Name: tracking drosophila larval behavior in response to optogenetic stimulation of olfactory neurons
Uploaded: 2018-03-21T14:30:00
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...

<ol>
	<li>Mathew, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversity+among+sensory+receptors+in+a+Drosophila+olfactory+circuit.">Functional diversity among sensory receptors in a Drosophila olfactory circuit.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 2134-2143 (2013).</li><li>Ramaekers, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+maps+without+cellular+redundancy+at+successive+levels+of+the+Drosophila+larval+olfactory+circuit.">Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit.</a> Current biology : CB. 15, 982-992 (2005).</li><li>Couto, A., Alenius, M., Dickson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular,+anatomical,+and+functional+organization+of+the+Drosophila+olfactory+system.">Molecular, anatomical, and functional organization of the Drosophila olfactory system.</a> Current biology : CB. 15, 1535-1547 (2005).</li><li>Kreher, S. A., Kwon, J. Y., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+odor+coding+in+the+Drosophila+larva.">The molecular basis of odor coding in the Drosophila larva.</a> Neuron. 46, 445-456 (2005).</li><li>Kreher, S. A., Mathew, D., Kim, J., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+sensory+input+into+behavioral+output+via+an+olfactory+system.">Translation of sensory input into behavioral output via an olfactory system.</a> Neuron. 59, 110-124 (2008).</li><li>Hallem, E. A., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+odors+by+a+receptor+repertoire.">Coding of odors by a receptor repertoire.</a> Cell. 125, 143-160 (2006).</li><li>Monte, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+larval+olfactory+response+in+Drosophila+and+its+genetic+basis.">Characterization of the larval olfactory response in Drosophila and its genetic basis.</a> Behav Genet. 19, 267-283 (1989).</li><li>Gershow, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+airborne+cues+to+study+small+animal+navigation.">Controlling airborne cues to study small animal navigation.</a> Nature methods. 9, 290-296 (2012).</li><li>Klapoetke, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nature methods. 11, 338-346 (2014).</li><li>Hernandez-Nunez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse-correlation+analysis+of+navigation+dynamics+in+Drosophila+larva+using+optogenetics.">Reverse-correlation analysis of navigation dynamics in Drosophila larva using optogenetics.</a> eLife. 4, (2015).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Kabra, M., Robie, A. A., Rivera-Alba, M., Branson, S., Branson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAABA:+interactive+machine+learning+for+automatic+annotation+of+animal+behavior.">JAABA: interactive machine learning for automatic annotation of animal behavior.</a> Nature methods. 10, 64-67 (2013).</li><li>Newquist, G., Novenschi, A., Kohler, D., Mathew, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contributions+of+Olfactory+Receptor+Neurons+in+a+Drosophila+olfactory+circuit.">Differential contributions of Olfactory Receptor Neurons in a Drosophila olfactory circuit.</a> eNeuro. 3, (2016).</li><li>Schulze, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamical+feature+extraction+at+the+sensory+periphery+guides+chemotaxis.">Dynamical feature extraction at the sensory periphery guides chemotaxis.</a> eLife. 4, (2015).</li><li>Tastekin, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Subesophageal+Zone+in+Sensorimotor+Control+of+Orientation+in+Drosophila+Larva.">Role of the Subesophageal Zone in Sensorimotor Control of Orientation in Drosophila Larva.</a> Current Biology. 25, 1448-1460 (2015).</li><li>Famiglietti, E. V., Kolb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+ON-and+OFF-center+responses+in+retinal+ganglion+cells.">Structural basis for ON-and OFF-center responses in retinal ganglion cells.</a> Science. 194, 193-195 (1976).</li><li>Luo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+thermotaxis+in+Caenorhabditis+elegans+is+mediated+by+distinct+sensorimotor+strategies+driven+by+the+AFD+thermosensory+neurons.">Bidirectional thermotaxis in Caenorhabditis elegans is mediated by distinct sensorimotor strategies driven by the AFD thermosensory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 2776-2781 (2014).</li><li>Berck, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wiring+diagram+of+a+glomerular+olfactory+system.">The wiring diagram of a glomerular olfactory system.</a> eLife. 5, (2016).</li></ol>

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。

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			<td height="40" style="height:40px;width:359px;">Video camera to capture larval movement</td>
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			<td height="33" style="height:33px;">CCD Camera&#160;</td>
			<td style="width:296px;">Edmund Optics</td>
			<td style="width:208px;">106215</td>
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			<td height="33" style="height:33px;">M52 to M55 Filter Thread Adapter</td>
			<td style="width:296px;">Edmund Optics</td>
			<td>59-446</td>
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			<td height="33" style="height:33px;">2&#34; Square Threaded Filter Holder for Imaging Lenses&#160;</td>
			<td style="width:296px;">Edmund Optics</td>
			<td>59-445</td>
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			<td height="33" style="height:33px;">RG-715, 2&#34; Sq. Longpass Filter</td>
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			<td>46-066</td>
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			<td height="33" style="height:33px;">Electronics for optogenetic setup</td>
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			<td height="33" style="height:33px;">Raspberry Pi 2B</td>
			<td>RASPBERRY-PI.org</td>
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			<td height="33" style="height:33px;">3 Channel programmable power supply</td>
			<td>newegg.com</td>
			<td>9SIA3C62037092</td>
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			<td height="33" style="height:33px;">8 Channel optocoupler relay</td>
			<td>amazon.com</td>
			<td>6454319</td>
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			<td height="33" style="height:33px;">630nm Quad-row LED strip lights</td>
			<td>environmentallights.com</td>
			<td>red3528-450-reel</td>
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			<td height="33" style="height:33px;width:359px;">850nm LED strips</td>
			<td>environmentallights.com</td>
			<td>wp-4000K-CC5050-60x2-kit</td>
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			<td height="33" style="height:33px;width:359px;">Software&#160;</td>
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			<td>Mathworks Inc.</td>
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			<td height="33" style="height:33px;width:359px;">Ubuntu MATE v16.04</td>
			<td>Nubuntu</td>
			<td>https://github.com/yslo/nubuntu</td>
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			<td height="33" style="height:33px;">Other items</td>
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			<td height="33" style="height:33px;">Plexiglass black acrylic</td>
			<td>Home Depot</td>
			<td style="width:208px;">MC1184848bl</td>
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			<td height="33" style="height:33px;">Nutrifly fly food</td>
			<td style="width:296px;">Genesee Scientific</td>
			<td>66-112</td>
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			<td height="33" style="height:33px;">Agarose powder</td>
			<td style="width:296px;">Genesee Scientific</td>
			<td>20-102</td>
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			<td height="33" style="height:33px;">22cm X 22cm square petri-dish</td>
			<td style="width:296px;">VWR Inc.</td>
			<td>25382-327</td>
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			<td height="33" style="height:33px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
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			<td height="33" style="height:33px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
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		<tr height="33">
			<td height="33" style="height:33px;">All trans-retinal</td>
			<td>Sigma-Aldrich</td>
			<td>R2500</td>
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			<td height="33" style="height:33px;">Flies</td>
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			<td height="33" style="height:33px;">UAS-IVS-CsChrimson&#160;</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>55134</td>
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			<td height="33" style="height:33px;width:359px;">Orco-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>26818</td>
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			<td height="33" style="height:33px;">Or42a-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>9970</td>
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			<td height="33" style="height:33px;">Or7a-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>23907</td>
			<td></td>
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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,...

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

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。

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

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Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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 ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

通过鼻腔活检获得联合激光捕获显微切割的嗅觉神经元：一种可能的方法来研究治疗反应的精神障碍

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Optogenetic Stimulation of the Auditory Nerve

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 ...