Name: quantification of macronutrients intake in a thermogenetic neuronal screen using drosophila larvae
Uploaded: 2020-06-11T14:45:00
Description: Watch this Scientific Journal Video about Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae at JoVE.com

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?...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:2112px;" width="2113">
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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">1.5 mL microtubes</td>
			<td style="width:271px;">Sarstedt AG &#38; Co.</td>
			<td style="width:247px;">72.690.001</td>
			<td style="width:1272px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10xPBS</td>
			<td style="width:271px;">Nytech</td>
			<td style="width:247px;">MB18201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2.0 mL microtubes</td>
			<td style="width:271px;">Sarstedt AG &#38; Co.</td>
			<td style="width:247px;">72.695.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">60 mm petri dishes</td>
			<td style="width:271px;">Greiner Bio-one, Austria</td>
			<td style="width:247px;">628161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">96 well microplates</td>
			<td style="width:271px;">Santa Cruz Biotechnology</td>
			<td style="width:247px;">SC-204453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Agar</td>
			<td style="width:271px;">Pr&#243;-vida, Portugal</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Bench cooler</td>
			<td style="width:271px;">Nalgene, USA</td>
			<td style="width:247px;">Labtop Cooler 5115-0032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Blue food dye</td>
			<td style="width:271px;">Rayner, Billingshurst, UK</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Cell disruption media</td>
			<td style="width:271px;">Scientific Industries, Inc.</td>
			<td style="width:247px;">888-850-6208</td>
			<td>(0.5 mm glass beads)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Dish weight boats</td>
			<td style="width:271px;">Santa Cruz Biotechnology</td>
			<td style="width:247px;">SC-201606</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Embryo collection cage for 60 mm petri dishes</td>
			<td style="width:271px;">Flystuff, Scientific Laboratory Supplies, UK</td>
			<td style="width:247px;">FLY1212 (59-100)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Featherweight forceps</td>
			<td style="width:271px;">BioQuip Products, USA</td>
			<td style="width:247px;">4750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Fly food for stocks maintenance</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></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 height="21">
			<td height="21" style="height:21px;width:323px;">Forceps #5</td>
			<td style="width:271px;">Dumont</td>
			<td style="width:247px;">0108-5-PS</td>
			<td>Standard tips, INOX, 11cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Incubator</td>
			<td style="width:271px;">LMS Ltd, UK</td>
			<td style="width:247px;">Series 2, Model 230</td>
			<td>For thermogenetic feeding assay (30&#8728;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Incubator</td>
			<td style="width:271px;">Percival Scientific, USA</td>
			<td style="width:247px;">DR36NL</td>
			<td>To stage larvae (19&#8728;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Janelia lines</td>
			<td style="width:271px;">Janelia Research Campus</td>
			<td style="width:247px;"></td>
			<td>Detailed information in Table 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Macronutrient balancing diets</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>Composition and nutritional information in Figure 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Methanol</td>
			<td style="width:271px;">VWR</td>
			<td style="width:247px;">CAS number: 67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nipagin (Methyl 4-hydroxybenzoate)</td>
			<td style="width:271px;">Sigma-Aldrich</td>
			<td style="width:247px;">H5501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nitrile gloves</td>
			<td style="width:271px;">VWR, USA</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Refrigerated centrifuge</td>
			<td style="width:271px;">Eppendorf, Germany</td>
			<td style="width:247px;">5804 R / Serial number: 5805CI364293</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Rubin Gal4 ines</td>
			<td style="width:271px;">Janelia Research Campus</td>
			<td style="width:247px;"></td>
			<td>Stoks available at Bloomington Drosophila Stock Center</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ShibireTS UAS line</td>
			<td style="width:271px;">Bloomington Drosophila Stock Center</td>
			<td style="width:247px;">BDSC number: 66600</td>
			<td>Provided by Carlos Ribeiro Group</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Soft brushes</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>For sorting anaesthetised fruit flies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Spectrophotometer plate reader</td>
			<td style="width:271px;">Thermo Fisher Scientific</td>
			<td style="width:247px;">Multiskan Go 51119300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Stereo microscope</td>
			<td style="width:271px;">Nikon</td>
			<td style="width:247px;">1016625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sucrose</td>
			<td style="width:271px;">Sidul, Portugal</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Third-instar larvae (L3) rearing diet</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>Composition and nutritional information in Figure 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Timer</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tissue lyzer / bead beater</td>
			<td style="width:271px;">MP Biomedicals, USA</td>
			<td style="width:247px;">FastPrep-24 6004500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">TRPA1 UAS line</td>
			<td style="width:271px;">Bloomington Drosophila Stock Center</td>
			<td style="width:247px;">BDSC number: 26264</td>
			<td>Expresses TrpA1 under UAS control; may be used to activate neurons experimentally at 25 &#8728;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Water bath</td>
			<td style="width:271px;">Sheldon Manufacturing Inc., USA</td>
			<td style="width:247px;">W20M-2 / 03068308 / 9021195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Yeast extract</td>
			<td style="width:271px;">Pr&#243;-vida, Portugal</td>
			<td style="width:247px;"></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 ...

Research

JoVE Journal

Neuroscience

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

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

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

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 ...

科研

神经科学

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

Appetitive Associative Olfactory Learning in Drosophila Larvae

欲求联想嗅觉学习<em&gt;果蝇</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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 ...

Quantification of Drosophila Grooming Behavior

果蝇美容行为的量化

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

Novel Assay for Cold Nociception in Drosophila Larvae

Novel Assay for Cold Nociception in Drosophila Larvae

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

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