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。

1. 准备送料室
注意:染料标记喂养试验的加料室由两部分组成: 外容器 (作为盖子) 和内容器 (作为食物来源)。
<ol><li>从玻璃瓶中修改外部容器以培养果蝇(内径为31.8 毫米, 高度为80毫米) (图 1A, 1C)。用一层1% 琼脂糖 (5 毫升) 盖住容器的底部, 以保持喂食装置的适当湿润, 并充当苍蝇行走的软基板 (图 1B)。容器从而提供一个受控但自然主义的设置, 对苍蝇进行最小的应力或扰动。<ol>
<li>准备外面的容器。将0.5 克琼脂糖悬浮在50毫升的双蒸馏水中, 加热时经常搅拌, 然后煮沸, 完全溶解在一个带有热板的磁力搅拌器上。</li>
		<li>当1% 琼脂糖冷却到约60摄氏度, 转移5毫升1% 琼脂糖到一个空的外部容器 (图 1B), 并让容器保持开放, 直到冷却到室温, 以产生琼脂糖垫。</li>
	</ol>
</li>
	<li>准备染料标签食品。染料标签食品的组成根据不同的实验目的而变化。这里是一个例子, 准备的染料标签食品只含有蔗糖。<ol>
<li>添加0.5 克琼脂糖到50毫升的双蒸馏水, 热频繁搅拌, 并煮沸完全溶解在一个磁性搅拌器与热板。</li>
		<li>当上述1% 琼脂糖冷却到约60摄氏度, 添加1.71 克蔗糖的琼脂糖, 以获得 100 mM 蔗糖。</li>
		<li>添加0.25 克蓝色染料 (erioglaucine 二钠) 到琼脂糖溶液中获得 0.5% (瓦特/v) 染料标签食品。</li>
	</ol>
</li>
	<li>准备里面的容器。将空内容器放在平坦的水平面上, 然后将750µL 的蓝色染料标签食品添加到单独的食品容器中, 让它凝固至少5分钟。 注意:内部容器是一个小的塑料碟, 内径为13.6 毫米, 高度为7.5 毫米 (图 1A, 1C)。它充满了染料标签的食物, 然后定位在琼脂糖垫的外部容器的中心。</li>
	<li>将填充的内容器转移到准备好的外部容器中, 并将其放置在琼脂糖垫的中心。让喂食室在室温下保持40分钟的开放, 直到外部容器的壁没有凝结。 注意:干墙是必要的, 以防止苍蝇被困在墙上, 接触水滴。</li>
	<li>用无染料食物 (相同成分但不含染料) 为背景减法准备喂食室。</li>
</ol>2. 苍蝇的制备
注意:成年女性飞行5至10天后, 羽化通常用于喂养化验。建议对不同基因型的苍蝇进行制备, 包括基因控制, 并进行平行试验。20飞行 (从一个哺养的房间) 生成一个数据点。
<ol><li>考虑到饲养成年苍蝇的人口密度。通常, 根据菌株和培养瓶的尺寸来改变亲本的数量来控制人口密度。举个例子, 将广州 S 的15男性和30女性转移到一个文化瓶 (直径31.8 毫米和高度80毫米), 让雌性产卵一天, 然后删除成年苍蝇。</li>
	<li>准备苍蝇 thermogenetic 激活。<ol>
<li>使用 Gal4/UAS 系统38 , 通过跨越 UAS-dTrpA1 与不同 GAL4 驱动程序线 (图 2B), 在不同的神经元中表达 dTRPA133 。</li>
		<li>保持成人飞行在标准食物在22°c, 60% 相对湿度和 12 h/12 h 光黑暗的周期。</li>
		<li>在喂食试验前两天, 在一个2飞垫上, 在一个立体显微镜下, 将苍蝇放在一个新鲜的食物瓶中, 并保持每瓶20只苍蝇的密度。</li>
	</ol>
</li>
	<li>准备苍蝇 optogenetic 激活。<ol>
<li>使用 Gal4/UAS 系统38 , 通过与不同 GAL4 驱动程序线交叉的无人驾驶 CsChrimson 苍蝇, 在不同的神经元中表达 CsChrimson36 。</li>
		<li>收集新的 eclosed (24 小时内) 苍蝇和转移到一个小瓶与标准食品含有400µM 全反式视网膜。</li>
		<li>为了防止过早激活, 用铝箔包裹饲养瓶, 然后放入一个黑暗的盒子里, 保护苍蝇不受不必要的光刺激。保持苍蝇在25摄氏度, 60% 湿度在黑暗中 4-6 天。</li>
		<li>在喂食试验前两天, 在 CO2飞垫上用立体显微镜对雌性苍蝇进行排序。收集20只苍蝇到一个食品瓶, 并保持他们在黑暗的喂养测试之前。</li>
		<li>尽可能快地在昏暗的灯光下进行所有操作, 以避免环境光线造成的影响。使用放置在远离工作区域的 LED 灯, 工作区的强度为5µW/厘米2。 注意:使用饿死的广州 S 苍蝇作为喂养试验的积极控制. 剥夺这些苍蝇的食物和维持在1% 琼脂糖24小时之前的喂养测试。这些苍蝇的食物消耗水平有助于评估日常的波动。</li>
	</ol>
</li>
</ol>3. Thermogenetic 激活
<ol><li>在当天的同一时间进行所有喂养实验, 大约授时时间 4-631,32, 以最小化由于昼夜节律的变化。</li>
	<li>准备一个孵化器 (或一个有温度控制的加热器的小房间) 作为供暖环境。</li>
	<li>在行为实验之前, 平衡在实验温度 (30 °c) 上准备好的进料室, 2 小时, 以确保进料室的温度相对均匀。</li>
	<li>要开始喂食过程, 小心地将20飞从他们的家小瓶到一个预热的喂食室通过温和的攻丝。继续以30°c 为60分钟的喂养过程 (图 3A) . 注意:频繁的鼓动, 如打开和关闭孵化器门, 不利地影响喂养行为, 因此应尽量减少。</li>
	<li>在-80 摄氏度 (或-20 °c) 冻结喂食室, 停止喂养60分钟, 30 分钟。 注意:冷冻有助于在所有房间同时停止喂养。结冰的苍蝇在-80 °c 服务二个目的, 为 euthanizing 苍蝇和为存贮直到均匀化为一个星期。</li>
	<li>对于温度控制组, 转移20飞入室温进给室与染料食品, 开始喂养过程在22°c 60 分钟。</li>
	<li>在-80 摄氏度 (或-20 摄氏度) 的温度控制组中, 停止喂养30分钟。</li>
</ol>4. Optogenetic 激活
<ol><li>在当天的同一时间进行所有喂养实验, 大约授时时间 4-631,32, 以最小化由于昼夜节律的变化。</li>
	<li>小心地将20只苍蝇从一小瓶移入进料室, 然后轻轻敲击, 然后将腔侧放在照明设置上 (图 4A, 4B)。<ol>
<li>对于 optogenetic 操作, 使用橙色 led 阵列 (607 nm) 作为光源的刺激, 并固定在 led 上方的水平有机玻璃板, 以支持在照明期间的喂食室。将安装程序保持在25摄氏度的孵化器中, 60% 的湿度 (图 4A, 4B)。</li>
		<li>刺激遗传控制苍蝇平行与饕餮 > CsChrimson苍蝇, 但在不同的喂养室。</li>
	</ol>
</li>
	<li>继续喂养过程为60分钟与背面照明由橙色光。<ol>
<li>确定光遗传学实验36的最佳光照强度.对于饕餮 > CsChrimson飞行, 强度为2.2 兆瓦/毫米2会诱发瘫痪 (停止运动和失去体位控制), 因此, 使用中等强度0.1 兆瓦/毫米2来刺激饕餮> CsChrimson在喂食测试期间飞行。</li>
	</ol>
</li>
	<li>在-80 摄氏度 (或-20 摄氏度) 的情况下, 通过冷冻喂养室停止喂食30分钟。</li>
</ol>5. 食品消费的视觉估计
注意:视觉评分法提供了对食品消耗量的半定量估计。虽然该方法不像光学方法那样客观, 但它能够及时地通过大量的飞行菌株, 使其适合于第一轮的基因筛, 迅速缩小候选名单, 以便进行进一步的测试。
<ol><li>在喂食过程之后, 麻醉在一个立体显微镜下的 CO2飞垫上的苍蝇。根据其腹部的染料色食物的体积和强度, 为每只苍蝇分别评分 (图 2A)。 注意: 视觉估计的标准: 评分系统有4水平的食物摄取量对应于不同体积和强度的彩色食物在腹部。<ol>
<li>无可察觉的染料色的食物的比分飞行比分 0;给那些微弱的 (几乎不被发现的) 蓝色染料或用染料色的食物占领少于一个第三容量腹部 a 比分1。</li>
		<li>得分与强烈的染料色的食物占领腹部的一半的分数为2。</li>
		<li>分数为 3 (图 2A) 的强烈染色的内容占据超过一半的腹部得分。</li>
	</ol>
</li>
	<li>在每个实验条件下计算不同分数的苍蝇的比例 (图 2B)。</li>
</ol>6. 食品消耗量的比色量化
<ol><li>停止进食后, 将所有20只苍蝇从喂食室倒入一张称重纸上, 然后用软刷子将其转移到1.5 毫升管中。 注意:不要把所有的房间的-80 °c 存储同时, 以避免苍蝇粘在房间壁, 因为水凝结在寒冷的房间。</li>
	<li>添加500µL 的 PBST 管和融汇的苍蝇使用研磨机在60赫兹 5 s。<ol>
<li>当身体部位没有可检测的片段时, 通过观察确认充分的均匀化。</li>
	</ol>
</li>
	<li>旋转管与组织匀浆30分钟, 在1.3万转, 以清除碎片。</li>
	<li>离心后, 将100µL 的上清液转移到96井板的井中。</li>
	<li>在所有样品被装载了之后, 把板材放入一个板材读者测量样品的吸光度在630毫微米。</li>
	<li>要去除背景吸收, 减去上清液的吸光度, 从吃的苍蝇的食物没有染料从上清的吸收从蓝色食物喂养的苍蝇。 注意:这一步是可选的, 这取决于所用食物的成分。当苍蝇食品 (没有染料) 产生 630 nm 的吸光度, 有必要执行这一步骤, 以消除背景。</li>
</ol>

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作者声明没有竞争的财政利益。

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?...

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。

<table border="0" cellpadding="0" cellspacing="0" width="942">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">UAS-CsChrimson</td>
			<td style="width:163px;">Bloomintoon</td>
			<td style="width:176px;">55135</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">UAS-dTrpA1</td>
			<td style="width:163px;">Bloomintoon</td>
			<td>26263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TDC1-GAL4&#160;</td>
			<td style="width:163px;">Bloomintoon</td>
			<td style="width:176px;">9312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">TDC2-GAL4</td>
			<td style="width:163px;">Bloomintoon</td>
			<td style="width:176px;">9313</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">sNPF-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Z. Zhao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NPF-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TH-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-HT-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AKH-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dip2-GAL4</td>
			<td></td>
			<td></td>
			<td>Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Taotie-GAL4</td>
			<td></td>
			<td></td>
			<td>Provided by J. Carlson</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Agarose</td>
			<td>Biowest</td>
			<td style="width:176px;">G-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Sucrose</td>
			<td>Sigma</td>
			<td style="width:176px;">S7903</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Erioglaucine disodium salt</td>
			<td>Sigma</td>
			<td>861146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">all-trans-retinal&#160;</td>
			<td style="width:163px;">Sigma&#160;</td>
			<td style="width:176px;">R2500</td>
			<td>stored in darkness</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td style="width:163px;">Amresco</td>
			<td>9002-93-01</td>
			<td></td>
		</tr>
		<tr height="146">
			<td height="146" style="height:146px;width:332px;">Fly food</td>
			<td style="width:163px;"></td>
			<td style="width:176px;">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&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;">&#160;1x PBS buffer&#160;</td>
			<td style="width:163px;"></td>
			<td style="width:176px;">1 L 1X PBS contains: 8 g Nacl, 0.2 g Kcl, 1.44 g Na2HPO4, 0.24 g KH2PO4, pH 7.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">PBST buffer</td>
			<td style="width:163px;"></td>
			<td colspan="2">1X PBS with 1% Triton X-100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Grinding mill</td>
			<td style="width:163px;">Shang Hai Jing Xin</td>
			<td>Tissuelyser-24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Incubator</td>
			<td style="width:163px;">Ning Bo Jiang Nan</td>
			<td>HWS-80</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stirrer with a heat plate</td>
			<td style="width:163px;">Chang Zhou Bo Yuan</td>
			<td style="width:176px;">CJJ 78-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrometer</td>
			<td style="width:163px;">Thorlabs</td>
			<td style="width:176px;">CCS200/M</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Microplate Spectrophotometer</td>
			<td>Thermo Scientific&#160;</td>
			<td style="width:176px;">Multiskan GO Type: 1510, REF 51119200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence stereo microscope&#160;</td>
			<td style="width:163px;">Leica</td>
			<td style="width:176px;">&#160;M205FA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereo microscope</td>
			<td style="width:163px;">Leica&#160;</td>
			<td style="width:176px;">S6E</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:332px;">Outside container</td>
			<td>Jiang Su Hai Men</td>
			<td style="width:176px;">glass vial with a diameter of 31.8 mm and a height of 80 mm (inside dimension)</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:332px;">Inside container&#160;</td>
			<td>Beijing Yi Ran machinery factory</td>
			<td style="width:176px;">plastic dish with a diameter of 13.6 mm and a height of 7.5 mm (inside dimension)</td>
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			<td height="20" style="height:20px;width:332px;">1.5 mL Eppendorf tubes</td>
			<td style="width:163px;">Hai Men Ning Mong</td>
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			<td height="20" style="height:20px;">&#160;96 well plate</td>
			<td style="width:163px;">Corning Incorporated</td>
			<td style="width:176px;">&#160;Costar 3599</td>
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			<td height="40" style="height:40px;width:332px;">LEDs</td>
			<td style="width:163px;">Xin Xing Yuan Guangdian</td>
			<td style="width:176px;">607 nm, 3W&#160;</td>
			<td>https://item.taobao.com/item.htm?id=20158878058</td>
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combining quantitative food-intake assays and forcibly activating neurons to study appetite in drosophila

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。

Thermogenetic 屏幕。
食欲异常增加会导致食物摄入量升高, 而不考虑生理需求。我们利用这个方案设计了一个高通量行为屏幕, 以获得与饥饿和饱足状态相关的神经元的遗传处理 (图 1)。屏幕产生Taotie-Gal424。当Taotie-Gal4神经元被强行激活为30摄氏度时,饕餮 > TrpA1苍蝇摄取?...

Watch this Scientific Journal Video about Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila at JoVE.com

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

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.

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, ...

combining-quantitative-food-intake-assays-forcibly-activating-neurons

Research

JoVE Journal

Behavior

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

8.2K 次观看.  University of Science and Technology of China. 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.

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

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

科研

行为学

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

揭开节拍耳聋:检测节律紊乱与同步手指敲击和感知定时任务

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

Design and Implementation of an fMRI Study Examining Thought Suppression in Young Women with, and At-risk, for Depression

Ruminative brooding is associated with increased vulnerability to major depression. Individuals who regularly ruminate will often try to reduce the frequency of their negative thoughts by actively suppressing them. We aim to identify the neural correlates underlying thought suppression in at-risk and depressed individuals. Three groups of women were studied; a major depressive disorder group, an at-risk group (having a first degree relative with depression) and controls. Participants performed a mixed block-event fMRI paradigm involving thought suppression, free thought and motor control periods. Participants identified the re-emergence of &#8220;to-be-suppressed&#8221; thoughts (&#8220;popping&#8221; back into conscious awareness) with a button press. During thought suppression the control group showed the greatest activation of the dorsolateral prefrontal cortex, followed by the at-risk, then depressed group. During the re-emergence of intrusive thoughts compared to successful re-suppression of those thoughts, the control group showed the greatest activation of the anterior cingulate cortices, followed by the at-risk, then depressed group. At-risk participants displayed anomalies in the neural regulation of thought suppression resembling the dysregulation found in depressed individuals. The predictive value of these changes in the onset of depression remains to be determined.

We aim to identify the neural correlates underlying sustained and transient thought suppression, and thought re-emergence in controls, at-risk and depressed individuals. Activation was greatest for controls compared to the at-risk and the depressed group in the dorsolateral prefrontal cortex during thought suppression and anterior cingulate cortex during thought re-emergence.

设计的功能磁共振成像研究与实现研究思想抑制在年轻妇女，以及在风险，为抑郁症

Ruminative brooding is associated with increased vulnerability to major depression. Individuals who regularly ruminate will often try to reduce the ...

Combining Multiple Data Acquisition Systems to Study Corticospinal Output and Multi-segment Biomechanics

Transcranial magnetic stimulation techniques allow for an in-depth investigation into the neural mechanisms that underpin human behavior. To date, the use of TMS to study human movement, has been limited by the challenges related to precisely timing the delivery of TMS to features of the unfolding movement and, also, by accurately characterizing kinematics and kinetics. To overcome these technical challenges, TMS delivery and acquisition systems should be integrated with an online motion tracking system. The present manuscript details technical innovations that integrate multiple acquisition systems to facilitate and advance the use of TMS to study human movement. Using commercially available software and hardware systems, a step-by-step approach to both the hardware assembly and the software scripts necessary to perform TMS studies triggered by specific features of a movement is provided. The approach is focused on the study of upper limb, planar, multi-joint reaching movements. However, the same integrative system is amenable to a multitude of sophisticated studies of human motor control.

The use of transcranial magnetic stimulation (TMS) to study human motor control requires the integration of data acquisition systems to control TMS delivery and simultaneously record human behavior. The present manuscript provides a detailed methodology for integrating data acquisition systems for the purpose of investigating human movement via TMS.

合并多个数据采集系统来研究皮质输出和多段生物力学

Transcranial magnetic stimulation techniques allow for an in-depth investigation into the neural mechanisms that underpin human behavior. To date, the use ...

Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in mice: the Feeding Experimentation Device (FED). FED is an open-source system that was designed to facilitate flexibility in food intake studies. Due to its compact and battery powered design, FED can be placed within standard home cages or other experimental equipment. Food intake measurements can also be synchronized with other equipment in real-time via FED's transistor-transistor logic (TTL) digital output, or in post-acquisition processing as FED timestamps every event with a real-time clock. When in use, a food pellet sits within FED's food well where it is monitored via an infrared beam. When the pellet is removed by the mouse, FED logs the timestamp onto its internal secure digital (SD) card and dispenses another pellet. FED can run for up to 5 days before it is necessary to charge the battery and refill the pellet hopper, minimizing human interference in data collection. Assembly of FED requires minimal engineering background, and off-the-shelf materials and electronics were prioritized in its construction. We also provide scripts for analysis of food intake and meal patterns. Finally, FED is open-source and all design and construction files are online, to facilitate modifications and improvements by other researchers.

Feeding Experimentation Device FED: Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

饲养实验装置（FED）:建筑和验​​证的开源设备的在鼠害测量摄食

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in ...

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

可逆冷却诱导 Deactivations 对行走猫的障碍记忆的皮层贡献研究

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

一种自动测定果蝇在空间和时间温度变化中的性能的方法

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

使用五分钟言语样本程序评估家长对孩子的简短叙述的一致性

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Concept Development and Use of an Automated Food Intake and Eating Behavior Assessment Method

The vast majority of dietary and eating behavior assessment methods are based on self-reports. They are burdensome and also prone to measurement errors. Recent technological innovations allow for the development of more accurate and precise dietary and eating behavior assessment tools that require less effort for both the user and the researcher. Therefore, a new sensor-based device to assess food intake and eating behavior was developed. The device is a regular dining tray equipped with a video camera and three separate built-in weighing stations. The weighing stations measure the weight of the bowl, plate, and drinking cup continuously over the course of a meal. The video camera positioned to the face records eating behavior characteristics (chews, bites), which are analyzed using artificial intelligence (AI)-based automatic facial expression software. The tray weight and the video data are transported at real-time to a personal computer (PC) using a wireless receiver. The outcomes of interest, such as the amount eaten, eating rate and bite size, can be calculated by subtracting the data of these measures at the timepoints of interest. The information obtained by the current version of the tray can be used for research purposes, an upgraded version of the device would also facilitate the provision of more personalized advice on dietary intake and eating behavior. Contrary to the conventional dietary assessment methods, this dietary assessment device measures food intake directly within a meal and is not dependent on memory or the portion size estimation. Ultimately, this device is therefore suited for daily main meal food intake and eating behavior measures. In the future, this technology based dietary assessment method can be linked to health applications or smart watches to obtain a complete overview of exercise, energy intake, and eating behavior.

This protocol shows and explains a new technology-based dietary assessment method. The method consists of a dining tray with multiple built-in weighing scales and a video camera. The device is unique in the sense that it incorporates automated measures of food and drink intake and eating behavior over the course of a meal.

自动食物摄入和饮食行为评估方法的概念开发和使用

The vast majority of dietary and eating behavior assessment methods are based on self-reports. They are burdensome and also prone to measurement errors. ...

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

摘要

探索更多视频

此视频中的章节

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

揭开节拍耳聋:检测节律紊乱与同步手指敲击和感知定时任务

设计的功能磁共振成像研究与实现研究思想抑制在年轻妇女，以及在风险，为抑郁症

合并多个数据采集系统来研究皮质输出和多段生物力学

饲养实验装置（FED）:建筑和验证的开源设备的在鼠害测量摄食

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

可逆冷却诱导 Deactivations 对行走猫的障碍记忆的皮层贡献研究

一种自动测定果蝇在空间和时间温度变化中的性能的方法

使用五分钟言语样本程序评估家长对孩子的简短叙述的一致性

自动食物摄入和饮食行为评估方法的概念开发和使用

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

摘要

探索更多视频

此视频中的章节

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

揭开节拍耳聋:检测节律紊乱与同步手指敲击和感知定时任务

设计的功能磁共振成像研究与实现研究思想抑制在年轻妇女，以及在风险，为抑郁症

合并多个数据采集系统来研究皮质输出和多段生物力学

饲养实验装置（FED）:建筑和验​​证的开源设备的在鼠害测量摄食

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

可逆冷却诱导 Deactivations 对行走猫的障碍记忆的皮层贡献研究

一种自动测定果蝇在空间和时间温度变化中的性能的方法

使用五分钟言语样本程序评估家长对孩子的简短叙述的一致性

自动食物摄入和饮食行为评估方法的概念开发和使用

饲养实验装置（FED）:建筑和验证的开源设备的在鼠害测量摄食