我们要感谢曼努埃尔-兹曼为我们提供的初步设计文件, 适合与男性使用;弗兰克施罗德为 ascr#3 的综合和供应;罗斯 Lagoy 为洞察和协助以成像和分析;劳拉 Aurilio 为大师制作, 并与克里斯托弗滑道, 贡献了对这份手稿的审查。这项工作的经费是在国家卫生研究所赠款 1R01DC016058-01 (国家科学基金会赠款本位 1605679 (D.R.A.), 和在科学接口 (D.R.A.) 的宝来惠康职业奖。

1. 设备制造 注意: 请参阅参考资料 1 。
 注意: 硅母模是使用标准的光刻技术制作的, 用于在硅母版上进行图案 SU-8 光刻胶的制造 1 , 7 。硅片图案的光印在 2.5万 dpi。该男性适应装置的特点是 Chronis 嗅觉芯片设计 19 与蠕虫加载端口的变化, 适应一个设计获得了 m. 兹 (个人通信, 2016)。一个回合包括控制动物的旋转。蜗杆加载端口通道的宽度缩小到50和 #956; m。所有通道都是32和 #956; 我很高。一旦用户使用了硅主模具, 用户就可以按照后面的协议进行操作, 如前所述 1 . 
<ol> <li> 按重量的比例将硅橡胶基体和硫化剂混合为 10:1. </li>
	<li> 与传输管完全混合. </li>
	<li> 将混合物放在真空干燥中1小时, 直到所有可见气泡都被移除. </li>
	<li> 将混合物倒入一个 150 mm 直径的硅模母盘中, 直到5毫米厚 (100 克)。使用巴斯德吸管移除已引入混合物的任何气泡或灰尘. </li>
	<li> 65 和 #176 烘烤; C 至少3小时, 或隔夜. </li>
	<li> 使用手术刀将其从模具中剪掉, 并使用刀片切割分离的设备. </li>
	<li> 穿孔入口和出口孔的 1 mm 真皮冲床. </li>
	<li> 冲洗与 dh 的孔 2 o, 乙醇, 并再次与 dh 2 O, 以清除粒子的冲床。用气流脉冲将装置烘干. </li>
	<li> 用胶带清洗通道两侧和设备的顶部, 清除设备上剩余的灰尘或碎屑, 以便成功接合. </li>
	<li> 等离子键将设备, 通道端向下, 到1盖玻璃. 
	<ol> <li> 将覆盖玻璃和设备 (通道端向上) 暴露到空气等离子体中, 使用允许适当粘合的条件, 如六十年代的三十年代或 24 w 的 100 w. 
		注: 可调整设置以提高粘合效率。在试图提高键合效率时, 等离子体结合条件不像适当的清洗那样重要。即使在理想的等离子体条件下, 一个未充分清洁的装置也无法粘合. </li>
		<li> 将盖玻璃倒置到设备的通道一侧, 并按拇指向下按5秒. </li>
	</ol> </li> </ol> 2。缓冲准备 <ol> <li> 从不育的10x 库存中稀释 1x S 基 (100 mM 氯化钠和0.05 米 KPO 4 , pH 6.0). </li>
	<li> 将1米咪的库存稀释到最终浓度为1毫米, 在 1x S 基础上的所有缓冲解决方案. </li>
	<li> 将荧光素添加到 #34; 流控制和 #34; 和 #34; 缓冲区和 #34; 水库. 
	<ol> <li> 在1x 的基础上创建一个100毫克/毫升的荧光素库存. </li>
		<li> 将库存稀释到1和 #181 的最终浓度; 流量控制中的 g/毫升; 缓冲区中的0.1 和 #181; g/ml. </li>
	</ol> </li> <li> 创建刺激。
	<ol> <li> 将甘油稀释到 1X S 基底的最终浓度为1米. </li>
		<li> 稀释 ascaroside #3 (ascr#3) 到最终浓度1和 #181; M 到 1X S 基部. </li>
	</ol> </li> </ol> 3。设备安装程序 注意: 请参见 1 。
 <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56026/56026fig1.jpg"/> 图1。微流体设备设置. ( A ) 水库和油管。30毫升注射器没有柱塞作为和 #34; 水库和 #34; 这是连接到一个口阀与三流量选项。一个插座连接到一个3毫升注射器与一个柱塞, 而另一个是连接到针 (橙色), 插入到油管连接到微流控装置。( B ) 微流体成像实验的总体设置。该装置放在一个倒置的荧光显微镜的舞台上, 高于物镜。#34; 流量控制和 #34; 缓冲器通过3路阀门, 该阀由安装上方的架子上的一个单元控制。然后将包含缓冲区的行插入相应的设备端口。( C ) 微流体设备的端口。#34; 流量控制和 #34; 端口侧面其他入口端口: #34; 刺激和 #34; 和 #34; 缓冲区和 #34; 端口。#34; 插座和 #34; 端口是最右边的端口。由于蜗杆加载竞技场的位置, #34; 蜗杆加载和 #34; 端口是设备中最集中的端口。<a href="//ecsource.jove.com/files/ftp_upload/56026/56026fig1large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
<ol> <li> 通过将30或60毫升注射器连接到3路口阀门, 并将3毫升注射器和针连接到口阀 (如 图 1A ), 准备三流体油藏。将针与延伸至微流体设备的油管连接 (如 图 1A -b )。
	<li> 从储油层和油管中除去气泡. </li>
	<li> 填充3毫升注射器与附加油管与 1x S 基础, 并插入到出口端口. </li>
	<li> 轻轻地将压力施加到注射器, 直到缓冲区出现在入口孔的顶部. </li>
	<li> 将流量控制、缓冲器和刺激油管连接到适当的入口孔 (如图 1B -c ), 确保在装货口孔和要连接的缓冲油管上均存在液滴. </li>
	<li> 再次, 轻轻地向连接到出口端口的注射器施加压力, 直到在蠕虫加载端口入口出现水滴. </li>
	<li> 在蠕虫加载端口上插入实心的锁定 pin. </li>
	<li> 将注射器从出水口卸下, 并将插座线连接到房屋真空 (-670 乇). </li>
	<li> 通过与所使用的照相机兼容的软件 (如开源软件的微型经理), 通过视觉和视频确认来检查流通道中的任何气泡的设备。有关使用微管理器的提示, 请参见步骤 6. 
	<ol> <li> 如果存在任何气泡, 请等待它们在加载任何动物之前将其取出或吸收到该墙上; 气泡的存在会扰乱流体通过该装置的正常流动. </li>
	</ol> </li> <li> 使用 GFP 滤镜, 通过执行3路阀门并观察缓冲器的切换, 在蠕虫加载之前确认设备内的适当流程动态. 
	<ol> <li> 确定正确的流程动态: 在流量控制和缓冲解决方案 ( 图 2D -2E ) 中, 观察荧光素的存在, 在通过按下控件更改流控制值时进行更改对应于阀门链接上的3通阀的按钮 ( 图 1B ). </li>
		<li> 打开微管理器后, 单击 #34; 实况和 #34; 观察设备的实时图像。打开荧光灯源以观察设备中的缓冲区流 ( 图 2D -2E )。
	</ol> </li> </ol> <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56026/56026fig2.jpg"/> 图 2: 一种男性适应的微流控嗅觉芯片。 ( ) 当蠕虫向缓冲区公开时, 该设备的流模式。缓冲区 (B) 以褐色显示, 流控制 (FC) 以黄色显示, 并以白色刺激 (S)。蠕虫加载端口已被改编为包含一条曲线, 从而可以更好地控制蠕虫的方向。( B ) 当蠕虫受到刺激时, 该设备的流模式。缓冲区 (B) 以褐色显示, 流控制 (FC) 以黄色显示, 并以白色刺激 (S)。( C ) 对已适应设备的测量 (如制造)。蠕虫加载端口以42和 #181; m 开头, 以50和 #181; m 通道为男性宽度设计。通道的测量高度为32和 #181; m, 尽管目标为25和 #181; m 在设计中。( D-E )表示 p sra-6 的被困男性:: GCaMP3。 sra-6 启动子不是特定于灰分的, 在 asi 神经元中可以观察到一些表达式, 尽管在 asi 中没有观察到钙的瞬变。该图像是 ( D ) 明亮场和荧光照明的组合, 而 ( E ) 只是荧光灯。刻度线表示42和 #181; m. ( F ) 灰神经元对1米甘油刺激有较强的神经活性反应。蓝色区域表示1米甘油刺激的时间。阴影区域表示标准误差, n = 20 脉冲来自七蠕虫。红色的痕迹表示极化的反应。Y 轴显示和 #916; f/f 0 。刻度栏表示 5 s. <a href="//ecsource.jove.com/files/ftp_upload/56026/56026fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 4. 动物准备 注意: 请参阅参考资料 23 。
<ol> <li> 对1米甘油的成像灰响应. 
	<ol> <li> 将约20的 C. 线虫 放置在 p sra-6 :: GCaMP3 阵列表达式的线虫生长培养基 (NGM) 琼脂板种子与 OP50 的草坪大肠杆菌 。使用荧光 GECI 和/或共标记物的表达来识别阵列阳性动物. 
		注: 阵列阳性动物将根据 GECI 使用的荧光 ( 即, 动物表达 GCaMP 将荧光绿色在蓝光刺激下, 而 RCaMP 动物将荧光红色在绿色光刺激下)。共注射标记可以从其他荧光蛋白, 如 GFP 和 RFP, 到表型标记, 如 rol-6 , 或者可以拯救显性的表现, 如 pha-1 突变 28 。
		<ol> <li> 如果在检测前立即采摘, 请选择年轻的成年雄性。如果选择前一天的化验, 选择 L4 幼虫雄性. </li>
		</ol> </li> </ol> </li> <li> 图像对1和 #181 的杰姆响应; ascr#3. 
	<ol> <li> 选取大约 20 L4 C. 线虫 雄性 (fkEx98 [p pkd-2 :: GCaMP::SL2::d 失调 + pBX-1]; pha-1 (e2123ts); him-5 (e1490); lite-1 (ce314))对 dsRed 共标记表达式是正的. 
		注意: dsRed 表达的男性故事中的射线神经元更容易观察和确认比 GCaMP 表达在四杰姆神经元. </li>
		<li> 在进行成像实验之前, 将这些雄性从雌雄同体上的 NGM 琼脂板上的 OP50 的草坪上的大肠杆菌中分离出来。
		注意: 不被隔绝的男性至少 5 h 不行为反应对 ascr#3, 因此可能陈列甚而少量钙瞬变对 ascaroside 比这里观察. </li>
	</ol> </li> </ol> 5。动物装入 注意: 请参见 refefence 1 。
<ol> <li> 使用标准的蠕虫维护技术在种子选手 NGM 琼脂板上选择一个蠕虫. 
	<ol> <li> 通过火焰采摘 (由扁平的铂线制成), 挑选细菌到采摘, 并 #34;d abbing 和 #34; 蠕虫来拾取蠕虫。轻轻地将蠕虫放到新的盘子上, 让它自己爬行. </li>
	</ol> </li> <li> 在种子选手板上添加大约5毫升的 1x S 基片, 这样会使板块被淹没. </li>
	<li> 将该蠕虫绘制到装入注射器 ( 即3毫升带有附加油管的注射器), 已预先填充 1x S 基. 
	<ol> <li> 一定要将蠕虫吸进油管, 而不是全部注入注射器. 
		注: 如果蠕虫进入注射器, 它几乎不可能得到它回到油管. </li>
	</ol> </li> <li> 关闭真空以通过打开插座口阀门来停止流量. </li>
	<li> 卸下阻止蠕虫加载端口的实心针脚 </li>
	<li> 将口阀门连接到插座端口 ( 图 1B ), 使其处于排气状态. 
	注: 在加载蠕虫时使用实时视频提要以确认动物的位置和方向 (步骤 5.8-5.13). </li>
	<li> 将蠕虫加载管插入蠕虫加载端口. </li>
	<li> 轻轻按压注射器直到蠕虫出现在加载通道中. </li>
	<li> 如果蠕虫开始进入通道尾部-首先, 拉动注射器柱塞以防止蠕虫进入通道. </li>
	<li> 在应用和反转压力之间切换, 直到头部先进入通道. </li>
	<li> 打开真空, 将3路口阀门连接到出口端口, 打开它到真空而不是大气. </li>
	<li> 手动施加压力, 通过压低注射器柱塞来定向和放置蠕虫头, 使其暴露在缓冲流通道上, 但到目前为止, 头部可以自由移动 ( 图 2 D-2E ). </li>
</ol> 6。刺激和获取 <ol> <li> 使用开放源码显微镜软件 (如微管理器), 通过在三十年代使用蓝光激发 (470 nm) 将图像捕获为 TIFF 堆栈, 记录为10帧/秒. </li>
	<li> 将主菜单上的曝光设置为100毫秒 <ol> <li> 打开和 #34; 多 Acq #34; 从软件的主菜单中。设置 #34; 数字和 #34; #34; 300, #34; 和 #34; 间隔和 #34; 到和 #34; 0. #34; 单击和 #34; 获取! 和 #34; 获取视频. </li>
	</ol> </li> <li> 在启动获取后, 应用刺激 5 s 的十年代脉冲。根据需要调整刺激应用的持续时间. 
	<ol> <li> 在获取5视频后, 更改控制流量控制缓冲区的3路阀, 以将刺激应用于被测试的动物。单击阀门链接上的最左侧按钮 ( 图 1B ). </li>
		<li> 在十年代的刺激曝光后 (此时间可以根据用户的需要进行调整), 通过再次按下阀门链接上的最左边的按钮来改变缓冲区的流量. </li>
	</ol> </li> <li> 仅在缓冲区中记录, 直到 30-s 窗口完成, 才能使 GECI 荧光返回到基线. </li>
	<li> 按需要重复。等待三十年代之间的收购结束和开始的下一个审判. </li>
</ol> 7。图像分析 <ol> <li> 通过将文件拖动到 ImageJ 窗口中, 打开与开源软件 ImageJ 的 TIFF 堆栈. </li>
	<li> 单击 "使用游标" 并拖动以设置感兴趣的神经元周围的感兴趣区域 (ROI)。将该区域设置为包含感兴趣的神经元的 soma (如 图 3A 中所示). </li>
	<li> 通过单击 "打开-#62; 图像和 #62; 堆栈和 #62; 绘制 z 轴轮廓, 绘制整个堆栈中的 ROI 的荧光强度的 z 堆栈. </li>
	<li> 单击和 #34; 列表和 #34; 在打开的窗口中。单击 "编辑-和 #62; 复制以复制这些值。粘贴值到电子表格程序中. </li>
	<li> 通过将 ROI 拖动到不包含 GCaMP 表达式的蠕虫区域, 分析每个脉冲的背景荧光. </li>
	<li> 通过从神经元荧光强度值中减去背景荧光值, 对每个脉冲执行背景减法. </li>
	<li> 为每个脉冲的每个帧计算和 #916; f/f 0 。
	<ol> <li> 计算 F 0 作为获取的前1个 s 的 ROI 的平均强度值 (如 帧 1-10). 
		<li> 计算和 #916; f/f 0 通过将利息框架的背景减去值除以计算的 f 0 值. </li>
	</ol> </li> <li> 对每个神经元成像和每个刺激脉冲重复. </li>
	<li> 对于具有一致响应剖面的神经元 (如灰), 每个神经元的平均所有脉冲, 并计算 SEM (如图 2F ). </li>
	<li> 绘制平均和 #916; f/f 0 随着时间的推移对每个神经元进行扫描. 
	注: 在这种情况下, 通常的做法是包括 heatmaps 的个别神经元反应的每个试验以及。在反复刺激时, 或在不同个体的 23 中, 不表现出持续的钙瞬变变化的神经元, 它可能更适用于显示单个脉冲痕迹 (如 图 4 )。有关确定如何显示数据的详细信息, 请参见 讨论 . </li>
</ol>

<ol>
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作者没有什么可透露的。

男性适应的嗅觉芯片结合了一个转弯到一个狭窄的装货口岸, 允许更多控制方向和为有效地诱捕男性C. 线虫。这使得对神经元双边对的左、右两个成员的可视化, 而无需 z 堆叠。这条曲线导致一个方向远离垂直100% 的时间在蠕虫, 只有一个双边对是目标与荧光标记, 如灰 (图 2D-E)29,30。然而, 在神经元类的四径向对?...

微流控设备提供了更高的访问精确控制环境, 其中动物, 如线虫的C. 线虫, 可以在实验上操纵1。这些研究包括行为分析, 钙显像研究, 甚至筛选特定的表型, 导致更精确的实验结果的测量1,2,3,4, 5,6。微提供 small-scale 的液体条件, 通过它可以在使用少量试剂的同时运行详细的实验。有一个不断生产的新的微流控装置设计, 并使用每一个不同的领域, 从允许的自然正弦运动的C. 线虫的行为分析和神经成像研究, 陷阱设备用于神经成像和嗅觉研究, 对设备, 允许高通量表型分析在遗传屏幕4,5,6,7。随着主模的制造, 微流控设备的构建成本低廉, 因为它可以重用主控器, 而且易于使用, 允许通过高通量的研究快速生成数据。使用烷 (聚烯烃) 等聚合物制造设备, 可以在数小时内创造出新的设备。
钙显像研究使用在靶细胞中表达的基因编码的钙指标 (GECIs) 实时测量这些细胞的神经动力学8,9,10,11。C. 线虫的透明性质允许在活体动物中记录这些蛋白质的荧光水平。传统上, GECIs 依赖于绿色荧光蛋白 (GFP) 为基础的传感器 GFP-Calmodulin-M13 肽 (GCaMP), 虽然最近的研究已经适应这些传感器, 以允许更好的信噪比和红色位移的励磁剖面。随着 GCaMP3 的发展, 与这些规格的蛋白质有不同, 包括传感器, 如 GCaMP6s 和 GCaMP6f (慢和快速荧光 off-rates, 分别), 以及 RFP-Calmodulin-M13 肽 (RCaMP), 其中有一个红色转移激活配置文件。这些 GECIs 与C. 线虫细胞特异基因启动子序列的组合可以针对感兴趣的细胞, 特别是感觉神经元12,13,14,15,16。
虽然在微流体研究中使用的线虫的易用性很明显, 但几乎所有的研究都集中在雌雄同体上。尽管男性只占 0.01-0. 02% 的野生类型的人口, 宝贵的发现可能会产生从他们的特征。虽然雌雄同体神经系统的物理连接已经被完全映射了几十年17, 雄性连接仍然不完整, 特别是在动物的头部区域18。使用钙显像在男性将有助于产生一个了解男性神经系统和差异, 出现在两性之间。较小的大小的C. 线虫成年男性防止有效和可靠的诱捕在加载端口的传统嗅觉设备设计为较大的雌雄同体。为了解决这个问题, 修改后的 Chronis 嗅觉芯片19的版本开发了一个较窄的加载端口, 一个较低的通道高度, 并在蠕虫加载端口 (旋转的动物), 允许可视化的双边左/右神经元对。这个设计允许: (1) 年轻成年雄性的有效诱捕, (2) 对双侧配对神经元的可视化的动物更可靠的定位, 以及 (3) 对雄性神经元的神经活动的精确成像。
越来越多的研究表明, 线虫雄性对各种 ascarosides (ascr) 或线虫信息素20、21 、22、23 的响应雌雄同体不同。 ,24。因此, 在雄性连接中培养对神经动力学和表达的理解变得更加贴切。雄性的线虫包含87性特异性神经元不存在于雌雄同体的25,26中, 改变连接的不确定的方式。能够想象这些独特的神经动力学将使我们能够更好地理解性特定的反应和神经表达。
本议定书描述了使用男性适应的嗅觉芯片的神经成像男性线虫chemosensation。伤害的神经元灰反应可靠地对 1 M 甘油在男性, 与早先两性研究一致27。暴露在 ascarosides 可能引起的反应, 从动物到动物的变化, 需要更多的动物进行测试。通过电生理学和钙显像研究表明, 雄性特异性的杰姆神经元的反应, 变对 ascaroside #323。

<table><tbody><tr><td>Silicon Wafer</td><td>University Wafer</td><td>452</td><td></td></tr><tr><td>SU-8 2035</td><td>MicroChem</td><td>Y111070-0500L1GL</td><td></td></tr><tr><td>Developer</td><td>MicroChem</td><td>Y020100-4000L1PE</td><td></td></tr><tr><td>Wafer Mask</td><td>Cad/Art Services</td><td>-</td><td>Custom order. Printed at 25,000 dpi.</td></tr><tr><td>Sylgard-184</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td></td></tr><tr><td>1.0 mm Dermal Punches</td><td>Acuderm Inc.</td><td>P150</td><td></td></tr><tr><td>Soft Tubing</td><td>Cole-Palmer</td><td>EW-06419-01</td><td></td></tr><tr><td>Hard Tubing</td><td>IDEX Health &amp;amp; Science</td><td>1622</td><td></td></tr><tr><td>Pins</td><td>New England Small Tube</td><td>NE-1027-12</td><td></td></tr><tr><td>Blocking Pins</td><td>New England Small Tube</td><td>0.415/0.425&quot; OD x .500 Long</td><td>Batch PB07027</td></tr><tr><td>3 mL syringes</td><td>BD</td><td>309657</td><td></td></tr><tr><td>30 mL syringes</td><td>Vitality Medical</td><td>302832</td><td>Used as buffer reservoirs.</td></tr><tr><td>Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer</td><td>Component Supply Company</td><td>NE-231PL-50</td><td></td></tr><tr><td>Stopcocks with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile</td><td>Cole-Palmer</td><td>EW-30600-07</td><td></td></tr><tr><td>Fisherfinest Premium Cover Glass</td><td>Fisher Scientific</td><td>12-548-5M</td><td></td></tr><tr><td>Mercator Control System LF-5 Plasma System</td><td>Mercator</td><td>LF-5</td><td></td></tr><tr><td>Scotch Tape</td><td>Scotch</td><td>BSN43575</td><td></td></tr><tr><td>Series 20 Chamber</td><td>Warner Instruments</td><td>P-2</td><td></td></tr><tr><td>Vacuum Desicator</td><td>Bel-Art Scienceware</td><td>420250000</td><td>24 cm inner diameter.</td></tr><tr><td>Weigh Boats</td><td>Cole-Palmer</td><td>EW-01017-27</td><td></td></tr><tr><td>Classic Plus Balance</td><td>Mettler Toledo</td><td>PB1501-S/FACT</td><td></td></tr><tr><td>Glass Pasteur Pipettes</td><td>Cole-Palmer</td><td>EW-25554-06</td><td></td></tr><tr><td>Transfer pipettes</td><td>Genesee Scientific</td><td>30-202</td><td></td></tr><tr><td>Oven</td><td>Sheldon Manufacturing Inc</td><td>9120993</td><td>Model Number: 1500E.</td></tr><tr><td>60 mm, non-vented, sharp edge Petri dishes</td><td>TriTech Research</td><td>T3308</td><td></td></tr><tr><td>Zeiss Axio Observer.A1</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Hammamatsu Orca Flash 4.0 Digital CMOS</td><td>Hammamatsu</td><td>C11440-22CU</td><td></td></tr><tr><td>Blue Fluorescent Light</td><td>Lumencor</td><td>SOLA SM6-LCR-SA</td><td>24-30V/7.9A DC.</td></tr><tr><td>Illumination Adaptor</td><td>Zeiss</td><td>423302-0000</td><td></td></tr><tr><td>Series 1 and 2 Miniature Inert PTFE Isolation Valve</td><td>Parker</td><td>001-0017-900</td><td>3-way valve for controlling flow.</td></tr><tr><td>ValveLink8.2&amp;reg;</td><td>AutoMate Scientific</td><td>01-18</td><td>Flow Switch Controller</td></tr><tr><td>Micro Manager</td><td>Micro-Manager</td><td>-</td><td>Free software, can be downloaded at: https://www.micro-manager.org/wiki/Download_Micro-Manager_Latest_Release</td></tr><tr><td>ImageJ</td><td>ImageJ</td><td>-</td><td>Free software, can be downloaded at: https://imagej.nih.gov/ij/download.html</td></tr><tr><td>Agar, Bacteriological Grade</td><td>Apex</td><td>9012-36-6</td><td></td></tr><tr><td>Peptone</td><td>Apex</td><td>20-260</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>VWR</td><td>BDH0224-1KG</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>230391-1kg</td><td></td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>A11470</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>270741-4L</td><td></td></tr><tr><td>Tetramisole</td><td>Sigma-Aldrich</td><td>L9756-10(G)</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Fluorescein</td><td>Sigma-Aldrich</td><td>FD2000S-250mg</td><td>Light Sensitive. Store in photoprotective vials.</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G6279-1L</td><td></td></tr><tr><td>Ascaroside #3</td><td>-</td><td>-</td><td>Synthesized in the Schroeder Lab (Cornell University).</td></tr><tr><td>NaCl</td><td>Genesee Scientific</td><td>18-215</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>BDH</td><td>BDH9268.25</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3252-025</td><td></td></tr><tr><td>ASH GCaMP3 line</td><td>-</td><td>-</td><td>CX10979 (KyEx2865 [p&lt;em&gt;sra&lt;/em&gt;-6::GCAMP3 @ 100 ng/uL]). Developed in Bargmann lab. Provided from Albrecht Lab library.</td></tr><tr><td>CEM GCaMP6 line</td><td>-</td><td>-</td><td>JSR49 (FkEx98[p&lt;em&gt;pkd&lt;/em&gt;-2::GCaMP::SL2::dsRED + pBX-1]; &lt;em&gt;pha&lt;/em&gt;-1(e2123ts); &lt;em&gt;him&lt;/em&gt;-5(e1490); &lt;em&gt;lite&lt;/em&gt;-1(ce314)). Developed by Robyn Lints. Provided from Srinivasan Lab library.</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; (OP50)</td><td>&lt;em&gt;Caenorhabditis&lt;/em&gt; Genetics Center</td><td>OP50</td><td></td></tr><tr><td>&quot;Reservoir&quot;</td><td>-</td><td>-</td><td>To create a Reservoir: A &quot;30 mL syringe&quot;, is connected to a &quot;Stopcock with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile&quot;, which is connected to a &quot;3 mL syringe&quot; and a &quot;Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer&quot;. The &quot;Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer&quot; is then inserted into &quot;Soft Tubing&quot; approximately 1/3 of the way down the needle.</td></tr></tbody></table>

using an adapted microfluidic olfactory chip for the imaging of neuronal activity in response to pheromones in male c. elegans head neurons

使用钙指标大大提高了我们对神经动力学和调节的认识。线虫线虫线虫, 其完全映射的神经系统和透明的解剖, 提出了一个理想的模型, 了解 real-time 神经动力学使用钙指标。结合微流控技术和实验设计, 使用这些指标的钙显像研究在自由和被困动物中都进行。然而, 以前的研究大多使用诱捕器, 如 Chronis et al.中描述的嗅觉芯片, 这些装置的设计用于更常见的雌雄同体, 因为不常见的雄性在形态和结构上都不同.设计和制作了一种适应的嗅觉芯片, 以提高男性神经元成像的效率, 使用年轻的成年动物。一个转弯被并入到蠕虫的装货港旋转的动物和允许分离的单个神经元内的一对双边对2D 成像。正如以前的雌雄同体研究中所描述的, 蠕虫被暴露在微流控装置内受控的气味流中。然后使用开源软件 ImageJ 分析钙瞬变。本文所述的过程应允许增加 male-based 的C. 线虫钙显像研究, 加深我们对性特异性神经元信号机制的理解。

在图 1A-b中可以看到整个设备设置的一个示例。图 1A描述了适当的油藏构造和设置。图 1B显示了储层与微流体设备的连接。图 1C描述了一个微流体设备, 其各个端口标记为清晰。
设计的男性适应微流控装置包含一条曲线在装?...

Watch this Scientific Journal Video about Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons at JoVE.com

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

在这里描述了使用一个适应的 "嗅觉芯片" 的有效钙显像的C. 线虫男性。还显示了男性接触甘油和信息素的研究。

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

using-an-adapted-microfluidic-olfactory-chip-for-imaging-neuronal

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

8.0K 次观看.  Worcester Polytechnic Institute. 在这里描述了使用一个适应的 "嗅觉芯片" 的有效钙显像的C. 线虫男性。还显示了男性接触甘油和信息素的研究。

视频: Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

科研

生物工程

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

在气味诱发反应的钙成像果蝇触角叶

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

神经科学

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

行为学

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

<meta charset="utf8"></head><body>秀丽隐杆线虫中挥发性信息素反应的提取和定量

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

<meta charset="utf8"></head><body>作者聚焦：检查挥发性信息素对雄性 秀丽隐杆线 虫行为的影响

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

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

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

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

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

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简单的微流控芯片的

在气味诱发反应的钙成像果蝇触角叶

基于线虫人群的化学感应优选测定的自动分析

使用微流体芯片实时成像和损伤研究的响应

长期的嗅觉适应性的分子读数

多单位记录方法来描述蝗虫的神经活动（

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

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