Name: a burrowing/tunneling assay for detection of hypoxia in drosophila melanogaster larvae
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Description: Watch this Scientific Journal Video about A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae at JoVE.com

凯伦. 羌是赖斯大学乔治 j 斯瑞普菲研究奖的2016接受者。范蠡是赖斯大学教学奖学金的接受者。布卢明顿果蝇库存中心, 哈佛旅行设施, 维也纳果蝇资源中心的服务, 感激地承认。

1. 幼虫的制备
<ol><li>两个或更多天在开始化验之前, 设置所需的实验和控制组合的十字架 (每一个至少20位女性和10个男性) 在蛋被放置的菜由 Wieschaus 和 Nusslein-佛尔哈德13的描述, 但使用50毫升聚丙烯烧杯和6.0 厘米一次性培养皿中含有葡萄琼脂, 涂上酵母膏, 然后才使用。将苍蝇放在蛋碗碟里, 在室温下, 直到需要的时候。</li>
	<li>葡萄琼脂食谱-结合100毫升冷冻100% 葡萄汁浓缩, 350 毫升 deioinized 水, 5 毫升冰川醋酸和13克D.黑腹琼脂在1L 玻璃烧杯。微波在1-2 分钟内爆发, 搅拌之间爆发, 直到琼脂全部溶解。冷却溶液几分钟, 然后加入10毫升 10% (w/v) 的 Nipagen (甲基对羟基苯甲酸) 在95% 乙醇。混合, 然后吸管7毫升每板成一次性 6.0 cm 培养皿, 允许在室温下凝胶和干燥3-4 小时, 在塑料袋 4 0C 存储。</li>
	<li>酵母膏食谱–7克干酵母, 10 毫升去离子水, 混合与铲, 直到一致的一致性。</li>
	<li>定时产卵产生实验和控制幼虫。使用新的, 酵母涂抹, 葡萄板, 收集鸡蛋在室温下4小时在黑暗中的早晨。卸下这 4 h 收集板, 并用新鲜的盘子替换。在 25 0C 一夜之间孵化4小时的收集板, 直到第二天下午, 当大多数幼虫孵化时。收集这些第一龄幼虫, 并用它们来建立实验。</li>
</ol>2. 设置化验盘
<ol><li>测定板的制备。在一次试验中, 准备五个化验板, 每10个实验幼虫和五盘10控制幼虫。用1.5 厘米的软木塞从每个盘子中取出琼脂的中心核心, 为食物创造一个洞。将酵母膏 (0.8-0.9 克) 放入孔中, 轻轻地用刮刀轻拍, 使土堆填满洞。</li>
	<li>琼脂板的制备-将700毫升去离子水与16克的2L 玻璃烧杯和微波结合在一起, 用5分钟的微波炉从微波炉中取出, 用玻璃棒搅拌, 将难溶琼脂放入溶液中。加入17.5 毫升的 10% (瓦特/v) Nipagen 在95% 乙醇, 混合, 然后继续微波加热在三十年代爆发后搅拌, 直到所有琼脂完全溶解。冷却一两分钟, 然后吸管15毫升整除数成10厘米塑料培养皿。允许琼脂凝胶, 盖上盖板, 让他们在室温下干燥几个小时或一夜。在18摄氏度的密封塑料袋中贮存板材。 注释 1:为了避免在板材表面形成 Nipagen 晶体, 由于过量的 dessiccation, 在几天的准备工作中使用板材。如有必要, 晶体可以通过滴几微升95% 的酒精重新溶解在它们上。 附注 2:琼脂批次可以具有不同的胶凝特性。干胶板应该是坚定的触摸和足够的弹性, 一个干净, 完整的琼脂环, 可以删除时, 使用软木塞创建食物孔 (见上文)。</li>
	<li>在化验盘中设置幼虫。使用机械压力曲线的塑料 microspatula 的尖端和蘸在酵母膏的尖端提供 "胶粘剂" 的采摘幼虫。单独捡起第一龄幼虫, 将它们放在靠近食物丘的琼脂盘上。为实验和控制基因型, 准备至少五个10幼虫的复制板。一旦完成, 盖和标签每个板块, 并设置在一个黑暗的空间在室温 (在我们的实验室, 这是22°c) 板 (盖子侧)。</li>
</ol>3. 监测化验盘
<ol><li>每天在解剖显微镜下检查盘子, 记录食物上方或琼脂表面上的幼虫数量。注意任何可见的死亡或垂死的幼虫。注意当和如果, 隧道进入琼脂基质被视为幼虫进入第三龄期。注意蛹开始形成的日期, 并注意任何蛹异常。继续每日观察直到所有幼虫死亡或 pupated。</li>
	<li>用弯曲的针刺针将蛹从盘子中取出, 然后转移到葡萄盘子里。如果需要, 继续监测蛹, 以确定多少 eclose 成人。</li>
</ol>4. 制备成像用检测板
<ol><li>小心地从每个盘子的中央井中取出和丢弃所有的酵母食品。</li>
	<li>用自来水轻轻地将每个盘子淹没, 用一个柔软的画家的画笔仔细地擦掉已经被跟踪到琼脂表面的碎片。更换水几次, 继续温和刷牙, 直到每个盘子完全干净。用去离子水给盘子最后的冲洗, 盖住它们, 在室温下过夜晾干。 注意:隧道进入琼脂是最激烈的周围的食物丘 (见图 2)。因此, 食物孔的边缘通常扩大超过最初的1.5 厘米直径。此外, 在这个地区的工作琼脂的脆弱性可能会导致小块的隧道琼脂在清理过程中从洞的圆周上丢失。增加凝胶琼脂浓度可能有助于这些问题, 但更正是可能的在图像 J 定量步骤 (见下文)。</li>
</ol>5. 隧道的定量
<ol><li>图像的盘子。在黑色背景上准备每个板块的图像, 以显示琼脂中的隧道为明亮的白色线条。 注意:我们使用的系统通常用于图像 DNA 凝胶的这一步骤 (见材料和试剂表)。能够生成 tiff 文件的可选成像系统, 如数码相机或手机摄像机, 可以调整以产生合适的图像。</li>
	<li>使用公共领域程序 NIH 图像 J 定量幼虫隧道。从 http://rsb.info.nih.gov/ij/download.html 下载该程序。有关该程序的详细信息, 则转到 http://imagej.nih.gov/ij/docs/intro.html。 注: 其他成像系统可用于定量隧道。下面的说明适用于 NIH 形象 J。</li>
	<li>打开隧道板的 tiff 文件图像后, 使用椭圆工具定义用于表示整个琼脂表面的定量的区域, 但将板材的塑性边缘裁剪出来。</li>
	<li>应用自动阈值产生的板的反向图像, 与隧道显示为黑色线。使用拾色器和画笔工具将代表对琼脂的损害而不是隧道的黑色区域白色。</li>
	<li>在分析下拉菜单下, 选择设置刻度 |单击以删除缩放, 然后在设置测量窗口 (也在分析下), 检查区域和限制阈值。</li>
	<li>按 Cntrl 键 + M 键以像素显示黑色区域 (隧道)。</li>
	<li>将每个板块的像素值复制并粘贴到电子表格程序 (如 Excel) 中, 以允许进行定量分析。</li>
	<li>由于该区域的强隧穿, 中心孔通常会膨胀。要定量缺少的隧道, 请取消选中 "限制到阈值", 然后使用魔杖工具定义中心孔。按 Cntrl 键 + M 键获取此区域的像素值。从该值减去1.5 厘米孔的像素值, 并将差异添加到步骤5.6 中获得的隧道值。</li>
	<li>对于某些板块, 中心孔可能缺少一条隧道琼脂, 其中包括与孔相邻的untunneled琼脂区域。当量化这些板块的中心孔时, 使用拾色器和画笔工具在缺少的区域中添加一个黑色条来定义隧道区域, 并将非隧道区域从定量中排除。 注释 1:如果实验幼虫执行任何隧道, 这定量允许计算一组板的平均隧穿值和控制和实验值的统计比较。</li>
</ol>

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作者声明他们没有竞争的金融利益。

我们在这里介绍了一种简单的检测方法, 用于测定黑腹鼠幼虫的组织缺氧. 诊断的基础是在早期幼虫生命中减少穴居入食物丘, 以及在幼虫生命后期没有基质隧道。幼虫的拥挤可能导致过早的迁移远离食物来源, 因此, 分析的一个关键方面是, 少量的幼虫在存在大量过剩食物的情况下进行测定。将杀菌剂甲磷羟基苯甲酸酯 (Nipagen) 加入琼脂板中, 对于防止霉菌在测定过程中的生长也是必不可少的。
我们发现, 琼脂板块可以是一个变异的来源, 在化验。通常, 同一基因型的幼虫, 从不同批次的父母, 或从不同的幼虫收集, 显示相对有限的变化, 在他们的行为在化验。相比之下, 在不同的天或不同批次的琼脂制成的琼脂可以产生不同的隧道。因此, 其中一个规定是, 控制和实验幼虫都应该使用琼脂板进行测试, 从同一批次准备。琼脂从不同的制造商, 甚至从同一制造的出货量可能会因其胶凝强度而异, 因此, 可能需要调整琼脂浓度从这里使用的 2.2%, 以达到最佳凝胶。我们发现野生型幼虫可以很容易地在3% 琼脂凝胶中挖洞。
为了证明这一检测的价值, 我们用它来研究在气管的uninflatable抑制功能的幼虫中潜在的组织缺氧.我们的研究结果有力地支持了这样的假说: 这种基因的气管表达丧失会产生缺氧, 如btl-Gal4 > 无人技术-uif rna 干扰幼虫显示在食物中无法挖洞, 完全没有基质隧道。第三龄。在我们先前对其他基因型的研究中, 我们观察到, 缺氧引起的食物挖洞的损失并不像底层隧道的损失那么完整, 而在这里研究的btl-Gal4 > 无人参与-uif rna 干扰幼虫的表现类似。因此, 这种化验失败的隧道成分提供了最强烈的缺氧迹象。
尽管btl-Gal4 > uif rna 干扰幼虫显示了缺氧的行为特征诊断, 但切口(ue)-Gal4 > 无人与uif rna 干扰没有表现出这些异常。btl和剪切(ue) Gal4 驱动程序以不同的阶段和不同的模式在幼虫气管中表示。btl-Gal4 驱动程序是在整个气管系统中表达的, 从胚胎发生的发育开始, 继续通过幼虫的生命。相比之下, 从切口(ue)-Gal4 驱动程序的 Gal4 表达式仅在胚胎生命结束后开始, 在气管发生后, 仅限于背树干的后部, 主要纵向血管气管系统。因此, 使用此 Gal4 行击倒的uif可能不会过早地减少uif表达式, 也不足以产生触发此检测中测量的行为所需的低氧阈值水平。
先前的研究发现, 第三龄幼虫暴露在低 (10%) 氧水平显示下降的增长和延迟发病的 pupariation 14。在这里研究的btl-Gal4 > 无人管理系统-uif rna 干扰幼虫进展到了第三个龄期, 但对它们的生长和蛹率的影响更明显: 它们比很少脂肪组织的控制小得多。表皮 (图 3), 并且只有次要分数 (~ 10%) 尝试 pupariation。这些差异表明btl-Gal4 > 无人参与-uif rna 干扰幼虫经历了更大程度的缺氧, 或者是因为uif在气管中的击倒在整个幼虫生命中存在, 或者因为它产生了更三龄时严重缺氧。在这一点上, 在气管中uif函数的丢失如何防止氧传输目前还不清楚。btl-Gal4 > 无人飞机-uif的气管通过角质层 (图 3) 可以很容易地看到, 这表明它们包含空气, 并没有被损坏到流体进入损害功能的地步。因此, 由于uif功能的丧失而造成的气管损害并不引起缺氧, 而是一些其他阻碍隧道的缺陷。对于先前研究的基因型, 我们确定, 隧道的失败与高浓度的 LDH mRNA7有关, 在晚期第三龄幼虫的糖酵解和缺氧的规范指标15。因此, 对btl-Gal4 > 无人参与-uif rna 干扰幼虫 (以及在今后使用本化验中检查的幼虫) 的缺氧的最后确认将涉及 rt-pcr, 以评估 LDH mRNA 水平或使用商业上可用的指标来衡量胞内氧水平 (例如, 请参见16)。

复杂的分子遗传工具阵列可在黑腹中使用, 这使它成为研究进化保守的生物过程的宝贵有机体. 在进化过程中, 对氧气可用性的关键分子反应被证明是保守的, 在D.腹腹中, 先前的研究已经产生了对这些信号通路的通用组件的洞察力1,2, 3,4,5,6。
作为一项研究, 目的是解剖D.黑腹幼虫的感官神经元功能, 我们确定了两种行为反应, 证明是在正常氧水平7的组织缺氧激活。其中之一, 没有挖洞到食物, 是高度相关的反应低氧水平报告的 Wingrove 和 O ' 法雷尔8。第二种行为是, 在晚期第三龄期流浪阶段, 未能进入软基底, 以前并没有发现与缺氧有关。我们确定, 暴露野生型流浪幼虫到低氧水平也抑制了基质隧道的7, 从而确定这两种行为都来源于缺氧-无论是由组织损伤或低氧摄入水平引起的。在这里, 我们描述了我们已经发展到定量这两种缺氧诱导行为, 这是从幼虫孵化后立即观察开始。
早期幼虫阶段的缺氧反应没有被检查过, 因此在整个幼虫生命中进行分析是我们化验的一个重要组成部分。大多数明显的缺氧表现--缓慢的发育、不良的生长和运动迟缓--与许多突变产生的幼虫表型重叠。但我们发现, 只有第三龄幼虫缺氧, 才显示隧道7完全失败。因此, 我们确定, 即使幼虫在生长和运动方面比我们的缺氧幼虫更受损害, 仍然执行了一些隧道, 而缺氧幼虫从来没有隧道7。这项化验的另一项有价值的因素是, 它提供了一种方法, 以建立当缺氧是特定的多效性表型的来源, 而不是其他一些压力或代谢故障。作为化验的演示, 我们在这里描述了它的使用, 以表征幼虫的反应, 减少气管表达的uninflatable, 一个基因, 功能在幼虫气道9。
我们设想, 这种化验将是有价值的研究人员的特性, 包括不良的生长和迟缓行为的幼虫表型。因此, 可以确定影响整个人体内氧气分布、使用或反应的新基因。此外, 将这一分析纳入突变体筛选协议, 将为确定产生缺氧的突变提供直接途径。这一检测也将有价值的分析电路, 诱发缺氧诱导的先天行为在这里描述。这种类型的神经网络分析是目前研究的热点, 而黑腹型幼虫的简单神经系统是解剖自动行为的重要系统. 已经确定了与幼虫氧知觉有关的感官神经元, 为确定缺氧诱发反应的完整电路提供了第一步10,11。使用我们的检测结合选择性神经击倒通过 GAL4-UAS 系统12是一个明确的路线, 以划定进一步的组成部分, 神经网络。

<table><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dehydrated yeast&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Frozen grape juice concentrate</td><td>Welch&#039;s</td><td></td><td>&amp;nbsp;Available at most large supermarkets</td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td></td></tr><tr><td>Drosophila agar</td><td>Apex Bioresearch Products</td><td>66-103</td><td></td></tr><tr><td>Methyl-para-hydroxybenzoate</td><td>Apex Bioresearch Products</td><td>20-658</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 ml polypropylene beakers</td><td></td><td></td><td></td></tr><tr><td>6.0 cm disposable Petri dishes</td><td>Falcon</td><td>08757100B</td><td></td></tr><tr><td>10 cm disposable plastic Petri dishes</td><td>E+K Scientific</td><td>EK-24104</td><td></td></tr><tr><td>Plastic microspatulas</td><td>Corning Incorporated</td><td>3012</td><td></td></tr><tr><td>Bent teasing needle</td><td>Nasco</td><td>S08848MH</td><td></td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>Any microscope with 10-30X magnification</td></tr></tbody></table>

a burrowing/tunneling assay for detection of hypoxia in drosophila melanogaster larvae

动物缺氧可能是由于暴露在低大气氧水平或内部组织损伤, 干扰氧分配。氧敏感神经元的异常行为也可能在正常氧水平存在时诱发缺氧样的行为。在黑腹中, 低氧水平的发育会在幼虫阶段抑制生长和迟缓行为. 然而, 这些已建立的氧缺乏表现与许多变异的表型重叠, 这些突变调节生长, 压力反应或运动。因此, 目前尚无可用于鉴别 i) 细胞缺氧的方法, 由突变或 ii. 类缺氧行为诱发的异常神经元行为所致。
我们最近确定了在正常氧水平下发生的对缺氧的内部检测的两种不同的行为. 首先, 在所有阶段, 这样的幼虫避免挖洞进入食物, 经常偏离远离食物来源。其次, 当幼虫缺氧时, 在游荡的第三龄期内通常发生的隧道进入软基底。本文所描述的检测方法旨在检测和定量这些行为, 从而提供一种检测内源性损伤而非低外氧诱发的缺氧的途径。用琼脂基质和酵母糊的中央插头的化验板, 用于支持动物通过幼虫生活。幼虫的位置和状态每天跟踪, 因为它们从第一个到第三个龄期开始。蛹使用 NIH ImageJ 后, 在游荡期进入琼脂基质的隧道量化。在确定缺氧是突变体表型的一个组成部分时, 该检测将具有重要的价值, 从而为有关基因的可能行动地点提供洞察力。

作为这项试验的价值的证明, 我们用它来研究在气管中基因uninflatable (uif) 功能受损的幼虫的潜在缺氧。uif对在幼虫气管细胞顶端表面强烈表达的大跨膜蛋白进行编码。前述uif突变体表现出异常行为, 可能表明由于气管故障9导致的组织缺氧。我们使用 Gal4-UAS 系统在幼虫气管中明确地抑制了uif表达式。使用了两个 Gal4 线: i)气喘吁吁(btl)-Gal4, 强烈表达在气管从他们的胚胎发育和 ii)切开(ue)-Gal4, 开始表达在一个小后区主背主干气管在胚胎发生末期, 并继续在整个幼虫生命区的强烈表达7。从维也纳果蝇研究中心 (VDRC ID #1050) 获得了一个无人服务的uif rna 干扰线。
如上所述, 五个新孵化的第一龄幼虫的复制板, 分别为四个十字架, 如下。
实验1 1) 控制 1-剪切(ue)-Gal4 x S (+) 2) 实验 1-剪切(ue)-Gal4 x 无人接入-uif rna 干扰
实验2 3) 控制 2- btl-Gal4 x S (+) 4) 实验 2- btl-Gal4 x 无人接入-uif rna 干扰
剪切(ue) 的行为-Gal4 > 无人参与-uif rna 干扰实验1的幼虫与控制切割(ue)-Gal4 > + 动物在挖洞, 隧道和生存到蛹, (图1和图 2)。相比之下, 从早期开发 (实验 2) 开始, 在整个气管系统中向下调节uif表达式产生了明显不同的反应。btl-Gal4 > 无人参与-uif rna 干扰幼虫显示了组织缺氧的行为表现-减少了食物挖洞和在幼虫生命后期完全没有基质隧道 (图 1 和图2).实验幼虫也明显小于对照组 (图 3) 的体积较小、较薄、较慢, 并在化验过程中显示出较高的死亡率。如上文所述, 减少生长, 迟缓和生物体死亡是已知的幼虫缺氧症状。此外, 大多数实验幼虫在控制幼虫 pupated 后, 未能尝试蛹, 并保持为第三龄幼虫。在没有化蛹的情况下, 一些幼虫存活了超过15天, 最终死亡 (图1A)。一些实验幼虫试图蛹, 但在所有情况下, 不正常的蛹形成, 没有产生可行的成年人。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57131/57131fig1.jpg"> 图1。定量的食物穴居和幼虫的发展。 (A) 在这里研究的四种基因型中, 食物丘外的活幼虫百分比。显示了用于每个基因型的五个检测板的平均值。孵化后的3天, 70% 的btl-Gal4 > 无人能的uif rna 干扰幼虫在食物之外, 而在食物以外或琼脂表面检测到其他三基因型的幼虫。btl-Gal4 > 无人合作的uif rna 干扰幼虫在4-20 天之间慢慢死亡, 15% 的人在孵化后15天仍活着。沿 x 轴的黑色箭头在这里和 (B) 表明三个其他基因型的所有幼虫 pupated 的那天。 (B) 四基因型的食物外可见的死亡幼虫百分比。显示了每种基因型的五个检测板的平均值。死的btl-Gal4 > 无人使用的uif rna 的幼虫随着时间的推移积累起来, 而在食物以外的大部分死亡。其他基因型都生存蛹 (C) 四基因型研究的蛹生存率。显示了每种基因型的五个检测板的平均值。无正常蛹的btl-Gal4 > 无人能的uif rna 基因型的产生。整个图中的误差线表示中小企业。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57131/57131fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57131/57131fig2.jpg"> 图2。幼虫隧道的定量。 (A) 在准备隧道定量后研究所有四种基因型的试验板的例子。注意btl-Gal4 > 无人操作uif rna 干扰幼虫完全没有隧道。检测板为10厘米的培养板。 (B) 四基因型的图像 J 中的隧道定量。每个基因型的五个检测板的平均像素值。未观察到btlGal4 > 无人操作uif rna 干扰幼虫的隧道。误差线 = 中小企业。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57131/57131fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57131/57131fig3.jpg"> 图3。btl-Gal4 > 无人接入uif rna 干扰和btlGal4 > 幼虫的比较 类似年龄的幼虫 (孵化后6天) 为btl-Gal4 > 无人能的uif rna 干扰 (A) 和btl-Gal4 > + (B) 基因型。红色箭头指向背主干气管。两个幼虫以相同的放大率成像。刻度条 = 0.5 毫米.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57131/57131fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae at JoVE.com

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

该议定书描述了一种简单的检测方法, 用于识别在正常大气氧水平下缺氧的果蝇幼虫。该协议允许低氧幼虫与其他突变体区别, 这些变种显示重叠的表型, 如迟缓或缓慢生长。

用于检测果蝇幼虫缺氧的挖洞/隧道法

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen ...

a-burrowingtunneling-assay-for-detection-hypoxia-drosophila

Universidad San Sebastian

Research

JoVE Journal

Behavior

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

7.8K 次观看.  Rice University. 该议定书描述了一种简单的检测方法, 用于识别在正常大气氧水平下缺氧的果蝇幼虫。该协议允许低氧幼虫与其他突变体区别, 这些变种显示重叠的表型, 如迟缓或缓慢生长。

视频: A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

科研

发育生物学

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

神经科学

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

果蝇黑色素血脑屏障完整性分析

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

<meta charset="utf8"></head><body>一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

行为学

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 ...

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

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

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

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

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 ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

生物学

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

光斑分析分析果蝇拉瓦尔光轴

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

用于检测果蝇幼虫缺氧的挖洞/隧道法

摘要

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此视频中的章节

诱导生活青蛙和斑马鱼胚胎缺氧

对检测方法果蝇行为

在幼虫馈电电路的功能分析果蝇

果蝇黑色素血脑屏障完整性分析

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

果蝇幼虫和成年脑的代谢分析

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

幼虫活动的测量

光斑分析分析果蝇拉瓦尔光轴

用于检测果蝇幼虫缺氧的挖洞/隧道法

摘要

探索更多视频

此视频中的章节

诱导生活青蛙和斑马鱼胚胎缺氧

对检测方法果蝇行为

在幼虫馈电电路的功能分析果蝇

果蝇黑色素血脑屏障完整性分析

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

果蝇幼虫和成年脑的代谢分析

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

幼虫活动的测量

光斑分析分析果蝇拉瓦尔光轴

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

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