我们感谢 Kopito, 罗和皮尔斯实验室的成员在这些方法开发过程中的许多有益的讨论。我们还感谢布赖恩 Temsamrit 对这篇手稿的批判性阅读。这项工作得到了科学大学和水史密斯慈善信托基金的资助。

1. 在果蝇中耦合 Gal4 和 Htt 的转基因表达
<ol><li>收集和/或生成含有组织特定 Gal4 或资历 "驱动程序" 的转基因果蝇线, 以及包含 Gal4-UAS38或 Htt QUAS39转基因下游的野生型或突变体的线。确保从这些转基因表达的蛋白质被融合到荧光蛋白或表位标记, 以允许分化的突变和野生型 Htt 转基因产品在同一苍蝇。请参见图 1。 注意: 我们通常使用的外显子1片段的人类 Htt 基因27。然而, 转基因苍蝇可以生成, 以表达较长的 Htt 片段或其他基因, 如果需要的话。</li>
	<li>计划基因策略, 如突变体和野生型 Htt 转基因将表达在不同的, 非重叠的细胞群体。<ol>
<li>如果任何表达重叠发生, 突变体和野生型 Htt 蛋白合成在同一细胞将共同聚集和防止检测朊病毒样的事件。为避免这种情况, 请分别使用 Gal4 和阻遏、Gal80 和 QS40(图 1)。选择发育控制的 Gal4 和/或有资历的驱动程序可以帮助限制突变体的表达 Htt 后有丝分裂细胞, 消除了细胞分裂可能有助于细胞对细胞聚集扩散的可能性。</li>
	</ol>
</li>
	<li>使用标准培养条件, 交配苍蝇产生的后代, 通过 Htt (或 Gal4) 在一个 "捐献者" 细胞种群和野生类型的 Htt 通过 Gal4 (或资历) 在 "接受" 细胞群体中表达突变体。有关显示可能的遗传组合的示意图, 请参见图 1 。 注意: 用于生成图 1-4和我们以前的出版物27中显示的数据的资历和 Gal4 驱动程序包括 DA1 嗅受体神经元 (ORN) 驱动程序、Or67d-QF 和泛胶质驱动程序 repo-Gal4。</li>
	<li>生成控制苍蝇平行, 表达野生型 Htt 在 Gal4-labeled 细胞种群。</li>
	<li>收集所需基因型的后代, 并适当地将动物年龄。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56906/56906fig1.jpg"> 图 1.利用 QUAS 和 Gal4-UAS 二元表达系统对突变体和野生型 Htt 转基因进行耦合表达的遗传方法.在 "细胞 A" 中, 一种含有致病长度 polyQ 拉伸 (Q91) 的 mCherry 标记突变 Htt 蛋白, 使用位于组织特定启动子 A ("PA") 下游的一个资历驱动程序表示。在 "细胞 B" 中, 含有正常 polyQ 拉伸 (Q25) 的 YFP 标记野生型 Htt 通过由组织特定启动子 b ("PB") 控制的 Gal4 驱动程序表示。在图 2-4中, Or67d-QF 用于在 DA1 ORNs 中驱动 QUAS-HttQ91-mCherry 表达式, repo-Gal4 用于在所有胶质细胞27中表示 UAS-HttQ25-YFP。重要的是, HttQ91-mCherry 只是表达了在表达的细胞, 凭借 QUAS 序列放置在上游的转基因。同样, HttQ25-YFP 只通过 Gal4 来表示, 它专门识别无人使用。如果检测到 Gal4 驱动程序的组织分布有任何重叠, 则可以介绍 Gal4-expressing 细胞中的 QS 转基因编码和 Gal80 的表达。将荧光蛋白标签添加到野生型和突变体 Htt, 可以在成像过程中对两种蛋白质进行分化, 并能够测量适当的供者/受体对 (例如、CFP/YFP 或 YFP/mCherry) 之间的焦虑。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56906/56906fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 成人果蝇脑的显微解剖和固定
注意: 此解剖过程已从以前的出版物44中进行了修改, 可用于为 Htt 荧光蛋白融合的成像直接荧光信号准备大脑。下一节将讨论对染色大脑的过程进行的修改。
<ol><li>收集下列材料和放置在冰上: 磷酸盐缓冲盐水含有0.03% 海卫 X-100 (PBS/T);离心管含有970µL 的4% 多聚甲醛 (粉煤灰) 固定液, 加入200µL 20% 粉煤灰至770µL PBS/T;含有良好的透明玻璃碟;一次性转移吸管;两个解剖钳 (一3和一 5)。</li>
	<li>麻醉使用 CO2的成人苍蝇, 并将它们转移到冰上的玻璃盘中的一个井中。</li>
	<li>使用转移吸管, 增加一个小数量 (~ 500 µL) 冷 PBS/T 对井包含苍蝇并且到空井。避免引入太多的气泡, 因为它们可以干扰解剖。</li>
	<li>将玻璃盘子放在解剖显微镜下的平坦表面上, 调整放大倍数, 直到飞体填充视野并聚焦。</li>
	<li>放置一对鹅颈光源, 使光线照亮玻璃盘子的两面。在玻璃盘子下锚定一个折叠的实验室组织, 在解剖过程中丢弃身体部位/角质层。</li>
	<li>在非显性手上使用3钳, 将一只苍蝇转移到含有 PBS/t 的井中, 通过抓住腹部与腹侧朝上, 将苍蝇固定在飞行中。</li>
	<li>保持苍蝇完全淹没在 PBS/T, 删除飞头与5钳在主手。在苍蝇头一侧的喙旁边的小空间中, 将这个钳的一个尖端插入到角质层下。通过捏紧钳子抓住两个侧面的眼睛, 以确保头部的抓地力。</li>
	<li>将两个钳拆开, 将飞头从其体内取出。在附近的实验室组织中处理飞行物体。保持主手钳的压力, 使飞头不会丢失。</li>
	<li>将3钳的一个尖端放在非显性的手里, 在喙另一侧的角质层下的同一小空间内。一旦定位, 捏钳, 抓住眼睛在同一位置两侧的头部。</li>
	<li>一旦对角质层的抓地力是安全的, 轻轻地拉钳在180°分开。这一行动将打破头部角质层, 而不损害大脑。在实验室组织中丢弃角质层残留物。<ol>
<li>虽然理想, 头部角质层可能不会完全删除一步。在这种情况下, 取出角质层, 直到大脑完全暴露。注意不要通过避免与镊子直接接触而损害大脑。</li>
	</ol>
</li>
	<li>从 PBS/T 中取出解剖过的大脑, 或者通过抓住连接的气管, 或者通过毛细管动作将大脑吸进钳尖之间的空间。 注: 气管切除是可选的, 因为它往往不会掩盖我们通常想象的大脑前表面的触角裂片。然而, 如果气管干扰成像, 仔细地删除它们, 这样大脑就不会被这种操作所破坏。</li>
	<li>将飞入的大脑转移到含有冰上固定液的离心管中。确保大脑与镊子分离, 并浸入固定液中。</li>
	<li>一旦所有的大脑被解剖, 将其置于一个 "短修复" 的 nutator, 在室温下将闭合的管子放置在黑暗中5分钟。 注意: 这个短的固定步骤确保大脑在随后的步骤中更容易处理, 不坚持离心管的两侧。</li>
	<li>用 P1000 吸管除去大多数固定液, 然后丢弃。<ol>
<li>避免在这段期间和任何随后的洗涤步骤中从管子中吸出大脑。将 P1000 设置为较小的卷 (例如, 650 µL), 并在两个步骤中移除上清。另外, 将离心管与光源 (如架空灯) 保持一致, 同时吸气以更好地可视化大脑。</li>
	</ol>
</li>
	<li>增加1毫升的新鲜 PBS/T 的大脑。在黑暗中迅速洗涤 (< 1 分钟), 从大脑中吸取上清, 然后丢弃。</li>
	<li>根据以下时间表在黑暗中重复洗涤与 PBS/T 在室温下: 2 快速 (< 1 分钟) 洗涤, 1 x 5 分钟, 3 X 20 分钟和 1 X 1 h 洗涤。在离心管上安全顶部, 并将管子放在洗涤间的 nutator 上。<ol>
<li>如果需要 DAPI 染色, 请用 1 x 30 分钟的孵化 DAPI 在 PBS/T 中稀释1小时, 然后用 2 x 快速和 1 x 20 分钟洗涤。</li>
	</ol>
</li>
	<li>最后洗完后, 取出大部分洗涤缓冲器上清, 注意不要扰乱大脑, 并添加30µL 甘油基 antifade 试剂给大脑。在黑暗中孵化4摄氏度, 不运动至少1小时, 最多24小时。</li>
</ol>3. 对成人大脑染色2节的修改
注: 使用本协议用于成像非荧光蛋白或荧光蛋白融合弱荧光。
<ol><li>每步增加10倍 (pbs + 0.3% 海卫 X-100) 在 PBS/T 中 X-100 的浓度。这确保了足够的洗涤剂存在 permeabilize 细胞膜和允许抗体进入细胞内的空间。</li>
	<li>在室温下将4% 只粉煤灰中的大脑固定在 PBS/T 中, 20 分钟。</li>
	<li>在完成所有的洗涤后, 孵化大脑在新鲜准备的阻断缓冲 (PBS/T + 5% 正常山羊血清 ()) 在室温下在黑暗中30分钟。</li>
	<li>删除并丢弃阻塞缓冲区。添加0.5 毫升的主抗体溶液每管准备作为一个主混合, 稀释原抗体适当的新鲜堵塞缓冲区 (见材料表, 用于常用抗体和稀释)。在 nutator 的主要抗体上孵育大脑至少24小时, 在4摄氏度的黑暗中。</li>
	<li>用 P1000 和储备在新鲜的试管中去除大脑中的主要抗体溶液。添加1毫升的 PBS/T 洗脑。 注意: 保留的主要抗体溶液可以储存在4摄氏度, 长达四周。在随后的实验中, 我们成功地将原抗体回收了两次。</li>
	<li>快速冲洗大脑 (< 1 分钟), 从大脑中吸取上清, 然后丢弃。再次重复此快速清洗。</li>
	<li>在室温下继续以1毫升的 PBS/T 洗涤, 按照以下时间表: 1 x 5 分钟, 3 x 20 分钟, 1 X 1 小时清洗。在离心管上安全顶部, 在洗涤过程中将管子放在 nutator 上。</li>
	<li>最后一次清洗后, 除去大多数的 PBS/T 上清, 并添加0.5 毫升的二级抗体解决方案, 作为一个主混合, 稀释的抗体适当的新鲜堵塞缓冲区 (见材料表常用抗体和稀释)。在黑暗中, 在4摄氏度的24小时内孵化大脑的二级抗体。</li>
	<li>在3.5、3.6 和3.7 步骤中重复清洗步骤, 然后再用二级抗体进行孵化。</li>
	<li>最后洗完后, 取出大部分的洗涤缓冲器, 确保所有的大脑都位于管子的底部。给大脑添加30µL antifade 试剂。在黑暗中孵化4摄氏度, 不运动16-24 小时。</li>
</ol>4. 全脑安装
<ol><li>将玻璃显微镜幻灯片放在解剖显微镜下, 用标识信息进行标签。 注: 或者, 大脑可以直接安装在盖玻璃上, 以便在成像过程中访问大脑的两侧。描述此安装过程的协议以前已发布45。</li>
	<li>使用钝吸管尖 (用剃刀刀片将底部 ~ 1 厘米从1-200 µL 尖上取下) 取出每个离心管的大脑, 并将它们转移到幻灯片中间。尽可能少的用大脑 antifade 试剂。</li>
	<li>使用镊子将大脑定位在所需的方向上 (例如, 背向指向幻灯片顶部和朝向触角叶成像的前表面)。在定向大脑时, 考虑显微镜的光路, 用于成像它们。</li>
	<li>使用折叠的实验室组织的尖角, 不干扰大脑, 从幻灯片中取出多余的 antifade 试剂。让幻灯片在黑暗中坐5-10 分钟, 让大脑坚持幻灯片。</li>
	<li>周围的苍蝇大脑与四小块的破碎盖玻璃放置在大脑的每一侧, 形成一个正方形, 是 ~ 19 毫米 x 19 毫米. 将 22 mm x 22 毫米1.5 盖玻璃的一个边放在这些小玻璃片的外面, 并轻轻地降低盖玻片的大脑, 以完成桥安装。</li>
	<li>慢慢地分配新鲜的 antifade 试剂填充盖玻片下的表面, 小心不要扰乱盖玻片或大脑。用折叠的实验室组织的尖角擦拭任何多余的 antifade, 而不直接接触盖玻片。</li>
	<li>添加一滴清晰, 快速干燥指甲油四角的盖玻片。允许干燥10分钟。然后, 用指甲油封住盖玻片的四边缘, 完全包围大脑。</li>
	<li>图像大脑立即, 或存储在4°c, 直到准备就绪。<ol>
<li>如果从 Htt 荧光蛋白融合成像内在荧光, 图像的大脑在24小时内的最佳信号。</li>
	</ol>
</li>
</ol>5. 影像学和量化的聚集体病毒样传播
<ol><li>使用装有40X 或 60/63 x 油的共焦显微镜图像安装的大脑目的是通过大脑区域收集 z 切片图像 (图 2a, B), 这些 Gal4 和资历驱动程序被表示出来。<ol>
<li>激发荧光蛋白融合或 immunolabeled 蛋白质使用适当的激光器 (例如, 488 毫微米为 GFP/YFP/FITC 或552毫微米为 mCherry/Cy3)。设置检测窗口, 捕捉最大的荧光信号, 同时消除通道交叉交谈使用的光谱检测系统或带/长通行证发射过滤器特定的每个荧光。 注意: 成像蛋白聚集时要细心。在每个聚合体中心的像素很容易变得饱和, 特别是在较大的骨料中。尝试尽量减少图像中的饱和度, 但要知道从较小的聚合种中丢失信号的风险 (图 2B、C、D)。过多的饱和度会增加图像的背景, 从而更难识别孤立的 puncta。在拍摄每个数据集的最终图像之前, 测试不同的成像设置以进行优化。</li>
	</ol></li>
	<li>通过通过单独的 z 切片 (例如、 图 2和图 4) 或在3维度中呈现共焦切片 (图 3 ) 后, 通过手动量化单个 puncta 来分析数据。A、B)。 注意: 这可以在图像 J 或其他图像处理软件中完成。<ol>
<li>如果聚合体分离良好并且有极小的背景荧光, 则使用图像分析软件系统地识别、量化和分析共焦栈3D 重建中的不同 "对象" 或 "曲面" (图 3B)。 注意: 所描述的软件操作特定于此处使用的仪器和软件 (请参阅材料表)。<ol>
<li>在3D 查看模式下可视化共焦 z 系列。使用 "分析" 向导标识选定通道中的各个点 (例如图 3中 HttQ91-mCherry 聚合的红色通道)。调整阈值和筛选器, 以准确地将所有异种地大小的聚合作为图像中的单个对象来表示。</li>
			<li>在 "二进制处理预筛选" 下启用 "拆分对象", 以分离与软件算法合并的 aberrantly 紧密关联的聚合。请注意, 在 "测量" 下报告的对象总数及其相关度量值。 注: 这种量化突变 Htt 骨料的方法不适合染色协议, 因为淀粉样体聚集的中心, 对抗体是坚不可摧的。结果表明, 聚集体在显微镜下作为环形结构出现, 图像分析软件无法准确识别和区分这些个体的 "斑点"。</li>
		</ol>
</li>
		<li>通过手动移动 z 堆栈和评分野生类型的 Htt 聚合来量化野生类型的 Htt 聚合, 确保如果它们出现在多个切片 (图 4A) 中, 则不会对聚合进行双重评分。 注意: 如果每个共焦堆栈中的聚合数合理 (例如20-50), 则可以使用此手动量化方法。当图像分析软件无法将单个 puncta 与周围的信号 (例如) 区分开来时, 它也很有用 (图 2B, C和图 4A). 对野生型 Htt 骨料进行量化也很有挑战性, 因为大脑中的许多正常结构出现点状 (例如、细胞过程和突触)。野生型和突变 Htt 信号的协同定位可以作为选择标准;然而, 某些突变体的 Htt "种子" 可能低于共焦检测的极限。不在接收单元 (例如、GFP) 中聚合的非 Htt 的蛋白质可用于标记大脑中不相关的点状结构。</li>
	</ol></li>
	<li>使用图像分析软件对单个骨料进行进一步的表征。例如, 确定聚合的大小分布 (图 3C)、突变体和野生型 Htt 蛋白之间的协定位百分比 (图 4A), 或使用烦恼直接测量蛋白质-蛋白质相互作用 (图 4B)。<ol>
<li>使用现场或表面检测算法确定单个 puncta 的大小分布, 以便准确地识别特定通道中的所有可见聚合。使用图像分析软件对点或曲面进行相关测量, 例如, 在步骤5.2.1 中描述的软件 "分析" 向导中, 从 "测量" 中获取 "总直径" (图 3C)、"体积" 或 "强度" 信息。 注意: 在图 3C中, 我们报告了在单个 DA1 肾小球中确定的所有 HttQ91-mCherry puncta 的直径分布。</li>
		<li>通过通过共聚焦 z 堆栈 (最精确的方法) 手动移动切片, 确定 HttQ25-YFP 和 HttQ91-mCherry 聚合之间的百分比。但是, 注意不要数两次聚合。为防止这种情况发生, 仅当焦点的平面通过聚合的中心 (图 4a) 时, 才计算特定 z 切片中的聚合。</li>
		<li>采用受体漂白法计算 colocalized HttQ91-mCherry 信号诱发 HttQ25-YFP 集料的烦恼效率。<ol>
<li>首先, 在 YFP 和 mCherry 通道之间消除潜在的交叉对话, 方法是建立检测窗口, 以便在其他信道中不产生信号的 mCherry 和仅 YFP 信号。使用这些设置, 请在单个 HttQ25-YFP puncta 及其关联的 HttQ91-mCherry 信号 (图 4B) 中采用 "前映像"。</li>
			<li>然后, 通过调整红色激光器 (例如, 552 nm) 到100% 的强度和扫描直到信号消失, photobleach mCherry 信号。还原为 "前映像" 所用的设置, 并在同一 puncta 中采取 "后映像" (图 4B)。</li>
			<li>用图像分析软件计算漂白前后各泪的荧光强度测量。</li>
			<li>通过在 "图像后" (YFPfinal) 中, 从 YFP 捐赠者荧光中减去在 "图像前" (YFP初始) 中测量的 YFP 捐赠者荧光来计算烦恼效率。通过 YFPfinal除以此值, 并乘以100。 注意: 对于每个 HttQ25-YFP 聚合 (图 4B), 可以按像素或整体计算 "烦恼" 效率。受体漂白是一个特别有用的技术, 以计算苦恼效率时, mCherry 信号与每个 HttQ25-YFP 泪是足够高, 以产生可检测 YFP dequenching 后, mCherry。</li>
		</ol>
</li>
	</ol></li>
</ol>

作者没有什么可透露的。

随着神经退行性疾病患者的数量继续增加, 迫切需要增加对这些疾病分子发病机制的了解, 以便更好地开发治疗方法。在这里, 我们描述的方法, 可以监测病毒样的传播致病蛋白聚集在不同的细胞类型之间的模型有机体,果蝇。我们最近使用这种方法来演示突变体 Htt 集的病毒样传播在体内, 并确定一种吞噬性受体, 介导这些聚合体从神经元扩散到胶质细胞27。我们的方法利用了使用果蝇研究遗传人类疾病的几个好处: 它的短生命周期和庞大的遗传工具, 它可以加速发现与治疗相关的基本生物学信息。
我们这里描述的方法提供了两个主要优势比其他现有的动物和细胞培养模式的朊病毒样的传播: (1) 聚集致病剂 (例如, 突变体 Htt) 是在一个完整的组织中产生的细胞, (2)同一蛋白质 (例如、野生型 Htt) 在单独的细胞群中的正常折叠版本的表达为类似于朊病毒的事件提供了一个易于访问的 "记者"。我们通过使用在果蝇40中建立的成熟的基因工具, 在同一生物体内的非重叠细胞种群中实现了突变体和野生型 Htt 蛋白的表达。由于许多不同的组织特定的 Gal4 和资历驱动程序是现成的, 检查朊病毒样转移本质上任何不同的细胞类型在飞行体是可行的。
该方法的一个关键组成部分是在同一动物体内的不同细胞群中实现突变体和野生型 Htt 蛋白的分离表达。任何表达式重叠都需要消除, 以便接收单元中的野生类型 Htt 聚集准确地报告来自捐献者细胞的朊病毒样集的细胞质渗透率9,12,27, 41。这可以通过引入附加的遗传工具 (例如、Gal80 和 QS 阻遏40) 来实现, 以缓解此问题。一旦理想的基因型被设计和选择, 就必须建立一个量化 puncta 的系统方法。这在很大程度上将取决于标记的单元格数、出现的聚合数以及样本的信噪比。正如我们在图 4中所描述的那样, 可以使用联合本地化和/或正烦恼等标准来分析数据。然而, 根据这些特征限制野生型 Htt 集料的选择可能导致对朊病毒样转移事件的低估, 因为某些突变 Htt 的聚合种子可能低于共焦显微镜检测的极限。
此处描述的体内方法并不排斥与 HD 或甚至其他 polyQ 疾病相关的聚合体的朊病毒行为。转基因苍蝇可以开发, 以检查朊病毒样的传播 synuclein 在 PD, 头在 AD, 和 SOD1 或 TDP-43 在 ALS 使用相同的实验范式。对于这些蛋白质中的每一种, 聚集易发突变体应在供体细胞中表达, 而同一蛋白的可溶性版本只有在有核的时候才能在受体细胞中表达。这一实验范式也有助于调查新的观点, 即与不同疾病相关的致病蛋白可能通过交叉播种机制47进行交互。最后, 在果蝇中可用的无数遗传工具可以用来调查和鉴定与这些致命疾病相关的致病性蛋白聚集物的细胞质对细胞质传播的分子机制。

朊病毒假说指出, 负责传染性海绵状性脑病(例如, 人克雅氏病, 羊瘙痒病绵羊, 鹿和麋鹿的慢性浪费疾病, 和牛的疯牛病) 的传染性剂) 完全由蛋白质组成, 缺乏核酸1。在朊病毒疾病中, 细胞朊蛋白 (prpC) 假设一个非本机、稳定的折叠 (prpSc), 它具有高度 beta 版, 可以通过将单体 PrPC分子转换和招募到稳定的淀粉样中进行自我传播。聚合.PrPSc聚合使用此自复制机制在有机体中的不同单元格之间进行传播, 甚至在单个有机体之间进行扩展2。
蛋白质错误折叠和聚集也是大多数神经退行性疾病 (阿尔茨海默病 (AD)、帕金森病 (PD)、亨廷顿氏病 (HD) 和肌萎缩侧索硬化症 (ALS)) 的中心特征 3.这些疾病中或超细胞内聚集蛋白的形成与细胞毒性4密切相关, 随着时间的推移, 通过大脑的高度可重现性和疾病特定的路径进展5,6. 这些传播模式表明, 与这些疾病相关的致病性聚集物具有类似朊病毒的性质。现在有强有力的支持, 因为朊病毒样的传播与 AD、PD、HD 和 ALS 有关的聚合体-它们从细胞到细胞和模板在以前未受影响的细胞中单体形式的构象变化7, 8。
迄今为止, 大多数研究都是利用哺乳动物细胞培养模型进行的, 调查蛋白质聚集蛋白样的扩散, 从细胞外空间或从另一细胞中转移到天真细胞的细胞质中。细胞质9,10,11,12,13,14,15, 或将含骨料材料注入鼠标大脑和监测聚合外观在注入站点的外部16,17,18,19,20,21,22, 23. 最近, 转基因动物被用来证明胞内聚集物扩散到完整大脑中的其他细胞中24,25,26,27, 28,29,30。在这里, 我们描述了一个方法, 直接可视化的总转移之间的单个细胞在完整的大脑中的果蝇。果蝇模型的 HD/polyglutamine (polyQ) 疾病的首次开发近两年前的31,32 , 并提供了许多宝贵的洞察力的致病机制, 这些疾病的根源33. HD 是一种遗传性神经退行性疾病, 由常染色体显性突变引起, 该基因的编码为蛋白杭丁顿蛋白 (Htt) 34.这种突变导致在 Htt 的 N 终点附近的 polyQ 伸展扩展超过 37 glutamines 的致病阈值, 导致蛋白质 misfold 和聚合35,36。野生型 Htt 蛋白含有 < 37 glutamines 在这个拉伸达到他们的本机褶皱, 但可以诱导聚合后, 直接物理接触 Htt 聚合 "种子"12,27,37。我们利用这一着, 有核聚集的野生型 Htt 作为一个读数的朊病毒样转移和细胞质进入的突变 Htt 聚合体起源于其他细胞。
确定像朊病毒样的聚集物在细胞间穿梭的机制, 可以为无法治愈的神经退行性疾病确定新的治疗靶点。我们利用快速生命周期, 易用性和基因驯良的果蝇, 以确定分子机制的细胞对细胞扩散的突变 Htt 集。我们的实验策略采用了两个二进制表达系统, 在果蝇, 建立良好的 Gal4-specific 上游激活序列 (Gal4-UAS) 系统38和最近开发的 QUAS 系统39。耦合这两个独立的系统允许限制突变体和野生型 Htt 转基因在同一飞行中的不同细胞群的表达40。利用这一方法, 我们通过监测胞质野生型 Htt 从其通常弥漫性、可溶性状态到聚集状态的分布, 对突变体的 Htt 的传播进行检查, 这是与预先形成的突变体物理接触的直接后果 Htt聚合 "种子"。利用突变体 Htt 的生化或生物物理技术, 可以证实野生型 Htt 的转化, 如荧光共振能量转移 (烦恼)9,27,41.
重要的是, 我们还可以访问大量的基因工具在果蝇, 以确定基因和/或途径, 介导朊病毒样的传播蛋白质聚集。最近, 我们使用这种方法来揭开细胞表面清道夫受体, 德雷柏42,43的关键作用, 将突变 Htt 聚合体从神经元轴突转移到果蝇中心附近的吞噬细胞中。神经系统 (中枢神经系统)27。因此, 我们在这里描述的基于遗传和影像学的方法可以揭示在简单使用但功能强大的模型有机体 (果蝇) 中有关疾病相关现象的重要基本生物学信息。

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Phosphate buffered saline (PBS), 10X, pH 7.4</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:344px;">AM9625</td>
			<td style="width:167px;">Dilute to 1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Triton X-100</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">T9284-1L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Kimwipes</td>
			<td style="width:123px;">Thomas Scientific</td>
			<td style="width:344px;">2904F24</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">20% paraformaldehyde (PFA)</td>
			<td style="width:123px;">Electron Microscopy Sciences</td>
			<td style="width:344px;">15713-S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Normal Goat Serum (NGS), filtered</td>
			<td style="width:123px;">Lampire Biological Laboratories</td>
			<td style="width:344px;">7332500</td>
			<td style="width:167px;">Aliquot and freeze upon receipt</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Chicken anti-GFP</td>
			<td style="width:123px;">Aves Labs</td>
			<td style="width:344px;">GFP-1020</td>
			<td style="width:167px;">Use at 1:500 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Rabbit anti-DsRed</td>
			<td style="width:123px;">Clontech</td>
			<td style="width:344px;">632496</td>
			<td style="width:167px;">Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse anti-Bruchpilot</td>
			<td style="width:123px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:344px;">nc82</td>
			<td style="width:167px;">Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">FITC anti-chicken</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:344px;">SA1-7200</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Alexa Fluor 568 anti-rabbit</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">A11011</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Alexa Fluor 647 anti-mouse antibody</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">A21235</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Slowfade Gold Antifade Reagent</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">S36936</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Microscope Slides (25 x 75 x 1.0 mm)</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">12-550-143</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cover Glass (22 x 22 mm)</td>
			<td style="width:123px;">Globe Scientific</td>
			<td style="width:344px;">1404-15</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont Biology Grade Forceps, Style 3</td>
			<td style="width:123px;">Ted Pella</td>
			<td style="width:344px;">503</td>
			<td style="width:167px;">use in non-dominant hand</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont Biology Grade Forceps, Style 5</td>
			<td style="width:123px;">Ted Pella</td>
			<td style="width:344px;">505</td>
			<td style="width:167px;">use in dominant hand</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">LAS X image analysis software</td>
			<td style="width:123px;">Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Imaris image analysis software</td>
			<td style="width:123px;">Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring cell-to-cell transmission of prion-like protein aggregates in drosophila melanogaster

蛋白质聚集是大多数神经退行性疾病的中心特征, 包括阿尔茨海默病 (AD)、帕金森病 (PD)、亨廷顿氏病 (HD) 和肌萎缩侧索硬化症 (ALS)。蛋白质团聚体与这些疾病中的神经病理学密切相关, 尽管不知道异常蛋白质聚集扰乱正常细胞稳态的确切机制。新出现的数据有力地支持了这种假说, 即 AD、PD、HD 和 ALS 中的致病性聚集物与普利子有许多相似之处, 它们是负责传染性海绵状性脑病的仅蛋白感染性药物。普利子自复制通过模板化同一蛋白质本机折叠的版本的转换, 导致聚集表型的传播。在 AD、PD、HD 和 ALS 中, 普利子和朊病毒样蛋白是如何从一个细胞移动到另一单元的, 目前是一个激烈调查的领域。在这里, 一个果蝇模型, 允许监测朊病毒样, 细胞对细胞的传播与 HD 相关的突变杭丁顿蛋白 (Htt) 聚集。该模型利用强大的工具来处理许多不同的果蝇组织中的转基因表达, 并利用荧光标记的细胞质蛋白直接报告突变 Htt 聚合体朊病毒样转移。重要的是, 我们在这里描述的方法可以用来确定新的基因和途径, 在不同的细胞类型之间, 介导蛋白质聚合体的传播在体内。从这些研究中获得的信息将扩大对神经退行性疾病的致病机制的有限理解, 并揭示治疗干预的新机会。

这里描述的方法可以产生强健的数据, 表明在完整的飞行中枢神经系统中, Htt 蛋白聚集物从一个细胞种群转移到另一个体细胞。在捐赠者 ORNs 中 HttQ91-mCherry 表达式 (图 2a-c和图 4a, B) 中, Htt 的 YFP 融合蛋白的直接荧光观察了野生型的从弥漫到点的转化。准确报告这两个细胞之间的朊病毒转移事件需要仔细选择转基因苍蝇和 Gal4/QF 驱动程序, 以产生强烈的表达水平突变和野生型 Htt 转基因没有任何重叠, 在发展或在成年。此外, 精心设计的荧光蛋白-Htt 融合转基因可以使强大的下游分析。例如, 变种人和野生类型的 Htt 聚合可以被量化为有点对象 (图 2C和图 4), 或者使用图像分析软件 (图 3A, B), 可以测量和作为聚合种群 (图 3C) 的进一步特征, 可以评估突变体和野生型蛋白质之间的协同定位 (图 4A), 并且可以分析烦恼27 (图4 B)。这些分析需要融合突变体和野生型 Htt 的荧光蛋白标签与充分分离的荧光性质, 但有足够的光谱重叠, 使焦虑之间的捐助者和受体对 (例如, CFP/YFP9或 YFP/mCherry27)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56906/56906fig2.jpg"> 图 2.神经 HttQ91-mCherry 聚合 HttQ25-YFP 胶质细胞朊病毒样转化的共焦图像.(A) 30 µm 共焦切片的最大强度投影, 显示一个触角叶, 在 DA1 ORN 轴突中使用 Or67d-QF 和 HttQ25-YFP (绿色) 的雄性苍蝇表达 HttQ91-mCherry (红色)。触角叶和 DA1 肾小球的近似边界, DA1 ORN 轴突终止, 分别由虚线和实线表示。(B) 从20µm 共焦切片的最大强度投影, 显示 DA1 肾小球区域从 a. (C) 的放大视图一个单一的0.35 µm 共焦切片显示单个 HttQ25-YFP puncta 及其相关 HttQ91-mCherry 信号 (由每个通道中的箭头指示)。在红色通道中的信号得到增强, 以可视化 HttQ25-YFP 和 HttQ91-mCherry 信号之间的协同定位。所有的图像都是使用 40X 1.4NA 的石油目标获得的。刻度条 = 10 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56906/56906fig2large.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/56906/56906fig3.jpg"> 图 3.DA1 ORN 轴突 HttQ91-mCherry 集料的三维分析(A) 通过 Or67d-QF 使用图 2B中显示的相同数据, 对 DA1 肾小球中表达的 HttQ91-mCherry 聚合进行3D 描述。(B) 使用图像分析软件包显示从 (A) 中的原始数据中标识的单个对象或 "斑点" 的屏幕截图。该软件在该通道/图像中确定了56个大小不同的对象。测量到 (B) 中箭头指示的点的直径为1.2 µm. 箭头指向两个对象在软件中不准确地合并到一个点的位置, 可能是由于单个 puncta 接近。为了克服这种情况, 应该在软件中测试不同的阈值设置, 并且/或合并点应该在可能的情况下手动分离。缩放条 = 10 µm (C) 直方图, 显示在 (B) 中显示的 HttQ91-mCherry "斑点" 软件测量的直径分布。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56906/56906fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56906/56906fig4.jpg"> 图 4.诱导 HttQ25-YFP 集料的共定位与烦恼分析(A) 在 DA1 ORNs 使用 Or67d-QF 和 HttQ25-YFP 在胶质细胞中表达 HttQ91-mCherry 的雄性飞脑中4个单独的0.35 µm 共焦 z 切片的蒙太奇。对信号进行了调整, 使小 HttQ91-mCherry 聚集物可见, 并诱导 HttQ25-YFP 聚集从周围弥漫信号中脱颖而出。所示切片分别由 ~ 1.0 µm (在合并图像的右下角显示切片编号), 以便观察多个聚合。箭头指示在该特定 z 切片中通过手动移动 z 堆栈来确定处于或接近焦点的 HttQ25-YFP puncta。在这里指出的七 HttQ25-YFP puncta 中, 有六个 detectably 相关 HttQ91-mCherry 信号 (即, 86% HttQ25-YFP 聚合与 HttQ91-mCherry 共同本地化)。请注意, 与 HttQ25-YFP puncta 相关的 mCherry 信号通常比 puncta 肾小球中 mCherry 阳性 DA1 的大多数弱。缩放条 = 5 µm (B) HttQ25-YFP/HttQ91-mCherry-co-localized 泪之前 (左面板) 和后 (右面板) mCherry (承兑人) 漂白。由此产生的 YFP (捐献者) 荧光的增加, 用于产生像素逐像素的烦恼效率 (烦恼) 图像使用的 AccPbFRET 插件 ImageJ 46.此特定聚合有一个总的烦恼 61%..缩放条 = 1 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56906/56906fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster at JoVE.com

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

积累的证据支持这种观点, 即与神经退行性疾病相关的致病蛋白聚集物在细胞间扩散, 与朊病毒类似。在这里, 我们描述了一种方法, 使可视化的细胞到细胞传播的朊病毒样聚集在模型有机体,果蝇。

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

monitoring-cell-to-cell-transmission-prion-like-protein-aggregates

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

7.9K 次观看.  University of the Sciences. 积累的证据支持这种观点, 即与神经退行性疾病相关的致病蛋白聚集物在细胞间扩散, 与朊病毒类似。在这里, 我们描述了一种方法, 使可视化的细胞到细胞传播的朊病毒样聚集在模型有机体,果蝇。

视频: Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

简单的检测方法的社交回避的量化<em&gt;果蝇</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

在条件影响社会空间<em&gt;果蝇</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

觅食路径长度协议<em&gt;果蝇</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

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 ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.