Name: tracking drosophila larval behavior in response to optogenetic stimulation of olfactory neurons
Uploaded: 2018-03-21T14:30:00.000+00:00
Duration: 6 min 49 s
Description: Watch this Scientific Journal Video about Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons at JoVE.com

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...

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。

<table><tbody><tr><td>&lt;strong&gt;Video camera to capture larval movement&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CCD Camera&amp;nbsp;</td><td>Edmund Optics</td><td>106215</td><td></td></tr><tr><td>M52 to M55 Filter Thread Adapter</td><td>Edmund Optics</td><td>59-446</td><td></td></tr><tr><td>2&quot; Square Threaded Filter Holder for Imaging Lenses&amp;nbsp;</td><td>Edmund Optics</td><td>59-445</td><td></td></tr><tr><td>RG-715, 2&quot; Sq. Longpass Filter</td><td>Edmund Optics</td><td>46-066</td><td></td></tr><tr><td>&lt;strong&gt;Electronics for optogenetic setup&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Raspberry Pi 2B</td><td>RASPBERRY-PI.org</td><td>RPI2-MODB-V1.2</td><td></td></tr><tr><td>3 Channel programmable power supply</td><td>newegg.com</td><td>9SIA3C62037092</td><td></td></tr><tr><td>8 Channel optocoupler relay</td><td>amazon.com</td><td>6454319</td><td></td></tr><tr><td>630nm Quad-row LED strip lights</td><td>environmentallights.com</td><td>red3528-450-reel</td><td></td></tr><tr><td>850nm LED strips</td><td>environmentallights.com</td><td>wp-4000K-CC5050-60x2-kit</td><td></td></tr><tr><td>&lt;strong&gt;Software&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Matlab</td><td>Mathworks Inc.</td><td></td><td></td></tr><tr><td>Ubuntu MATE v16.04</td><td>Nubuntu</td><td>https://github.com/yslo/nubuntu</td><td></td></tr><tr><td>&lt;strong&gt;Other items&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plexiglass black acrylic</td><td>Home Depot</td><td>MC1184848bl</td><td></td></tr><tr><td>&lt;strong&gt;Fly food and other reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nutrifly fly food</td><td>Genesee Scientific</td><td>66-112</td><td></td></tr><tr><td>Agarose powder</td><td>Genesee Scientific</td><td>20-102</td><td></td></tr><tr><td>22cm X 22cm square petri-dish</td><td>VWR Inc.</td><td>25382-327</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr><tr><td>All trans-retinal</td><td>Sigma-Aldrich</td><td>R2500</td><td></td></tr><tr><td>&lt;strong&gt;Flies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;UAS-IVS-CsChrimson&amp;nbsp;&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>55134</td><td></td></tr><tr><td>&lt;em&gt;Orco-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>26818</td><td></td></tr><tr><td>&lt;em&gt;Or42a-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>9970</td><td></td></tr><tr><td>&lt;em&gt;Or7a-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>23907</td><td></td></tr></tbody></table>

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,...

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 ...

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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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

科研

生物学

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

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 ...

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 ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

通过计算机跟踪检测大鼠嗅觉行为<em&gt;果蝇</em&gt;在四象限嗅

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

黑腹果蝇神经回路光遗传学作的行为测定

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Navigational Behavior of Drosophila Larvae in Response to Optogenetic Stimulation of Olfactory Sensory Neurons

Source: Clark, D. A., et al. <a href="https://app.jove.com/v/57353">Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons.</a> J. Vis. Exp. (2018).
This video demonstrates a study of the navigational behavior of Drosophila larvae in response to optogenetic stimulation of olfactory sensory neurons (ORNs). Transgenic Drosophila larvae expressing light-sensitive ion channels in the ORNs are placed on agarose within a behavior arena. Optogenetic stimulation with light opens the channels, causing a cation influx that triggers the transmission of the signal to the brain, and the navigation behavior is recorded.

果蝇幼虫对嗅觉感觉神经元光遗传学刺激的响应的导航行为

1. Building a Behavior Arena and Preparing Hardware to Enable Optogenetic Stimulation in the Behavior Arena

	To build a light-deprived behavior arena, ...

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

摘要

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此视频中的章节

高分辨率测量的气味驱动在果蝇幼虫的行为

在气味诱发反应的钙成像果蝇触角叶

欲求联想嗅觉学习

Optogenetic刺激的逃避行为

光遗传学的扰动激光照射半完好的神经活动

在气味引导行为的高分辨率定量

通过计算机跟踪检测大鼠嗅觉行为

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

黑腹果蝇神经回路光遗传学作的行为测定

果蝇幼虫对嗅觉感觉神经元光遗传学刺激的响应的导航行为