这项工作得到了内华达大学、雷诺分校和国立卫生研究院研究院 P20 GM103650 的启动资金的支持。

1. 建立行为竞技场, 准备硬件, 使 Optogenetic 在行为舞台上得到刺激
<ol><li>要构建一个光被剥夺的行为竞技场, 构造一个尺寸为 89 x 61 x 66 cm3 (35 "L x 24" W x 26 "H) 的框, 由黑色有机玻璃丙烯酸片 (3 毫米厚) 制成 (见材料表)。在当地的五金店里, 应该有制作这种盒子的材料。将此框放在行为室的桌面上 (图 1a)。</li>
	<li>安装一个单色 USB 3.0 CCD 摄像机, 装有红外长通 830 nm 过滤器和一个8毫米 F1.4 C 安装透镜 (请参见材料表) 到黑盒天花板的中心。将两个红外线 LED 带 (参见材料表) 放在桌面上, 以照亮黑暗竞技场中的幼虫 (图 1).</li>
	<li>要建立 LED 平台, 获得一个22厘米 x 22 厘米方形铝板 (最好喷涂与哑光黑色完成, 以消除任何反射)。在板的中心, 使用金属切割器, 切割一个足够大的孔, 以适应 CCD 相机。</li>
	<li>用红色 LED 带灯覆盖金属板 (请参阅材料表)。焊料 LED 灯带线串联, 并将带状导线馈入由覆盆子 Pi 2B 微处理器控制的光耦继电器 (请参阅材料表) (图 1B和2)。</li>
	<li>在将光电耦合器中继连接到 LED 带之前, 在树莓 Pi 处理器上安装和配置 Ubuntu 伙伴/Raspian 基于 Linux 的操作系统。连接电源以通电 LED 带和光耦 (图 2) (请参阅材料表)。在 CCD 摄像机周围安装 LED 平台 (图 1B)。 注意: Ubuntu 队友 v16.04 操作系统是免费提供的。一组简单的基于 Python 的命令可以很容易地适应 LED 光刺激的程序模式 (参见语法文件)。</li>
	<li>确保行为舞台上各点的均匀辐照度。在分光计的帮助下, 测量竞技场表面的绝对辐照度, 并确定它在整个竞技场表面为1.3 瓦特/m2 。 注: 在这种强度下, 实验过程中的温度没有明显的变化。另一项研究10使用了1.9 瓦特/m2的较高辐照度, 并观察到实验过程中的温度变化。</li>
</ol>2. 为行为分析准备果蝇幼虫
<ol><li>在标准飞行食品上保持苍蝇 (参见材料表), 25 摄氏度, 50-60% RH, 12 h/12 h 光/暗循环。</li>
	<li>
为了在单对 ORNs 中表达CsChrimson , 从无人参与 IVS CsChrimson行到从OrX-Gal4线 ("X") 到雄性的交叉处女雌性, 对应于每种唯一表达的21种幼虫气味受体 (或) 基因之一。21对 ORNs)9,11。
	<ol>
<li>交替地, 为了在所有21幼虫 ORNs 中表达CsChrimson , 从无人参与的 IVS CsChrimson线向雄性从Orco-Gal4线 ("萨拉") 中的杂交处女雌性是在所有 21 ORNs 中表达的共同受体。</li>
		<li>在这些实验中, 使用无人 IVS CsChrimson行本身作为控件。 注: 此处列出的苍蝇库存均可在布卢明顿果蝇库存中心 (参见材料表) 提供。</li>
	</ol>
</li>
	<li>
一旦雄性和雌性苍蝇在十字架上被允许交配和产卵48小时, 转移成人到一个新鲜的瓶子。
	<ol>
<li>在含有鸡蛋的食物瓶的表面, 加入400µL, 其中含有400µM 的全反式视网膜 (ATR) 溶解在二甲基亚砜 (亚砜) 和89毫米蔗糖溶解在蒸馏水中。 注: 少量蔗糖促进了 ATR 溶液的幼虫喂养。ATR 是植入 CsChrimson 表达式9、10所需的余子。ATR 是光敏感的。</li>
		<li>一旦 ATR 被添加到含有鸡蛋的食物瓶中, 在黑暗中孵化出一小瓶额外的72小时。 注: 虽然本研究和上述两项研究未观察到对 atr 喂养对幼虫行为的影响, 但建议通过将试验线置于不含 ATR 的上述混合物的相同数量上, 控制 atr 喂养的效果。</li>
	</ol>
</li>
	<li>用高密度 (15%) 蔗糖溶液将三龄幼虫 (产卵后120小时) 从苍蝇的表面提取出来。使用 P1000 微分离在蔗糖溶液表面漂浮的幼虫成1000毫升的玻璃烧杯。</li>
	<li>每次通过在玻璃烧杯中交换800毫升新鲜蒸馏水来洗涤幼虫3–4倍。允许幼虫休息10分钟之前, 使他们的行为化验。</li>
</ol>3. 行为分析法
<ol><li>保持一致的温度 (介于22–25°c 之间) 和湿度 (介于50和 60% RH 之间) 在行为室。</li>
	<li>
将150毫升的熔融琼脂糖 (1.5%) 倒入22厘米 x 22 厘米方培养皿中, 准备幼虫爬行培养基。每项试验的每项试验都要浇一个培养皿, 8–10试验。允许琼脂糖在培养皿中凝固和冷却, 然后用在行为测定中。
	<ol>
<li>将未准备的第三龄幼虫的20转移到培养皿的中心 (图 1C)。用盖子盖住培养皿。将培养皿放在 CCD 摄像机下的行为竞技场中。 注意: 根据实验, 行为分析通常是为 3–5 min 进行的。如果在检测中使用气味提供指导线索, 则观察到产生的气味梯度保持稳定大约5分钟1。不建议使用更长的检测时间。在这些时间点内没有观察到因脱水或长时间 630 nm 的红光暴露而对幼虫造成的有害影响。</li>
	</ol>
</li>
	<li>打开 850 nm 红外线 LED 光源, 使视频中的幼虫可视化。启动 CCD 摄像机记录幼虫运动。</li>
	<li>使用与树莓 Pi 处理器相关的软件, 编程程序来管理适当的红光刺激模式。 注意: 一组简单的基于 Python 的命令可以很容易地适应 LED 光刺激的程序模式 (参见语法文件)。</li>
</ol>4. 数据处理和分析
<ol><li>将每个试用录制的视频导入到任何可用的编程软件 (如 Matlab) 中。</li>
	<li>获取电影中每个幼虫的 XY 坐标作为时间的函数。根据跟踪软件的限制, 可以在单个影片1、8中跟踪15–20第三龄幼虫。 注意: 提供了一组简单的 Matlab 代码 ("Tracklarva"), 可以方便地适应适当的条件 (参见语法文件)。这个程序结合了实验中的所有试验, 并输出每个幼虫的 XY 坐标, 在整个测试期间 (请参见下面的代码语法)。另外, 可以使用几个开放源码的程序, 为研究人员免费提供。例如JAABA (http://jaaba.sourceforge.net/)12。</li>
	<li>
使用生成的 XY 坐标绘制幼虫轨迹并进一步分析幼虫的运动。
	<ol>
<li>对于行为分析, 使用导航统计 (如速度、路径曲率、航向角) 将单个幼虫轨迹分割成交替序列的奔跑和转弯。</li>
		<li>运行定义为向前移动的连续期间。轮流连续运行。当对轨迹方向角的更改为 > 45° (参见语法文件) 时, 将标记为旋转。</li>
	</ol>
</li>
	<li>根据需要计算运行速度、运行长度、运行方向和其他参数的平均值。 注意: 提供了一组从幼虫轨迹中提取 "运行" 和 "停止" 的简单语法 (参见语法文件)。可以将简单的 Matlab 或基于 Excel 的函数应用于提取的数据, 以计算 "运行速度"、"运行长度"等的值。</li>
	<li>表示每个行为度量的数据为平均值。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们描述了一个方法, 允许测量的果蝇幼虫行为的反应, 同时 optogenetic 激活嗅觉神经元。以前描述的幼虫跟踪方法1,8使用不同的气味传递技术来激活 ORNs。然而, 这些方法不能控制 ORN 激活的特异性或时间模式。我们的方法克服这些缺陷, 通过使用光刺激, 而不是气味刺激, 以更精确的控制 ORN 活化。
构建行为舞台所需的材料可以很容易地在本地硬件存储区获得, 并且在组装过程中需要最小的工作量。准备光遗传学模块所需的电子设备也很容易获得和构建。此处描述的方法使用红光激活在特定神经元中表达的红移通道紫红 (CsChrimson) 。所产生的幼虫行为响应相应的 ORN 激活是记录使用 CCD 相机和测量使用定制的书面软件, 这里提供。我们的方法允许研究人员问几个以前不可能的问题: 1) 不同的嗅觉刺激模式对动物嗅气味的能力有什么影响？2) 类似于哺乳动物视网膜的中心和离中心神经节细胞16, 是否有 ORNs, 除了 ORNs, 对嗅觉刺激的增加作出反应之外, 还特别对嗅觉刺激的减少作出反应？最后, 我们的方法允许各种未来的应用, 包括测量下游神经元在嗅觉电路 (LNs) 对幼虫导航的影响。
虽然我们的方法有几个优点, 但我们承认某些限制。目前还不清楚使用该系统是否可以很容易地复制气味观察到的浓度效应。虽然我们目前的设置不允许这样做, 光遗传学模块可以很容易地修改, 以适应增加或减少光刺激的强度。在未来, 我们将检查是否变化强度的光刺激模拟气味的浓度效应。简单的苍蝇遗传技术可用于在所有21对 ORNs (使用 Orco-Gal4) 或单对 ORNs (使用单独的 Or-Gal4s) 中表达CsChrimson 。然而, 需要复杂的遗传学来表达CsChrimson在 ' 1 < n < 21 ' 神经元。由于这种情况, 很难用气味混合物来复制观察到的效果, 因为混合物的单个成分会引起来自多个 ORN 的反应。虽然幼虫导航行为被认为是一个低维行为, 我们承认, 我们的幼虫跟踪计划可以进一步改善, 在未来考虑更多的行为描述, 基于动物的姿态 (例如,头部转弯、身体弯曲等的概率)8,17。我们的研究仅限于幼虫的一阶感官神经元。在我们的方法可以应用于大脑的脑叶区域的第二阶投射神经元和局部神经元之前, 需要进一步的研究18。
总之, 我们的方法提供了能够解剖的功能, 每个 ORN 在简单, 易于操作的嗅觉电路的果蝇幼虫。通过这样做, 我们的方法将使开发更精确的计算模型, 描述如何将气味信号转化为不同的行为输出。

果蝇幼虫环境中的嗅觉信息仅由21功能分明的 ORNs 感知, 其活动最终决定幼虫行为1,2,3,4。然而, 在这 21 ORNs 的活动中, 对感官信息进行编码的逻辑所知甚少。因此, 需要实验性地测量每个幼虫 ORN 对行为的功能贡献。
尽管果蝇幼虫 ORNs 的整个曲目的感官反应剖面已经详细研究了1,4,5, 个人 ORNs 对嗅觉电路的贡献, 从而导航行为仍然很不清楚。困难在幼虫行为研究, 到目前为止, 出现由于不能空间地和世俗地激活单一 ORNs。最近描述了一个专门激活 21果蝇幼虫 ORNs 19 的气味的面板1。小组中的每一个气味, 在低浓度下, 只能从同源 ORN 引起生理反应。但是, 在通常用于常规行为检测的较高浓度下, 每个气味从多个 ORNs1、5、6中引出生理响应。此外, 气味在这个小组有不同的波动性, 复杂的解释的行为研究, 依赖于形成稳定的气味梯度7,8。最后, 自然发生的气味刺激有一个时间成分难以复制在实验室条件下。因此, 发展一种能在空间和时间上同时激活个体 ORNs 的方法可以测量幼虫的行为是很重要的。
在这里, 我们演示了一种比以前描述的幼虫跟踪分析1,8更有优势的方法。Gershow et . 中描述的跟踪检测使用电子控制阀, 在行为竞技场8中保持一种稳定的气味梯度。然而, 由于构建恶臭刺激装置所涉及的复杂工程水平, 这种方法在其他实验室难以复制。此外, 与使用气味专门激活单个 ORNs 有关的问题仍未解决。马修et . 中描述的跟踪分析使用了一种更简单的气味传递系统, 但由此产生的气味梯度依赖于测试气味的挥发性, 并且在检测的长时间内是不稳定的1。因此, 通过用光刺激代替气味刺激, 我们的方法具有特异性和 ORN 活化的精确时间控制的优点, 不依赖于不同强度的气味梯度的形成。
我们的方法是容易建立和适合的研究人员感兴趣的测量方面的果蝇幼虫导航。这种技术可以适应其他模型系统, 前提是研究人员能够在他们喜爱的系统的神经元中驱动CsChrimson的表达式。CsChrimson是通道紫红的红色移动版本。它被激活在波长是不可见的幼虫的趋光性系统。因此, 我们能够利用特异性、可靠性和再现度9来操作神经元的活动。通过修改自定义编写的软件来考虑受试者的尺寸变化, 这种方法可以很容易地适应其他昆虫种类的爬行幼虫。

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tracking drosophila larval behavior in response to optogenetic stimulation of olfactory neurons

昆虫对气味来源的导航能力是基于它们的一级嗅觉受体神经元 (ORNs) 的活动。虽然在对气味的 ORN 反应方面产生了大量的信息, 但具体 ORNs 在驾驶行为反应方面的作用仍然不甚了解。行为分析的并发症产生的原因是不同的气味的波动性, 激活个体 ORNs, 多个 ORNs 激活单气味, 和难以复制自然观察到的嗅觉刺激的时间变化使用实验室常规的气味传递方法。在这里, 我们描述了一个协议, 分析了果蝇幼虫行为, 以响应同时 optogenetic 刺激其 ORNs。这里使用的 optogenetic 技术允许 ORN 活化的特异性和精确控制 ORN 活化的时间模式。对相应的幼虫运动进行跟踪、数字记录, 并使用自定义书面软件进行分析。通过用光刺激代替气味刺激, 这种方法可以更精确地控制个体的 ORN 活化, 以研究其对幼虫行为的影响。我们的方法可以进一步扩展, 以研究二阶投射神经元 (三七) 和局部神经元 (LNs) 对幼虫行为的影响。这种方法将使嗅觉回路功能得到全面的解剖, 并补充了嗅觉神经元活动如何转化为行为反应的研究。

为了证明 ORN 激活的特殊性, 我们的方法成功地应用于确定两个不同的 ORN (ORN::7a & ORN::42a) (ORNs 表示 Or7a 或 Or42a) 激活幼虫行为 (图 3) 的影响。与最近的研究表明, 单个幼虫 ORNs 在功能上不同的1,10,13, 我们的代表性数据表明, 当 ORN::7a 表达 CsChrimson 是由光刺激, 有一个与控制动物相比, 跑步长度显著降低。反之, 当 ORN::42a 表达 CsChrimson 被光刺激时, 与控制动物相比, 运行长度显著增加 (图 3)。从对每种基因型进行的试验 (n = 8) 得到了从100-120 幼虫轨迹分析得出的集体数据。误差线代表 SEM。虽然我们只描述一个行为参数 (运行长度) 在这里, 我们注意到, 每个幼虫轨道可以进一步分析, 以计算速度, 路径曲率和身体弯曲的参数1,8,13, 14,15。如果在竞技场的一侧 (1、8、13) 上提供了异味源, 则可以获得更多与方向性相关的参数, 如标题角、运行长度和向异味方向的运行速度。
为了证明我们的方法改变 ORN 激活的时间模式的能力, 我们改变了我们的刺激, 以交替之间的灯光和打开。我们在 ORN::42a 中对三种不同的光刺激时间模式 (0.04 赫兹、1赫兹和常数) 进行了 CsChrimson 的幼虫的表达, 它们的作用是:在时间上, 在光照下, 对光线的激发然后我们测量在灯关闭期间发生的行为参数的变化→在阶段和在灯期间→关闭阶段。我们发现, 对于 ORN::42a, 不同的时间模式的光刺激引起不同的行为反应 (图 4)。在任何 ORNs 中未表示CsChrimson的控制幼虫中未观察到这种变化.这些结果突出了理解 ORN 激活时间模式对动物行为的重要性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57353/57353fig1.jpg"> 图 1: 行为竞技场和幼虫爬行培养基。(A)黑盒行为竞技场的前视图。竞技场的敞开的门显示了从盒子天花板上悬浮下来的 CCD 摄像机。(B)在 CCD 摄像机周围安装了一个金属平台的底部视图, 其中包含用于 optogenetic 刺激的红色 LED 灯条。(C)检测中使用的幼虫爬行培养基的顶部视图。在记录幼虫运动开始之前, 20 只被洗涤的幼虫沿22厘米 x 22 厘米培养皿的中心放置, 1.5% 琼脂糖。含有幼虫的培养皿置于 CCD 摄像机下的竞技场中心, 在两个红外线 LED 光条之间, 用作照相机的光源。<a href="//ecsource.jove.com/files/ftp_upload/57353/57353fig1large.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/57353/57353fig2.jpg"> 图 2: 光遗传学安装程序。显示光遗传学设置的电子安排的图表。简单地, LED 光 (630 毫微米) 条连接在系列和导线从小条被哺养入一个光耦连接到树莓 PI 2B 微处理器。LED 灯条和光耦都由电源供电。<a href="//ecsource.jove.com/files/ftp_upload/57353/57353fig2large.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/57353/57353fig3.jpg"> 图 3: 单个 ORNs 的光激活对幼虫行为的影响.在 ORN::7a 和 ORN::42a 中表达CsChrimson的幼虫的运行长度与控制幼虫在光激活时的影响不同。每个条形图代表平均 RI (n = 8)。ORN::7a 激活时幼虫的运行长度明显低于对照。ORN::42a 激活时幼虫的运行长度明显高于对照。条形表示平均值的电子扫描电镜 (n = 8, 学生 t 测试;"*" 为p < 0.05, "**" 为p < 0.001).<a href="//ecsource.jove.com/files/ftp_upload/57353/57353fig3large.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/57353/57353fig4.jpg"> 图 4: ORN 激活的不同时间模式对幼虫行为的影响。(A)三用于激活 ORNs 的刺激时间模式。刺激 a: 1 分钟恒定的光在期间, 刺激 b: 0.04 赫兹轻的刺激, 刺激 c: 1 赫兹光刺激在期间在分钟2的期间 LED。(B)幼虫受三种不同的光刺激模式所描述。每个点代表幼虫行为的变化, 在每种模式下的光刺激, 当光激活从上到下切换。在 X 轴上绘制 "运行长度" (av、运行长度 (OFF)-av. 运行长度 (ON)) 的更改。"运行速度" (av, 运行速度 (关闭)-av. 运行速度 (ON)) 绘制在 Y 轴上。左图 (灰色点) 代表来自控制幼虫的测量, 右图 (红点) 代表 ORN::42a 中表示CsChrimson的幼虫的测量结果。<a href="//ecsource.jove.com/files/ftp_upload/57353/57353fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Final Code PDF" src="/files/ftp_upload/57353/57353code.jpg"> 语法补充文件:一组简单的 Matlab 代码 ("Tracklarva"), 可以方便地适应适当的条件.<a href="https://www.jove.com/files/ftp_upload/57353/Final_Code.pdf" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons at JoVE.com

追踪果蝇幼虫响应嗅觉神经元光遗传学刺激的行为神经科学 |视频

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

该协议分析了果蝇幼虫对其嗅觉神经元同时 optogenetic 刺激的导航行为。光的 630 nm 波长是用来激活单个嗅神经元表达一个红色转移通道紫红。幼虫运动是同时跟踪, 数字记录, 并分析使用自定义编写的软件。

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

tracking-drosophila-larval-behavior-response-to-optogenetic

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Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

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7.2K 次观看.  University of Nevada. 该协议分析了果蝇幼虫对其嗅觉神经元同时 optogenetic 刺激的导航行为。光的 630 nm 波长是用来激活单个嗅神经元表达一个红色转移通道紫红。幼虫运动是同时跟踪, 数字记录, 并分析使用自定义编写的软件。

视频: Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

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 ...

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神经科学

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Methods to Assay Drosophila Behavior

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

Methods to Assay Drosophila Behavior

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

对检测方法果蝇行为

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

欲求联想嗅觉学习<em&gt;果蝇</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

果蝇嗅觉成人学习的冲击

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including 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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

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3幼虫节段性神经的可视化^RD龄果蝇幼虫的筹备工作

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

对检测方法果蝇行为

欲求联想嗅觉学习

果蝇嗅觉成人学习的冲击

Optogenetic刺激的逃避行为

嗅觉受体神经元在全山免疫标记

在啮齿动物中枢神经系统的体内光遗传学刺激

听觉神经的光遗传学刺激