所不同，特别是关于蛋白质与碳水化合物（P:C）的比例:1:1（富含蛋白质的饮食），1:4（中间饮食）和1:16（蛋白质贫乏的饮食），如 图1A所示。简言之，使用这三种异种糖-酵母（SY）为基础的饮食用蓝色食物染料染色，建立了定量无选择喂养检测。由于酵母提取物和蔗糖被用作蛋白质和碳水化合物来源，并且都含有碳水化合物，因此通过改变这两个成分的平衡（如图16 和 图1B所示）获得了P:C比率的变化。 图 2中提供了协议的示意图概述，其中显示了主要的实验步骤。
该协议的建立旨在调查特定神经元人群在调节幼虫喂养水平在不同P:C比率的饮食中以及在恒温遗传神经元屏幕中的作用。一个特征良好的神经遗传学工具被使用从瞬态受体潜力（TRP）家族:嗜异性瞬态受体潜力通道（dTRPA1），这是一个温度和电压门的晶体通道，允许在环境温度上升到25°C17以上时激发行动潜力。为了表达dTRPA1转基因，我们利用了Gal4线基于从罗宾实验室建立的d罗索菲拉基因组的cis-监管区域，在Janelia研究校园18，19的FlyLight项目的背景下。
虽然协议，这里描述，已经建立在激活屏幕的背景下，它可以很容易地适应实验者的其他特定需求或兴趣，即执行抑制屏幕使用温度敏感神经元消音器ShibireTS20，替代dTRPA1。此适应和其他适应性在协议和讨论部分进行讨论。

<table><tbody><tr><td>1.5 mL microtubes</td><td>Sarstedt AG &amp;amp; Co.</td><td>72.690.001</td><td></td></tr><tr><td>10xPBS</td><td>Nytech</td><td>MB18201</td><td></td></tr><tr><td>2.0 mL microtubes</td><td>Sarstedt AG &amp;amp; Co.</td><td>72.695.500</td><td></td></tr><tr><td>60 mm petri dishes</td><td>Greiner Bio-one, Austria</td><td>628161</td><td></td></tr><tr><td>96 well microplates</td><td>Santa Cruz Biotechnology</td><td>SC-204453</td><td></td></tr><tr><td>Agar</td><td>Pr&amp;oacute;-vida, Portugal</td><td></td><td></td></tr><tr><td>Bench cooler</td><td>Nalgene, USA</td><td>Labtop Cooler 5115-0032</td><td></td></tr><tr><td>Blue food dye</td><td>Rayner, Billingshurst, UK</td><td></td><td></td></tr><tr><td>Cell disruption media</td><td>Scientific Industries, Inc.</td><td>888-850-6208</td><td>(0.5 mm glass beads)</td></tr><tr><td>Dish weight boats</td><td>Santa Cruz Biotechnology</td><td>SC-201606</td><td></td></tr><tr><td>Embryo collection cage for 60 mm petri dishes</td><td>Flystuff, Scientific Laboratory Supplies, UK</td><td>FLY1212 (59-100)</td><td></td></tr><tr><td>Featherweight forceps</td><td>BioQuip Products, USA</td><td>4750</td><td></td></tr><tr><td>Fly food for stocks maintenance</td><td></td><td></td><td>1 L food contains: 10 g Agar, 100 g Yeast Extract, 50 g Sucrose, 30 mL Nipagin, 3 mL propionic acid</td></tr><tr><td>Forceps #5</td><td>Dumont</td><td>0108-5-PS</td><td>Standard tips, INOX, 11cm</td></tr><tr><td>Incubator</td><td>LMS Ltd, UK</td><td>Series 2, Model 230</td><td>For thermogenetic feeding assay (30?C)</td></tr><tr><td>Incubator</td><td>Percival Scientific, USA</td><td>DR36NL</td><td>To stage larvae (19?C)</td></tr><tr><td>Janelia lines</td><td>Janelia Research Campus</td><td></td><td>Detailed information in Table 2</td></tr><tr><td>Macronutrient balancing diets</td><td></td><td></td><td>Composition and nutritional information in Figure 1</td></tr><tr><td>Methanol</td><td>VWR</td><td>CAS number: 67-56-1</td><td></td></tr><tr><td>Nipagin (Methyl 4-hydroxybenzoate)</td><td>Sigma-Aldrich</td><td>H5501</td><td></td></tr><tr><td>Nitrile gloves</td><td>VWR, USA</td><td></td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf, Germany</td><td>5804 R / Serial number: 5805CI364293</td><td></td></tr><tr><td>Rubin Gal4 ines</td><td>Janelia Research Campus</td><td></td><td>Stoks available at Bloomington Drosophila Stock Center</td></tr><tr><td>ShibireTS UAS line</td><td>Bloomington Drosophila Stock Center</td><td>BDSC number: 66600</td><td>Provided by Carlos Ribeiro Group</td></tr><tr><td>Soft brushes</td><td></td><td></td><td>For sorting anaesthetised fruit flies</td></tr><tr><td>Spectrophotometer plate reader</td><td>Thermo Fisher Scientific</td><td>Multiskan Go 51119300</td><td></td></tr><tr><td>Stereo microscope</td><td>Nikon</td><td>1016625</td><td></td></tr><tr><td>Sucrose</td><td>Sidul, Portugal</td><td></td><td></td></tr><tr><td>Third-instar larvae (L3) rearing diet</td><td></td><td></td><td>Composition and nutritional information in Figure 1</td></tr><tr><td>Timer</td><td></td><td></td><td></td></tr><tr><td>Tissue lyzer / bead beater</td><td>MP Biomedicals, USA</td><td>FastPrep-24 6004500</td><td></td></tr><tr><td>TRPA1 UAS line</td><td>Bloomington Drosophila Stock Center</td><td>BDSC number: 26264</td><td>Expresses TrpA1 under UAS control; may be used to activate neurons experimentally at 25 ?C</td></tr><tr><td>Water bath</td><td>Sheldon Manufacturing Inc., USA</td><td>W20M-2 / 03068308 / 9021195</td><td></td></tr><tr><td>Yeast extract</td><td>Pr&amp;oacute;-vida, Portugal</td><td></td><td>51% Protein, 15% Carbohydrate</td></tr></tbody></table>

quantification of macronutrients intake in a thermogenetic neuronal screen using drosophila larvae

觅食和喂养行为使动物能够获得对其发育、健康和健身至关重要的能量和营养来源。研究这些行为的神经元调节对于理解营养平衡背后的生理和分子机制至关重要。使用遗传可牵引的动物模型，如蠕虫、苍蝇和鱼，极大地促进了这些类型的研究。在过去的十年里，果蝇果蝇褪黑素被神经生物学家用作一种强大的动物模型，研究喂养和觅食行为的神经元控制。虽然毫无疑问是有价值的，但大多数研究都对成年苍蝇进行了研究。在这里，我们描述了一个协议，利用更简单的幼虫神经系统来研究神经元基板控制喂养行为时，幼虫暴露在饮食不同的蛋白质和碳水化合物含量。我们的方法基于定量色度无选择喂养检测，在神经元恒温激活屏幕的背景下执行。作为读出，幼虫在1小时间隔内食用的食物量被用于暴露在三种染料标签饮食之一，这些饮食的蛋白质与碳水化合物（P:C）的比例不同。通过识别调节不同宏量营养素质量饮食中食用食物量的候选神经元人群，在幼虫果蝇的神经遗传学筛查中证明了此协议的有效性。我们还能够将测试的基因型分类并归类为表型类。除了简要回顾文献中目前可用的方法外，还讨论了这些方法的优点和局限性，并就如何使该方案适应其他具体实验提出了一些建议。

果蝇幼虫以摄入过量碳水化合物23（图2E中的示意图图）为代价调节其蛋白质摄入量。事实上，这种蛋白质摄入的优先顺序已经观察到在许多其他动物，被称为蛋白质杠杆24，25。
利用这种强大的喂养行为反应，设计了一个基于行为的屏幕，旨在识别参与宏量营养素平衡的神经元群体。?...

Watch this Scientific Journal Video about Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae at JoVE.com

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

这里描述的是一个协议，使颜色定量在定义的时间间隔内食用的食物量，由 德罗索菲拉梅拉诺加斯特 幼虫暴露在不同宏量营养素品质的饮食。这些检测是在神经元恒温屏幕的背景下进行的。

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

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Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

3.7K 次观看.  Monash University. 这里描述的是一个协议，使颜色定量在定义的时间间隔内食用的食物量，由 德罗索菲拉梅拉诺加斯特 幼虫暴露在不同宏量营养素品质的饮食。这些检测是在神经元恒温屏幕的背景下进行的。

视频: Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

科研

遗传学

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

神经科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

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

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

行为学

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

用于果蝇消费量化的高吞吐量微板馈线分析

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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.

毛细管馈线分析测量的摄食<em&gt;果蝇</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

果蝇的快速食物偏好分析

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

使用 FRET 传感器监测 果蝇 中的脑代谢物运输

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the production of ATP from glucose and its metabolites, such as the monocarboxylates lactate and pyruvate. It is still unclear how this process is regulated or who the key players are, particularly in Drosophila.
Using genetically encoded F&#246;rster resonance energy transfer-based sensors, we present a simple method for measuring the transport of monocarboxylates and glucose in glial cells and neurons in an ex-vivo Drosophila larval brain preparation. The protocol describes how to dissect and adhere a larval brain expressing one of the sensors to a glass coverslip. 
We present the results of an entire experiment in which lactate transport was measured in larval brains by knocking down previously identified monocarboxylate transporters in glial cells. Furthermore, we demonstrate how to rapidly increase neuronal activity and track metabolite changes in the active brain. The described method, which provides all necessary information, can be used to analyze other Drosophila living tissues.

作者聚焦:使用 果蝇 模型的脑能量代谢研究进展

Here we present a protocol to visualize the transport of monocarboxylates, glucose, and ATP in glial cells and neurons using genetically encoded F&#246;rster resonance energy transfer-based sensors in an ex-vivo Drosophila larval brain preparation.

使用德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

摘要

探索更多视频

高脂膳食喂养和高通量三测定在果蝇中的含量

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

从查明的神经元和取食行为的钙信号的同时记录果蝇

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

局部和全局的方法评估热痛觉

用于果蝇消费量化的高吞吐量微板馈线分析

毛细管馈线分析测量的摄食

果蝇幼虫和成年脑的代谢分析

果蝇的快速食物偏好分析

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

摘要

探索更多视频

高脂膳食喂养和高通量三测定在果蝇中的含量

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

从查明的神经元和取食行为的钙信号的同时记录果蝇

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

局部和全局的方法评估热痛觉

用于果蝇消费量化的高吞吐量微板馈线分析

毛细管馈线分析测量的摄食

果蝇幼虫和成年脑的代谢分析

果蝇的快速食物偏好分析

使用基因编码传感器在 离体果蝇 幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物