我们感谢桑德拉 n. Manosalvas-Kjono, 普珥节 Ladpli, 法拉梅蒙, Divya Gopisetty, 和维罗妮卡支持在设备设计和产生变种动物。这项研究得到了 NIH 赠款 R01EB006745 (BLP)、R01NS092099 (MBG)、K99NS089942 (至 MK)、F31NS100318 (氮化铝) 的支持, 并获得欧洲联盟2020研究和创新计划 (协调委员会) 的资助 (授予 MK 715243 号协议)。

1. 设备制造
<ol><li>下载附加的掩码文件 (补充文件 1), 并使用商业服务或内部设施生成 chrome 掩码。由于器件的最小尺寸为10µm (执行器膜厚度), 确保掩模具有足够高的分辨率, 在0.25 µm 内, 可靠地产生特性。</li>
	<li>
遵循标准的 SU-8 光刻方法 (例如, 引用24,25,26), 以制造随后生产的注塑设备的模具;下面列出了步骤的摘要. 注: SU-8 是一种光敏材料, 在紫外线照射下 crosslinks (最大吸收量约 365 nm)。使用制造商说明作为起点, 确定软烘烤、曝光 (UV) 和后烘烤的加工参数。<ol>
<li>将 SU-8 2002 存入硅片, 将其纺成大约2µm 的厚度, 以更好地粘合小结构。软烘烤在热的板材为1分钟在95°c, 轻微地暴露 (UV) 整个表面 (~ 100 mJ/cm2) 和在热板材烘烤在2摄氏度的95分钟。</li>
		<li>自旋涂层 47-µm 厚层 SU-8 2050 用于定义设备功能。使用 500 rpm 的旋转速度为十五年代 (130 rpm/秒加速), 然后 2000 rpm 九十年代 (260 rpm/秒加速)。如有必要, 重复并调整旋转速度, 以获得适当的光刻胶厚度。</li>
		<li>软烘烤在热的板材2分钟在65°c, 然后7分钟在95°c。根据制造商指令 (〜160兆焦耳/cm2) 公开光刻胶, 使用铬面具和长通滤波器确保直边墙。</li>
		<li>在热板上取出硅片, 后烘烤1分钟, 65 摄氏度, 7 分钟95摄氏度。</li>
		<li>溶解未暴露的 SU-8 与开发商, 和清洁与2丙醇。</li>
		<li>在180摄氏度的热板上烘烤硅片30分钟 (硬烘烤) 以稳定光刻胶的特性。</li>
	</ol>
</li>
	<li>
使用标准的副本成型方法27, 从 SU-8 模型中制作一个来自该产品的。
	<ol>
<li>用 trichloromethylsilane (中药) 蒸气处理 SU-8 霉菌, 以降低其附着力 (硅烷化)。 注意: 中药毒性和水反应。<ol>
<li>将图案化的晶片放在一个钟罐真空干燥内的晶片架内, 不含水或水溶性试剂。</li>
			<li>在引擎盖下, 用吸管将1滴中药在玻璃盘子里, 放在干燥内。</li>
			<li>关闭干燥盖, 允许中药蒸气将晶片涂上至少20分钟。</li>
			<li>通风口, 然后打开干燥。打开钟罐盖子, 用塑料镊子取下晶片。将其放入培养皿或铝箔船上, 用于塑料制品的复制成型。在适当的危险废物中处理中药涂层材料。</li>
		</ol>
</li>
		<li>采用基聚合物40克和4克聚硅固化剂进行混合 (比例为 10:1)。</li>
		<li>将混合物倒入 SU-8 模具上, 将混合物加在真空室至少30分钟。</li>
		<li>在烤箱中至少6小时的70摄氏度的固化。</li>
	</ol>
</li>
	<li>将晶圆片水平放在平坦的表面上, 并仔细剥离该硅烷, 将其从晶片上提起。在这一步, 不要弯曲晶片向上, 因为它可以打破模具。 注意: SU-8 与晶片或不完全硅烷化的不完全粘附会导致 SU-8 结构在这一步骤中被破坏。</li>
	<li>将这些设备上的盖玻片切割成单个芯片, 这样设备就能适应24毫米 x 60 毫米的产品。</li>
	<li>使用1毫米活检冲床, 在入口, 两个插座, 和六执行机构使用1毫米活检打孔。瞄准设备边缘周围2毫米直径的圆圈。</li>
	<li>
将该芯片与玻璃罩片结合在一起。
	<ol>
<li>将两个表面暴露在 80 W 氧等离子体中 (三十年代)。</li>
		<li>轻轻地将暴露在外的表面表面放在盖板滑动面上, 用于保形密封。</li>
		<li>在热板上退火设备 (100 °c, 10 分钟)。 注: 粘结不足会导致设备因渗漏而失效。</li>
	</ol>
</li>
</ol>2. 显微镜的制备
注: 转基因动物: 表达钙指示器, 如 GCaMP6s28或其他基因编码活动探头 (例如, TRN);与活动无关的荧光蛋白发射在不同的波长, 以纠正小的横向和外焦点运动工件出现由于机械刺激。自动分析软件跟踪和补偿活动探针强度的运动引起的变化。蠕虫菌株 GN69223 (或 AQ323629) 表示 GCaMP6s (或 GCaMP6m) 和 tagRFP mec-7 控制下的钙独立助剂。此外, GN692 包含突变简装-1 (ce314), 它防止由于蓝光传感器lite-130在 GCaMP6s 荧光23的激发期间激活 TRNs。
<ol><li>
建立了 GCaMP 和 RFP 同时激发的显微系统。
	<ol>
<li>使用连续光源和双波段励磁过滤器, 仅传输青色和黄色光, 或者使用光源, 仅在定义的带宽中发出波长, 如同时青色 (0.77 兆瓦) 和黄色 (1.21 兆瓦) LED 励磁源, 用于同时激发钙依赖性和独立荧光, 分别。</li>
		<li>使用数码相机来记录显微图像。 注: 大多数照相机都带有用于图像采集的软件。另外, 也有免费的软件, 可以用来控制相机和可能也有其他部分的显微系统。</li>
		<li>根据荧光强度调整励磁强度, 避免相机饱和。</li>
	</ol>
</li>
	<li>
使用一个荧光立方体, 根据激发源的选择需要配备一个励磁过滤器。 注: 488 纳米 GFP 和580纳米 mCherry 励磁过滤器在这里使用。<ol>
<li>将分束器添加到立方体中, 以反射青色和黄色光, 并传送绿色和红色光。</li>
		<li>用10X 目标和一个高放大目标 (例如, 63 x/1.32 NA 油) 装备显微镜, 将励磁灯聚焦到样品上。</li>
	</ol>
</li>
	<li>在摄像机前安装一个光束分配器, 同时记录 GCaMP 和与钙无关的信号。确保光束分配器有一个分色镜 (长通行证, 切割在 570 nm), 以分离绿色和红光使用一个发射过滤器为绿色 (通径中心在525毫微米以50毫微米宽度) 和一个发射过滤器为红灯 (通入中心在632毫微米与60毫微米 宽度)。</li>
	<li>将绿色和红色荧光投影到上半部分 (绿色) 和下半 (红色), 分别为摄像机芯片 (请参见图 3)。此方向是提供的分析软件的先决条件。</li>
</ol>3. 动物制剂
<ol><li>准备年龄同步的年轻成人或成年日一个线虫, 如前所述的 "门"-- . 31</li>
	<li>准备微流控芯片。
	<ol>
<li>将重力流储层 (60 厘米以上的芯片级) 连接在一起, 包含过滤 (0.2-µm 聚醚砜注射器过滤器) M9 缓冲器到芯片的一个出口。将另一个插座连接到两个出口废物容器的一个出口,即, 一个过滤瓶。将废物容器的其他出口连接到蠕动泵。 注: 对所有连接使用聚乙烯 (PE) 油管, 并使用金属管接头将 PE 油管连接到芯片上。这样, 所有的废物解决方案都将从芯片中冲出, 并收集在废物容器中。通过出口通道的流量创建一个温和的吸力, 将保持蠕虫在以后的诱捕过程中舒适。</li>
		<li>用金属管 (规格尺寸 23TW, 0.635 毫米外径) 在两端准备 50 mm 长 PE 油管 (0.9652 毫米外径, 0.5842 毫米 ID) 的互连线。按下-将这些互连插入到六个驱动手指和蠕虫入口 (请参见图 1)。将这些联网装置放在芯片中, 因为重复的拆卸会导致在其上的孔上磨损。</li>
	</ol></li>
	<li>把芯片放在显微镜上。将2–5蠕虫放入一滴过滤 (0.2-µm 注射器过滤器) M9 缓冲液中, 并使用1毫升注射器将其绘制成 PE 油管 (0.9652 毫米外径, 0.5842 毫米 ID), 通过轻轻拉动活塞将其连接到1毫升注射器上。把动物放在 PE 管里, 而不是放在注射器里。 注意: 芯片中过多的蠕虫或未过滤的解决方案会导致堵塞。</li>
	<li>将注射器的 PE 油管连接到芯片的蠕虫入口 (图 1和图 2) 的互连。打开阀门启动蠕动泵, 激活重力流。然后轻轻按注射器的注射器柱塞, 以移动动物进入诱捕通道, 同时观察在显微镜下与10X 透镜在 brightfield 模式下的通道。 注: 偶尔出现气泡不构成问题;它们通常会自动移到插座中。一些动物可以 "停放" 在候诊室, 并按顺序使用。<ol>
<li>将动物装入陷印通道后 (请参见图 2), 通过轻轻推或拉动注射器的柱塞来调整其位置, 将动物头部定位在通道前面的锥形形状中。</li>
		<li>确保蠕虫 (成人日 1) 有合适的大小被困在芯片。<ol>
<li>如果蠕虫不填充通道的整个横断面从鼻子到接近身体的末端 (不包括尾巴尖端) 或蠕虫沿渠道的轴移动, 不用移动注射器的柱塞, 请按注射器的柱塞, 直到它从陷印通道中消失, 然后加载一个新的蠕虫 (参见步骤 3.8)。</li>
		</ol>
</li>
		<li>切换到显微镜的荧光模式和更高的放大透镜 (40X 或更高), 并检查感兴趣的神经元的突起是否位于一个执行器的膜片上。如果没有, 通过拉或推活塞来调整动物的位置。如果这没有帮助, 请删除该蠕虫并加载一个新的。</li>
	</ol></li>
	<li>聚焦于感兴趣神经元的细胞体, 并将与细胞体前侧神经元最接近的传动器的互连连接到可编程压力泵, 使用 PE 管。
	<ol>
<li>如果实验需要测量执行器和神经元之间的距离, 请移动视图场, 以便两者都在视图领域, channelwall 与图像的上下边缘平行。</li>
	</ol></li>
	<li>使用可编程压力泵定义压力协议。 注意: 此协议可以调整为所需的实验。<ol>
<li>以0帕的恒定压力开始, 时间对应于图像序列的至少50幅图像 (规范化的必要)。为 10 Hz 的成像率这对应于 5 s. 添加一个期望的刺激波形和压力, 并定义刺激的长度作为第二步。 注: 为刺激 TRNs, 建议在生成一个大的神经元响应时, 使用一个与步骤 (即, 275 帕) 叠加的正弦波形 (即, 75 帕, 10 Hz)。</li>
		<li>如果实验需要额外的刺激, 包括至少十年代的期间在0帕在刺激之间的恒定的压力。在开关压力泵之前, 要确保仪器压力在恒定的0帕, 以避免在实际实验之前意外地刺激蠕虫。</li>
	</ol></li>
	<li>运行成像和压力协议。
	<ol>
<li>在相机的图像采集软件中, 在压力协议的持续时间内, 使用100毫秒的曝光时间在10赫兹上设置图像序列。调整激发强度, 使最大荧光增加不饱和相机。将图像另存为 *. tif 文件, 在第一次规范化刺激之前至少有50张图像。</li>
		<li>在可编程泵软件中, 启动图像采集软件和压力协议中的成像协议。在录制过程中, 观察感兴趣的神经元是否是 10 x 10 像素区域中最亮的点, 在序列图像中不超过10个像素, 并且在录制期间停留在视图领域。 注意: 如果未完成此操作, 分析软件将失败。神经元周围的明亮斑点 (即自发荧光) 使神经元的清洁记录变得困难。要纠正此错误, 请从陷阱中取出蠕虫并将新蠕虫加载到芯片中。如果蠕虫移动太多, 尝试一个新的记录相同的蠕虫和观察蠕虫运动。如果问题仍然存在, 则蠕虫可能太小而无法被捕获。在这种情况下, 删除此蠕虫和加载一个稍大的蠕虫进入陷阱。</li>
		<li>如果需要, 只要神经元在整个录音过程中至少被10像素隔开, 就能同时在视场中的多个神经元记录信号。</li>
		<li>为了调查习惯, 对动物进行反复实验。</li>
	</ol></li>
	<li>从陷印通道中移除蠕虫。
	<ol>
<li>如果希望将蠕虫用于后续的研究, 请断开芯片的插座朝向重力流和废物容器。轻轻按压注射器的柱塞, 直到整个蠕虫通过陷印通道进入流道。</li>
		<li>继续按注射器柱塞直到动物出现在芯片外的水滴。从蠕虫入口拔下包括 PE 油管在内的注射器, 用它来吸入液滴中的蠕虫并将其转移到琼脂盘上。</li>
		<li>如果不需要保留蠕虫, 则通过按注射器的柱塞来去除蠕虫, 直到将整个蠕虫通过陷印通道推入流通道;蠕动泵的重力流和吸力会将动物从芯片中取出, 进入废物容器。</li>
	</ol></li>
</ol>4. 分析
<ol><li>下载并安装最新的斐济版本32。</li>
	<li>下载并安装最新的 java SDK 版本。 注意: 如有必要, 请删除或重命名斐济文件夹中的 java 文件夹, 以使用新安装的 java 编译器。</li>
	<li>
打开斐济软件。
	<ol>
<li>通过打开 "插件" 来检查软件是否正在运行新安装的 java 编译器 |公用事业 |ImageJ |属性 "。</li>
	</ol>
</li>
	<li>下载 pokinganalyzer 文件 (https://github.com/HFehlauer/Poking-Analyzer, 请参阅补充文件 2)。</li>
	<li>将. java 文件拖放到 "软件" 窗口中, 将其作为插件打开。</li>
	<li>
通过在斐济按 Ctrl + R 键编译并运行 pokinganalyzer 文件;将打开图形用户界面 (戳分析器) (图 3)。
	<ol>
<li>单击 "帮助" 选项卡, 阅读有关软件的要求以及如何执行分析。要开始, 请选择 "打开视频" 选项卡. 指定分析器的视频文件位置: 单击 "打开视频" 按钮, 导航到视频的位置, 然后打开它。 注意: 软件从打开该选项卡开始。如果在启动新分析时未打开, 请单击 "打开视频" 选项卡。如果视频是. tif 格式, 程序将在内部打开视频并在界面下部显示第一个图像。如果图像不是需要分析的视频, 请单击 "打开视频" 按钮打开另一个视频。</li>
		<li>要继续, 请单击 "定义 ROIs" 选项卡。在此选项卡中, 定义 TRN 的位置。请注意, 打开的视频的第一个图像显示在该选项卡的下部. 点击 "定义 TRN" 按钮, 点击显示图像上部的神经元, 显示 GCaMP 荧光。</li>
		<li>可选: 如果执行器在图像的上半部分可见, 程序可以自动跟踪神经元和执行器之间的距离 (如果需要)。为此, 请检查 "定义执行器"。-框中, 单击 "定义执行器" 按钮, 然后单击图像上半部分的执行器。</li>
		<li>单击 "分析" 选项卡. 定义图像获取速率。请在字段中输入一个数字, 或单击向上或向下按钮以增加或减少数字。如果在上一个选项卡 "定义驱动器？-复选框, 定义相机单元格大小、放大倍数和 binning 因子, 这样程序就可以计算出距离。</li>
		<li>点击 "开始分析" 按钮。 注: 该程序现在将跟踪神经元的荧光在图像序列中的上半部分 (钙依赖) 和在较低的一半 (钙独立) 的图像序列。为了解释在记录平面中的神经元运动, 该程序将调查前一幅图像中神经元位置周围 10 x 10 像素的区域, 以查找下图中神经元的位置。它将通过校正背景荧光 (Fbg) 并除以前50图像 (F0) 中的荧光来计算两半中的荧光: <img alt="Equation 1" src="/files/ftp_upload/56530/56530eq1.jpg"> 在绿色、与活动相关的通道 (FCa2 +) 中由神经元运动引起的荧光变化, 然后使用红色的、与活动无关的通道中的荧光来校正 (f对) 和依赖钙 ( sdCa2 +) 和独立荧光 (sd) 的预刺激标准偏差通过: <img alt="Equation 2" src="/files/ftp_upload/56530/56530eq2.jpg">
</li>
		<li>该程序将显示其在接口下部状态栏中的进度;当状态栏达到100% 时, 单击 "结果" 选项卡。如果状态栏在100% 之前停止, 程序将给出一条错误消息, 指出程序无法分析视频的原因。<ol>
<li>如果信噪比过低, 则程序无法识别神经元;调整后续录制中的成像参数。</li>
			<li>如果神经元在录制过程中离开了视野, 程序就无法跟踪它, 并且会停止。对于后续录制, 将神经元放置在该记录开始处的视图区域中间。</li>
			<li>如果视图领域中的某些区域是饱和的, 则自动分析将产生无效结果。对于后续记录, 请确保荧光信号不饱和相机传感器。</li>
		</ol>
</li>
		<li>在 "结果" 选项卡中, 结果显示在上半部分。通过单击 "保存结果表", 保存结果表的制表符分隔的 .txt 文件。 注: 该表包括时间以秒为单位, 规范化的钙依赖性荧光 (由钙无关荧光校正)。如果先前定义, 它还包含在µm 中的 TRN 和执行器之间的总距离以及与µm 中的通道壁垂直的 TRN 与执行器之间的距离. 荧光强度增加后的代表性结果在图 4中绘制了刺激与单元体与最接近的驱动器的距离。</li>
		<li>如果一个记录中有多个神经元 (用超过10个像素隔开), 请再次单击 "定义 ROIs" 选项卡, 然后返回到第二个神经元的步骤4.6.2。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

该协议说明了一种方法, 提供精确的机械刺激的皮肤的蛔虫被困在微流控芯片。它的目的是促进物理刺激的综合回答生物学问题, 并旨在简化生物实验室的美国匹茨堡研究。这种方法扩展了以前的化验, 以评估 mechanosensory 神经元在C. 线虫中的作用。以前测量的定量和半定量技术力1、34和行为35, 但很难与神经元活动的高分辨率成像相集成。因此, 我们认为使用这个芯片可以扩展当前的应用, 它的性能被优化为刺激的神经元需要温和的触摸。我们优化了执行器的挠度达到5µm, 这是一个足够强大的刺激, 以达到几乎最大的神经元反应。由于细微的变化, 我们相信这种设计可以用来刺激苛刻的触摸感受器, 如支配体表36的 PVD 痛觉受器。
修改和疑难解答 如果没有洁净室可用于 SU-8 光刻, 则可以使用台式清洁盒或 SU-8 软光刻站, 配有 UV 灯、可编程烤盘和自旋镀膜机。为避免使用洁净室设施而生产微流控设备的低成本替代方案也可以遵循26。
在设计中, 我们实现了一个现有的捕获技术, 力场一个动物在微流控通道。陷阱设计是根据用于研究蠕虫36中嗅觉感觉的设备改编的, 但它并不是抑制蠕虫运动的唯一可行选项。据报道, 在微流控设备中保存蠕虫的其他策略有: CO2、电泳和压缩固定化11、37、38已被使用, 并可集成到修改过的设计.
我们使用一种商用的压电压力泵向驱动通道施加压力, 它可以控制和交付多达800帕从外部压力源。我们把水泵与压缩空气连接在研究楼里, 它能提供450帕。如果没有可用的压缩空气源, 则可以使用一罐压缩的惰性气体 (例如, N2)。这将有额外的好处, 提供更高的压力和产生较大的变形, 但也需要高压配件连接到压力控制器的芯片。用较软的聚硅烷制备芯片是提高压力诱发变形的替代方法。
在本实验中, 压力泵的出口通过1毫米接收开口 (例如、冲孔孔) 连接到该芯片上, 并插入 PE 中的压合20克金属管, 由无粘结和可逆的联网装置插入。油管.这些已被证明可以抵抗多达700帕的压力39。但是, 在重复插入过程中必须注意, 因为在孔周围的聚硅烷会撕裂, 从而限制性能或使芯片无法使用。由于这个原因, 金属油管插入一次, 左插入芯片, 没有删除和插入它反复。
关键步骤 刺激是由加压驱动通道提供的, 它使皮肤缩进。由此产生的变形和 mechanoreceptor 神经元活化可以成像高分辨率的光学荧光显微镜和/或共焦设置。有几个因素会阻碍 GCaMP 反应的检测。首先, C. 线虫在许多神经元中表达蓝光受体30 , 包括 TRNs21、23, 它在光照后激活细胞, 蓝色 (488 nm) 光。因此, 使用基因编码的钙传感器 GCaMP6s 的成像C. 线虫将在光照下激活其神经元, 遮盖由机械刺激所激活的钙瞬变23。在lite-1突变体背景下工作或使用红色转移的钙活性传感器可避免这种混淆。第二, 如果刺激剖面与 mechanoreceptors 的灵敏度不符, 机械诱导的钙瞬变可能无法检测到。我们未能检测到高振幅的步骤和坡道的活化, 但刺激后诱发极高的信号 (如例如, 200 帕巴斯)。最后, TRNs 习惯的重复刺激提供短 (< 六十年代) interstimulus 间隔。因此, 重要的是设计刺激序列, 最大限度地减少习惯, 除非在适应的情况下正在研究。
我们建议过滤所有解决方案, 因为缓冲区中的碎片和灰尘可以很容易地堵塞小的陷印通道。此外, 动物必须是年龄同步和大小匹配的芯片的设计;小于年轻成年人的动物将滑过该装置或无法被限制, 较大的动物不能进入诱捕通道, 并有爆裂和/或堵塞芯片的危险。在堵塞的情况下, 对蜗杆或重力流进气道施加过大的压力可能会清洗通道, 但也可能导致接头失效。
如结果部分所述, 几个因素可能导致分析程序失败。如果编译代码时遇到问题, 请验证最近的斐济软件正在运行, 并且建议使用最新的 Java 编译器。如果程序正在运行, 但有意想不到的结果, 请确保录制的影片在进行实际的机械刺激之前有一个50帧的缓冲区, 以确保对基线和背景荧光的正确估计。此外, 绿色、活动相关 (GCaMP6s) 和红色、活动无关 (tagRFP) 通道的视野被假定为投射到摄像机芯片的上下半部分。如果光束分配器将图像投射到传感器的左、右半部分, 请将相机90°转到在录制过程中获取所需的方向, 或在预处理步骤中旋转图像堆栈。此外, 该程序通过荧光识别神经元, 如果信噪比或对比度过低, 则会失败。在这种情况下, 改变励磁强度和/或曝光时间可以解决问题。如果神经元在录音过程中离开视野, 这个程序也会停止, 在这种情况下, 神经元应该定位在视野的中心, 然后再开始新的习得。
技术的局限性 目前的设计可以刺激身体壁 mechanoreceptor 神经元, 如 PVD 痛觉受器和 TRNs, 但不 mechanoreceptors 支配蠕虫的头部区域, 如 FLP 和灰。它仅限于压力 < 700 帕。对于较大的压力, 需要使用不同的管件。因为这里的压力供应没有超过500帕, 可能适用的最大压力被限制了到450帕。这里观察到的钙瞬变标记了预期信号的上边界。我们的执行器设计的另一个限制是它们的纵横比。原则上, 较薄的隔膜会使较大的变形, 但高纵横比的结构难以可靠地制造。如果更大的变形是必要的, 更广泛的执行器或平行变形从双方可能是有用的, 如 Cho et 等。29
虽然我们可以监测膜片的变形, 但很难测量在气动激励过程中施加的作用力。原理的局限性是, 执行机构的接触面积没有定义良好。这是因为执行器本身在驱动过程中是弹性和变形的, 这改变了接触半径, 增加了挠度。考虑到这些注意事项, 我们可以估计5µm 压痕 (一个空的芯片大约300帕驱动压力) 预计将根据 Petzold et 等的蠕虫刚度估计对蠕虫应用约3.8 µN。34由于在不同刚度1、34的蠕虫中, 神经元电流的力依赖性各不相同, 因此建议对缩进进行监测以刺激强度而不是强制。
我们希望, 该协议的传播将帮助用户利用微流体作为模型系统进行美国匹茨堡研究, 从而加快科学在这一重要学科领域的发展.

触觉为动物提供了关于环境的关键信息。根据施加的力量, 触摸被认为是无害的, 愉悦的, 或痛苦的。在接触过程中的组织变形是由嵌入在皮肤中的专门 mechanoreceptor 细胞检测到的, 表达受体蛋白, 最常见的离子通道。在接触和疼痛过程中, 连接力知觉与离子通道活化的步骤没有完全理解。对于皮肤组织如何过滤机械变形以及 mechanoreceptors 检测应变或应力变化的情况 (如1、2、3), 更甚了解。这种理解上的差距, 部分是由于缺乏适当的工具, 在观察细胞水平的反应时, 对活着的动物的皮肤表面施加精确的机械刺激。虽然原子力显微镜已广泛用于应用和测量孤立细胞中的作用力4,5和激活 Piezo1 受体在活细胞6, 类似的实验使用活体动物, 特别是C. 线虫, 由于这个主题的内在流动性, 而臭名昭著的挑战性。这一挑战传统上绕过使用兽医或外科级氰胶水固定在琼脂垫上的个别动物,1,7,8,9。这种方法是有成效的, 但有限制与固定的技能所需的胶合和软琼脂表面上的机械依从性。微流体策略是一种免费的替代方法, 可以避免一些与胶合有关的并发症。
线虫线虫是一个基因模型有机体与完全映射神经系统, 由于动物的大小, 是一个很好的适合微流体技术. 微流体的设备提供的优势, 否则极端移动动物可以克制, 而执行高分辨率成像和提供相关的神经调节刺激。在微流控技术的帮助下, 活着的动物可以不受伤害地固定化10,11, 启用对整个生存期1213和高分辨率的行为活动的监视神经元活动的成像14,15,16,17。此外, 许多 mechanoreceptor 神经元需要的触觉和疼痛的特点, 可以在他们的生理1,8, 机械4,18,19, 和分子级别20,21,22。
C. 线虫用六 TRNs 对其体壁进行柔和的机械刺激, 其中三支配动物的前 (ALML/r 和 AVM), 三支配动物的后部 (PLML/r 和 PVM)。在它的 TRNs8中广泛研究了传感所需的离子通道分子在生化信号中的应用。本文介绍了一个微流控平台23 , 使研究人员能够将精确的机械力应用于固定化的C. 线虫蛔虫的皮肤, 同时通过光学成像读取其内部组织的变形。除了提供良好定义的机械刺激, 钙瞬变可以记录在 mechanoreceptor 神经元的亚细胞分辨率和相关的形态学和解剖特征。该设备由一个中央补漏白通道组成, 它包含一个动物, 并在六个气动驱动通道 (图 1和图 2) 旁边呈现其外观。六通道沿陷印通道定位, 为蠕虫的六 TRNs 提供机械刺激。这些通道通过薄的可由外部气压源 (图 1) 驱动的薄壁膜片与陷印室隔开。我们对压力的偏转进行了校准, 并提供了本文的测量结果。每个执行器可以单独解决, 并用于刺激 mechanoreceptor 的选择。压力是通过压电驱动的压力泵提供的, 但任何替代装置都可以使用。我们表明, 压力协议可以用来激活 TRNs在体内和演示的操作设备, 以提供机械刺激成人C. 线虫, 加载成年动物进入设备, 执行钙成像实验, 分析结果。器件制造包括两个主要步骤: 1) 光刻, 使模具从 SU-8;2) 模压成型机制造设备。为了简洁明了, 读者可以参考以前发布的文章和协议24,25 , 了解如何生成模具和设备的说明。

<table>
	<tbody>
		<tr>
			<td>Chrome mask</td>
			<td>Compugraphics (http://www.compugraphics-photomasks.com/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Mitani-Micronics (http://www.mitani-micro.co.jp/en/)</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>Chrome mask</td>
			<td>Kuroda-Electric (http://www.kuroda-electric.eu/</td>
			<td></td>
			<td>5&#39;&#39;, designed in AutoCAD (Autodesk, Inc.)</td>
		</tr>
		<tr>
			<td>4&#39;&#39; Silicon wafer (B-test)</td>
			<td>Stanford Nanofabrication Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2002</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Laurell Technologies</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Exposure timer</td>
			<td>Optical Associates, Inc</td>
			<td>OAI 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumination controller</td>
			<td>Optical Associates, Inc</td>
			<td>2105C2</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A426F-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A18-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethylsilane (TCMS)</td>
			<td>Sigma-Aldrich</td>
			<td>92361-500ML</td>
			<td>Caution: TCMS is toxic and water-reactive</td>
		</tr>
		<tr>
			<td>Sylgard 184 Elastomer Kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td>PDMS prepolymer</td>
		</tr>
		<tr>
			<td>Biopsy punch, 1 mm</td>
			<td>VWR</td>
			<td>95039-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Plasma Asher</td>
			<td>Branson/IPC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small metal tubing (0.635 mm OD, 0.4318 mm ID, 12.7 mm long); gage size 23TW</td>
			<td>New England Small Tube Corporation</td>
			<td>NE-1300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene syringe filter, 0.22 &#956;m</td>
			<td>Thermo Scientific</td>
			<td>725-2520</td>
			<td>to filter all solution, small particles would clog the chip</td>
		</tr>
		<tr>
			<td>Polyethylene tubing; 0.9652 mm OD, 0.5842 mm ID</td>
			<td>Solomon Scientific</td>
			<td>BPE-T50</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 ml</td>
			<td>BD Scientific</td>
			<td>309628</td>
			<td>for worm trapping and release</td>
		</tr>
		<tr>
			<td>Syringe, 20 ml</td>
			<td>BD Scientific</td>
			<td>309661</td>
			<td>for gravity-based flow</td>
		</tr>
		<tr>
			<td>Gilson Minipuls 3, Peristaltic pump</td>
			<td>Gilson</td>
			<td></td>
			<td>to suck solutions and worms out of the chip</td>
		</tr>
		<tr>
			<td>Microfluidic flow controller, equipped with 0&#8211;800 kPa pressure channel</td>
			<td>Elveflow</td>
			<td>OB1 MK3</td>
			<td>pressure delivery</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Poly- urethane Tubing, 4 mm ID and 6 mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T52</td>
			<td>connection from house air to pressure pump</td>
		</tr>
		<tr>
			<td>Water-Resistant Clear Polyurethane Tubing, 2.6mm ID and 4mm OD</td>
			<td>McMaster-Carr</td>
			<td>5195 T51</td>
			<td>connect pressure pump to small tubng</td>
		</tr>
		<tr>
			<td>Push-to-Connect Tube Fitting for Air</td>
			<td>McMaster-Carr</td>
			<td>5111K468</td>
			<td>metric - imperial converter</td>
		</tr>
		<tr>
			<td>Straight Connector for 6 mm &#215; 1/4&#8243; Tube OD</td>
			<td>McMaster-Carr</td>
			<td>5779 K258</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI 4000 B microscopy system</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>63&#215;/1.32 NA HCX PL APO oil objective</td>
			<td>Leica</td>
			<td>506081</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu Orca-Flash 4.0LT digital CMOS camera</td>
			<td>Hamamatsu</td>
			<td>C11440-42U</td>
			<td></td>
		</tr>
		<tr>
			<td>Lumencor Spectra X light engine</td>
			<td>Lumencor</td>
			<td></td>
			<td>With cyan and green/yellow light source</td>
		</tr>
		<tr>
			<td>Excitation beam splitter</td>
			<td>Chroma</td>
			<td>59022bs</td>
			<td>in the microscope</td>
		</tr>
		<tr>
			<td>Hamamatsu W-view Gemini Image splitting optics</td>
			<td>Hamamatsu</td>
			<td>A12801-01</td>
			<td>to split green and red emission and project them on different areas on the camera chip</td>
		</tr>
		<tr>
			<td>Emission beam splitter</td>
			<td>Chroma</td>
			<td>T570lpxr</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters GCamp6s</td>
			<td>Chroma</td>
			<td>ET525/50m</td>
			<td>in the image splitter</td>
		</tr>
		<tr>
			<td>Emission filters mCherry</td>
			<td>Chroma</td>
			<td>ET632/60m</td>
			<td>in the image splitter</td>
		</tr>
	</tbody>
</table>

using a microfluidics device for mechanical stimulation and high resolution imaging of c. elegans

美国匹茨堡的一个中心目标是了解机械应力对蛋白质和细胞的相互作用。尽管它的重要性, 机械应力对细胞功能的影响仍然是不太清楚。在某种程度上, 这种知识差距之所以存在, 是因为很少有工具能使组织和细胞同时变形, 活动物的细胞活动成像, 以及在其他高度流动的模型生物体中有效限制运动, 如线虫秀丽线虫。小的C. 线虫使它们与基于微流体的研究设备非常匹配, 并利用微流控装置提出了固定化解决方案。虽然这些设备允许高分辨率成像, 该动物是完全包裹在烷和玻璃, 限制物理访问提供机械力或电生理记录。最近, 我们创建了一个设备, 集成气动执行器与陷阱设计兼容高分辨率荧光显微镜。所提出的驱动通道是由一个薄的, 由一个小的反式的双膜片分离的蠕虫捕获通道。这种隔膜通过施加外部源的压力偏转到蠕虫的一侧。该装置可以针对单个 mechanosensitive 神经元。这些神经元的活化是用基因编码的钙指标进行高分辨率成像的。本文介绍了在触摸受体神经元 (TRNs) 中, 使用线虫菌株表达钙敏感活性指标 (GCaMP6s) 的一般方法。然而, 该方法并不局限于 TRNs 或钙传感器作为探针, 但可以扩展到其他机械敏感的细胞或传感器。

SU-8 光刻与芯片粘合 光刻协议和注塑工艺遵循标准程序。详细信息可在其他地方找到23,24,25,26。在固化后, 应将硅片剥离, 无问题。如果 SU-8 的特性在其剥离过程中脱落, SU-8 黏附层或硅烷化是不够的。如果等离子活化的玻璃盖玻片和非晶硅芯片是不够的, 弱粘结可能导致泄漏的陷阱通道或气动执行器导致性能下降和缺乏刺激。如果流体泄漏是明显的, 芯片是不可用的。
执行器性能 所有的聚硅烷执行器和油管连接到气藏必须正确密封, 以确保适当的气动驱动膜片。反复插入和拆卸 PE 油管 (管道) 将会损坏塑料孔, 并导致压力降低。这种压力的损失将限制执行机构的偏转和施加在皮肤的固定化动物的力量。膜片的偏转可以用一个空的芯片 (没有动物存在) 测量通过执行几个驱动周期以期望压力和比较定量价值与理论预测。由于膜和蠕虫的边界难以可视化, 所以在诱捕通道中没有动物的存在来测量隔膜的偏转。这使精确校准与被困的动物不可能。表 1列出了预期的挠度作为压力函数。这些值是用弹性板理论计算的, 细节可以在 Nekimken et . 中找到。23此关系预计会因在芯片浇铸过程中产生的隔膜厚度和其他因素 (如芯片与玻璃之间的粘结完整性和连接芯片与压力源的密封件) 的不同而异。在这里, 值是可重现的, 轻微的变化不超过1µm 在450帕的压力。我们的特点是膜片的性能为所有三种不同的刺激配置文件, 并参考感兴趣的读者 Nekimken et al。23在摘要中, 如果值偏离表 1: 1) 在油管或油管连接器中的泄漏 (2), 则有几个因素需要考虑, 这可能是由于基聚合物和固化剂的替代比率,或 3), 该项已老化, 并继续在一段时间内交联。因此, 建议使用在一个月内准备好的芯片。
补漏白性能 将蠕虫插入芯片的入口后, 使用注射器的温和压力应将一只动物置于诱捕通道内, 并将其皮肤与六执行器 (图 1) 一起呈现。重要的是, 动物的鼻子不一定需要凸出到缓冲储层中。相反, 更重要的是定位的动物, 使突起是相邻的最接近的执行机构, 其细胞体是在视野中的显微镜。为了实现最佳的神经元活化, 应调整执行器与细胞体之间的距离, 使执行器位于感兴趣的细胞体前。根据我们的经验, 神经元刺激后的细胞体, 不会发生激活, 钙瞬变将缺席。适当的固定化可以实现与年轻的成人或一天老雌雄同体 (为这两个年龄优化的设备可在同一面具)。较小的动物将滑入收集水库或横向移动在陷印渠道之内。太大的动物将被挤入通道, 这可能导致其 TRNs 过早活化, 随后无法检测 mechanoreceptor 瞬态。此外, 清除大蠕虫是一个挑战, 导致动物死亡和/或堵塞的设备。通过这种设计, PLM 神经元通常不能完全固定, 所以可以自由地横向和垂直移动。尽管我们成功地激活了 PLM 神经元, 但由于尾部的垂直运动, 记录这些神经元中的钙瞬变是困难的。另一个补漏白设计已用于从 PLM33中进行记录。
钙瞬变的影像学分析 一旦到位, 动物可以被刺激使用六执行器之一。六执行机构的位置, 以提供机械刺激到每一个六 TRNs。一个与450帕内部压力源相连的微流控流控制器, 用于向动物传递一项由2至275帕步组成的刺激协议, 一个 2 s 0–275人民军坡道和 2 s 嗡嗡声 (75 帕, 10 赫兹正弦叠加与275帕步), 每隔10秒等待一段时间。在斐济, 使用在我们的 GitHub 帐户上提供的定制的软件, 对钙瞬态的同步记录图像序列进行了分析 (见上文和 https://github.com/HFehlauer/Poking-Analyzer)。我们发现只有当刺激达到400帕以上的压力时, 压力坡道和压力步骤才会激活 TRNs。相比之下, 在压力 < 275 帕的正弦、10赫兹嗡嗡声刺激下, 所有 TRNs 的鲁棒活化都被观察到 (图 4)。一般而言, TRNs 对一个简短的正弦刺激 (称为 "嗡嗡声") 做出了更好的响应, 与以前的报告使用不同的方法 1,9一致。刺激与嗡嗡声是高效率的, 导致90% 激活在所有神经元和试验测试 (图 4)。令人惊讶的是, 没有观察到刺激是否应用于背部或腹侧的强烈相关性, 并且独立于细胞体与突起的刺激位置之间的距离。未来的研究将不得不研究最大反应的延迟是否与刺激距离有关。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56530/56530fig1.jpg"> 图 1: 芯片设计概述.(A) 光掩模和生成的芯片的示意图布局, 并显示所有连接器。有六个驱动手指, 三在中央陷印通道的两侧。缩放条 = 3 毫米 (B) 微流控装置的照片, 其连接到盖玻片, 并与蠕虫入口和蜗杆插座中的油管相连。只有一个驱动手指连接到压力源, 而其他五没有占领。图形的部分已从参考23中复制到具有创造性的通用许可证下。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56530/56530fig1large.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/56530/56530fig2.jpg"> 图 2: 关闭中央补漏白通道和补漏白过程.(A) 补漏白通道的示意图。蠕虫入口侧的柱子有助于使蠕虫定向, 并便于动物的装载。驱动手指可以加压和缩进一个薄薄的隔膜进入被困标本。缩放条 = 200 µm (B) 在水平通道中捕获蠕虫的典型视频帧。该通道的设计, 使蠕虫来铺设横跨六气动执行器。尾巴在左边, 头在右边。单个触点受体神经元 (TRNs) 的位置由箭头表示, 并标记在图中。刻度条 = 100 µm (在所有面板中)。图形的部分已从参考23中复制到具有创造性的通用许可证下。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56530/56530fig2large.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/56530/56530fig3.jpg"> 图 3: 提供的斐济插件概述, 用于分析通过此协议获取的钙跟踪.软件启动图形用户界面。用户界面的第一个选项卡 ("打开视频") 显示打开视频的按钮, 它显示打开的视频的路径和第一帧。第二个标签 ("定义 ROIs") 包括按钮来定义触摸受体神经元, 一个用于激活按钮 "定义执行器" 和按钮 "定义执行器" 的复选框。在下部, 显示视频的第一帧。在第三个选项卡的上部 ("分析") 中, 将显示图像分析的可调整参数。中间部分显示 "开始分析" 按钮。在下部, 进度条显示当前分析的进度。在第四个标签, "结果", 结果显示为一个图表的相对荧光强度随着时间的推移。在下部, 有一个按钮来保存结果表。在第五个选项卡 "帮助" 中, 将显示帮助文本, 以显示分析软件的要求以及如何使用它的说明。在具有创造性的通用许可证下, 从参考23中改编的图。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56530/56530fig3large.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/56530/56530fig4.jpg"> 图 4: 单个神经元响应机械刺激.(A) 具有代表性的荧光显微图像, GCamP6s 在机械刺激前和之后用嗡嗡声标记了 AVM 触碰受体神经元。刻度条 = 10 µm. 色标 = 1,500-3, 500 灰色值。(B) 刺激协议, 包括 2 s 膜片励磁, 代表275帕步, 275 帕斜坡, 正弦 (75 帕; 10 Hz) 叠加与275帕步 (嗡嗡声)。(C) 规范化的 GCamP6s 强度跟踪 (均为 "灰色"), 记录自反车辆地雷 (绿色、n = 14) 和 mechanoreceptor 通道亚单位mec-4 (蓝色、n = 10) 中的动物突变体所激发的 AVM, 即以促进对机械应力的反应。这表明瞬态是由应用的机械刺激引起的。图形的部分已从参考23中复制到具有创造性的通用许可证下。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56530/56530fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>压力 (人民军)</td>
			<td>平均偏转 (µm)</td>
			<td>sd</td>
			<td>预期挠度 (µm)</td>
		</tr>
<tr>
<td>0</td>
			<td>-0.01</td>
			<td>0.01</td>
			<td>0</td>
		</tr>
<tr>
<td>50</td>
			<td>0.77</td>
			<td>0.26</td>
			<td>0.607143</td>
		</tr>
<tr>
<td>100</td>
			<td>1.57</td>
			<td>0.58</td>
			<td>1.21429</td>
		</tr>
<tr>
<td>150</td>
			<td>2.32</td>
			<td>0.65</td>
			<td>1.82143</td>
		</tr>
<tr>
<td>200</td>
			<td>3.13</td>
			<td>0.74</td>
			<td>2.42857</td>
		</tr>
<tr>
<td>250</td>
			<td>3.86</td>
			<td>0.79</td>
			<td>3.03571</td>
		</tr>
<tr>
<td>300</td>
			<td>4.61</td>
			<td>0.79</td>
			<td>3.64286</td>
		</tr>
<tr>
<td>350</td>
			<td>5。3</td>
			<td>0.82</td>
			<td>4.25</td>
		</tr>
<tr>
<td>400</td>
			<td>5.92</td>
			<td>0.82</td>
			<td>4.85714</td>
		</tr>
<tr>
<td>450</td>
			<td>6.43</td>
			<td>0.75</td>
			<td>5.46429</td>
		</tr>
</tbody></table>表 1: 预期的试验挠度值, 适用压力范围从0–450人民军和他们的理论预测.为步骤刺激而导出的数据。意思是。这些数据是用空补漏白通道获取的。

Watch this Scientific Journal Video about Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans at JoVE.com

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

美国匹茨堡研究的新工具需要了解机械应力是如何激活生化通路并引起生物学反应的。在这里, 我们展示了一种新的方法, 选择性机械刺激固定化动物与微流控器陷阱, 允许高分辨率成像的细胞反应。

利用微流体装置进行机械刺激和高分辨率成像的研究C.线虫

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

using-microfluidics-device-for-mechanical-stimulation-high-resolution

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

9.7K 次观看.  Stanford University. 美国匹茨堡研究的新工具需要了解机械应力是如何激活生化通路并引起生物学反应的。在这里, 我们展示了一种新的方法, 选择性机械刺激固定化动物与微流控器陷阱, 允许高分辨率成像的细胞反应。

视频: Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

含量测定β-淀粉样蛋白的毒性，使用转基因 C。线虫模型

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

科研

神经科学

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

结合经颅磁刺激和功能磁共振成像检查默认模式网络

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS:使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

依赖国家对TMS的:一看动机幻视行为

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS的设备进行美国FDA批准用于治疗抑郁症的议定书"

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments in vision research require displays with more than 12 bits of luminance resolution. Several solutions are available. Bit++ 1 and DataPixx 2 use the Digital Visual Interface (DVI) output from graphics cards and high resolution (14 or 16-bit) digital-to-analog converters to drive analog display devices. The VideoSwitcher 3 described here combines analog video signals from the red and blue channels of graphics cards with different weights using a passive resister network 4 and an active circuit to deliver identical video signals to the three channels of color monitors. The method provides an inexpensive way to enable high-resolution monochromatic displays using conventional graphics cards and analog monitors. It can also provide trigger signals that can be used to mark stimulus onsets, making it easy to synchronize visual displays with physiological recordings or response time measurements.
Although computer keyboards and mice are frequently used in measuring response times (RT), the accuracy of these measurements is quite low. The RTbox is a specialized hardware and software solution for accurate RT measurements. Connected to the host computer through a USB connection, the driver of the RTbox is compatible with all conventional operating systems. It uses a microprocessor and high-resolution clock to record the identities and timing of button events, which are buffered until the host computer retrieves them. The recorded button events are not affected by potential timing uncertainties or biases associated with data transmission and processing in the host computer. The asynchronous storage greatly simplifies the design of user programs. Several methods are available to synchronize the clocks of the RTbox and the host computer. The RTbox can also receive external triggers and be used to measure RT with respect to external events. 
Both VideoSwitcher and RTbox are available for users to purchase. The relevant information and many demonstration programs can be found at <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a>.

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

启用高灰度分辨率的显示器和传统计算机准确的响应时间测量

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...