这项工作是由研究经费JWW从美国国立卫生研究院（R01DK092640）和美国国家科学基金会（0920668）的支持。

1。粉煤灰收集和饥饿<ol><li>后面的实验苍蝇在控制温度和湿度条件下（ 如 21℃，50〜60％相对湿度）在12小时亮/暗周期。 </li><li>收集雌蝇在羽化的日子，把它们摆放，伴随着4-5的男性，成为新的食品小瓶（最多30个每小瓶）。年龄过得2-5天。 </li><li>准备室的苍蝇饥饿。 <ol><li>推单一组织（4.8×8.4英寸）下降到一个空的塑料小瓶的底部。完全浸泡的组织用蒸馏水冲洗。使用对象向下推纸巾，轻轻挤出多余的水分。 </li><li>倒置小瓶丢弃多余的水。应该有足够的水，以保持肌肤的苍蝇和饥饿室湿润，但不能足以淹没苍蝇。 </li></ol></li><li>从食品小瓶传送苍蝇进入饥饿室，然后将小瓶约18-24小时，在实验开始之前。存储在受控的温度和湿度条件下的小瓶过夜，直至实验开始的第二天。 </li></ol> 2。食物气味的制备<ol><li>加入0.1克低熔点琼脂糖在一个玻璃烧瓶中加入10ml蒸馏水制备1％琼脂糖溶液。热火在微波琼脂糖溶液只是直到它开始沸腾，但沸腾了好之前。 <ol><li>停止微波和摇动烧瓶一次。重复此步骤两次，直至琼脂糖完全溶解。通过保持烧瓶温暖的加热板上设置在50℃保持琼脂糖溶液在液体状态</li></ol></li><li>加入990微升1％琼脂糖溶液和10μl的苹果醋来1.5ml离心管中，制成1％苹果醋的解决方案。涡旋该溶液直至混合，并装在一个干浴恒温箱设定为50℃。 </li></ol> 3。测试室和行为商会设置<ol><li&gt;将测试室到所需的条件（ 如温度和湿度）。 </li><li>打开LED面板（660纳米）上。 </li><li>冲洗筛和用热水的测试板和加热它们在干燥炉中，直到所有的湿气蒸发。实验开始前，冷却筛和板块下降到检测室温度。 </li><li>定位在扩散板的顶部上的浅盘中并灌满水，以增加局部湿度和掩膜的水在琼脂糖液滴。 </li><li>定位筛过水菜。 </li></ol> 4。飞装载到测试板图与规格测试板可以在补充文件一节中找到。该测试板是由透明的丙烯酸和由6测试领域。一个简单的滑块包含控股室，允许动态加载，暂时的遏制，并同时发布6苍蝇到他们各自在室在实验的开始。十字蚀刻在每个舞台在板中央指示在那里的气味应该吸取。 <ol><li>将滑块到丙烯酸测试板。 </li><li>轻轻吸气泵滑入小瓶过去的棉塞，并允许约6苍蝇走进aspirator.it关键是要尽可能轻柔处理它们。有人可能会采取的趋光飞行为好处，以诱使苍蝇指向一个昏暗的光源小瓶开放抓取朝着吸引。如果有必要，还可以申请温柔的吸力吸出约6雌性果蝇。 </li><li>插入抽吸器的尖端插入测试板的第一孔中。允许单个飞通入牢房，轻轻推动滑块向前加载一只苍蝇进入下一洞。继续，直到苍蝇占据板的所有6个控股细胞。 </li><li>吸取5微升1％，苹果醋琼脂糖溶液直接喷在CR的中心在测试板的内表面OSS-毛发。 </li></ol> 5。定位板测试<ol><li>到中心的检测板，打开名为“定位Tool.vi.”文件“LabVIEW的VI的定位工具。六，可以在补充文件一节中找到。通过点击白色箭头在屏幕的左上角运行该文件。 </li><li>将测试板在筛上的顶部，使得竞技场开口面对筛地板和气味的目标是在板的顶。对准蚀刻到测试板的背面与显示器屏幕上的十字线的十字星。 </li><li>当校准完毕后，通过点击红点靠近显示器的左上角中止执行。 </li></ol> 6。记录飞的位置在实验<ol><li>每个食品搜索审讯过程中跟踪和记录苍蝇的坐标，打开采集软件文件“楼Ÿ跟踪 - 六Zones.vi“的LabVIEW VI为”飞跟踪 - 六Zones.vi“可以在补充文件一节中找到通过点击白色箭头在显示器的左上角运行该文件。 。 </li><li>指定文件名，然后单击“确定”。 </li><li>推动滑块在测试室释放苍蝇进入测试领域。要小心，不要移动检测室，因为这会导致不正确的对齐与分析软件坐标。 </li><li>点击“开始”（开始记录），并确保光在测试室的唯一来源是660 nm的LED面板。 </li><li>当试验结束后，取出筛子和行为室。从筛提起的测试板和淹没板冰取出苍蝇。轻轻擦拭板用热水和删除任何琼脂糖碎片。将测试板在干燥炉中以除去水分。 </li><li>通过打开一个SMaL通风试验区升风扇为约2分钟。关闭风扇和负载苍蝇下一组进入下一个测试板。 </li></ol> 7。使用自定义软件数据分析 “对飞跟踪，六个区域的数据分析”，可以在补充文件一节中找到。在数据采集，采集软件记录个人动态位置坐标在文本文件中的每个时间点。一个单一的数码相机定位在所述检测板获得的图像在0.5 Hz的帧速率。该分析软件程序“的飞跟踪，六个区域的数据分析”中提取信息，从该文本文件一）计算出平均车速，b）决定的时间点了一只苍蝇成功地找到气味源，和c）建立图形窗口允许用户查看：飞位置，距离飞从气味源随着时间的推移和平均飞行速度随着时间的推移。它也能格式化，方便的数据导出到一个SPREadsheet程序。在这个宏，食品搜索延时被定义为在其拥有舞台的中心5毫米的半径范围内的花费至少5秒的时间点。 <ol><li>打开分析软件文件“为飞跟踪数据分析 - 六区”。在“窗口”选项卡，点击“创建新表。”直到已建立六个表重复此步骤。 </li><li>在“宏”选项卡，点击“Foodfinding。” A主面板应该会出现以下选项：打开原始数据文件进行布局;打开原始数据文件的数据文件;飞位置，距离，速度，布局; FormatDataFile。 </li><li>要查看原始数据，而不附加值到一个文本文件，点击“打开原始数据文件的布局。”找到并选择在浏览器窗口中显示的实验数据文件。点击“打开”。 </li><li>点击“飞的位置”在所有六个领域（6 XY坐标图描绘每个飞“中查看每个苍蝇的位置随着时间的推移地位应该出现在屏幕上）。 </li><li>点击“距离”，从气味源（6宗地块从气味源随着时间的推移描绘了苍蝇的距离应该出现在屏幕上）查看每个飞的距离。水平线在y =5毫米表示其上飞被认为是定位在食物源的阈值。 </li><li>点击“速度”的审判（6宗地块描绘了苍蝇的速度随着时间的推移应该会出现在屏幕上）中查看每个苍蝇的平均速度。 </li><li>点击“布局”与所有除了平均速度（第50秒时），并找到气味源的每个飞（图1）的延迟蝇位置，距离和速度的图表显示的布局。以正确观赏的布局，它可能有必要调整边界。要做到这一点，首先单击布局窗口。在“文件”选项卡，点击“为布局页面设置”。重置边距0.2英寸并单击“确定”。马上离开的每个位置情节是一张小桌子与标题“速度”和“延迟”。每个标题下输入的数值表示在毫米/秒，并在几秒钟内食品搜索延迟的平均速度。潜伏在空白项表示飞未能找到气味源。食品搜索延迟定义为时间点的苍蝇花了至少5秒从腔的中心有5 mm的半径之内。 </li><li>若要打印布局，点击布局区域（更新布局，当前文件）。点击“文件”，然后点击“打印布局”。 </li><li>要查看下一个文件，只需点击“打开原始数据文件的布局。”单击要查看和点击下原始数据文件“确定”。单击布局窗口上，用新的数据文件更新窗口。 </li><li>该设置可以保存供以后使用的实验文件。 </li></ol> 8。从出口数据数据分析软件到电子表格程序<ol><li>要导出速度和延迟数据为每个文件，点击“打开原始数据文件的数据文件。”选择一个实验数据文件并单击“打开”。将出现一个新的浏览器窗口。 </li><li>在新的浏览器窗口中，在“新建”单击鼠标右键，然后单击“文本文档”。命名新的文本文档。选择新命名的文本文件并单击“打开”。该存储数据从原始数据文件转换成文本文件。 </li><li>若要从另一个文件导出数据，点击“打开原始数据文件的数据文件。”单击另一个文件，然后单击“打开”。选择从步骤8.2文本文件）。继续这个过程要导出剩余的数据文件。 </li><li>一旦所有所需的数据文件已被添加到文本文件，单击“格式数据文件。”选择用于存储从前面的步骤数据，然后单击“打开”（新windo的文本文件W将自动打开）。 </li><li>在窗口中新建一个文本文件，指定文件的名称，然后单击“保存”。这将创建一个包含文件名，平均车速，和延迟对于每个飞，可导入到电子表格程序的文本文件。 </li><li>累积曲线是从上苍蝇到达食物气味靶作为时间（图3）的函数的总数量数据构成。 </li><li>实验数据集被用于使用z检验对比例的统计显着性分析。 </li></ol>

<ol>
	<li>Dethier, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The hungry fly : a physiological study of the behavior associated with feeding. , (1976).</li><li>Root, C. M., Ko, K. I., Jafari, A., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+facilitation+by+neuropeptide+signaling+mediates+odor-driven+food+search.">Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search.</a> Cell. 145, 133-144 (2011).</li><li>Root, C. 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=A+presynaptic+gain+control+mechanism+fine-tunes+olfactory+behavior.">A presynaptic gain control mechanism fine-tunes olfactory behavior.</a> Neuron. 59, 311-321 (2008).</li><li>Semmelhack, J. L., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Select+Drosophila+glomeruli+mediate+innate+olfactory+attraction+and+aversion.">Select Drosophila glomeruli mediate innate olfactory attraction and aversion.</a> Nature. 459, 218-223 (2009).</li><li>Faucher, C., Forstreuter, M., Hilker, M., de Bruyne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+responses+of+Drosophila+to+biogenic+levels+of+carbon+dioxide+depend+on+life-stage,+sex+and+olfactory+context.">Behavioral responses of Drosophila to biogenic levels of carbon dioxide depend on life-stage, sex and olfactory context.</a> J. Exp. Biol. 209, 2739-2748 (2006).</li><li>Quinn, W. G., Harris, W. A., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+behavior+in+Drosophila+melanogaster.">Conditioned behavior in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. U.S.A. 71, 708-712 (1974).</li><li>Fishilevich, E., Domingos, A. I., Asahina, K., Naef, F., Vosshall, L. B., Louis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+behavior+mediated+by+single+larval+olfactory+neurons+in+Drosophila.">Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila.</a> Curr. Biol. 15, 2086-2096 (2005).</li><li>Venken, K. J., Simpson, J. H., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulation of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Hamada, F. N., 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=An+internal+thermal+sensor+controlling+temperature+preference+in+Drosophila.">An internal thermal sensor controlling temperature preference in Drosophila.</a> Nature. 454, 217-220 (2008).</li><li>Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+modification+of+behavior+in+Drosophila+by+targeted+expression+of+a+temperature-sensitive+shibire+allele+in+defined+neurons.">Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.</a> J. Neurobiol. 47, 81-92 (2001).</li><li>Sweeney, S. T., Broadie, K., Keane, J., Niemann, H., O'Kane, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+expression+of+tetanus+toxin+light+chain+in+Drosophila+specifically+eliminates+synaptic+transmission+and+causes+behavioral+defects.">Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.</a> Neuron. 14, 341-351 (1995).</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></ol>

作者宣称没有竞争的经济利益。

在这个协议中，我们描述了食物搜索行为分析一步一步的过程。除了食物有关的气味，它也可适于用于苍蝇的定位其他气味物体的能力的研究。例如，它可能会朝雄蝇3在队友的本地化行为的研究应用还有几个额外的考虑这个协议，我们将在这里提到有关此过程： 首先，饲养温度决定实验苍蝇多久应该进行老化测试之前。建议年龄的范围进行检查，以确定最合适的年?...

饥饿的国家促进两种欲求行为：寻找食物和食品消费1。这个简单的行为测定法是与觅食2,3相关的趋化行为的研究非常有用。具体来说，它跟踪飞位置，行走速度和延迟定位食物气味的目标。食品发现的延迟作为度量测量的变化在苍蝇的气味探测系统的变化在其内部欲求状态的下游的灵敏度。此法的手动版是以前用来显示GABA-B受体信号是很重要的气味本地化行为，成蝇3。检测的电流自动版本是有助的在多短神经肽F（sNPF）信令重塑地图嗅觉的果蝇和影响欲求行为2的研究。 测试是在黑暗的，温度和湿度控制的房间进行。数字上面的透明亚克力板的测试设置摄像机跟踪苍蝇由660 nm的LED照明背光。从相机信息是实时由驻旁边的测试区域的计算机进行处理。我们使用的数据采集软件在测试期间录制和保存苍蝇位置的坐标。 在这个范例中，受试者被释放到包含在该中心食品气味的舞台;气味对象创建诱导苍蝇的食物搜索行为竞技场内食品串味梯度。类似的气味搜索协议已对chemosensation在单一果蝇幼虫7的研究应用。而其他行为分析，如四场嗅觉4,5或T型迷宫6评估厌恶的气味或退房的行为，这种模式是最适合评估嗅觉的灵敏度和趋化行为。 几个关键的优势伴随着这个ASSA年。首先，它允许快速获取大数据集，因为数据收集和分析大多是自动化的。第二，该测定分离和测量单个苍蝇的行为，从而消除社会嗅觉可能影响他们的行为。三，协议和简单的实验设计的简单性使检测效率，容易教别人。 另外，此法可用于进一步探讨神经回路基本食品的搜索行为由它提供给果蝇 8的广泛的遗传工具箱结合。转基因的靶向表达，沉默或激发的神经元可以与如GAL4-UAS系统，以及无人机，shibire TS1，UAS-破伤风毒素，和UAS-TRPA1（B）工具来实现转基因9-12。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Apple Cider Vinegar</td>
			<td>Spectrum</td>
			<td></td>
			<td>commercially available</td>
		</tr>
		<tr>
			<td>Agarose, Type VII</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td>low gelling temperature agarose</td>
		</tr>
		<tr>
			<td>Acrylic Testing Plate</td>
			<td></td>
			<td>custom</td>
			<td>Plate contains 6 arenas. Each arena is 60 mm in diameter 6 mm in height. See testing plate diagrams for specific measurements.</td>
		</tr>
		<tr>
			<td>LabVIEW V.8.5</td>
			<td>National Instruments</td>
			<td>776670-09</td>
			<td>platform for programs: PositioningTool.vi, FlyTracking--Six Zones.vi NOTE: &#34;elapsed time.vi&#34;, &#34;time into file.vi&#34;, and &#34;two object detect.vi&#34; are included subroutines that must be available in order for the main data acquisition program &#34;FlyTracking--Six zones.vi&#34; to run.</td>
		</tr>
		<tr>
			<td>LabVIEW Vision 8.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Vision Acquisition Software 8.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Vision Builder AI 3.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Igor Pro V.6</td>
			<td>Wavemetric, Inc.</td>
			<td></td>
			<td>platform for macro: Data Analysis for Fly Tracking--Six Zones</td>
		</tr>
		<tr>
			<td>Basler scA1390-17fm</td>
			<td>National Instruments</td>
			<td>779980-01</td>
			<td>Digital Camera NOTE: driver for camera available at Baslerweb.com</td>
		</tr>
		<tr>
			<td>8 mm lens</td>
			<td>National Instruments</td>
			<td>780024-01</td>
			<td>Lens for Basler Digital Camera</td>
		</tr>
		<tr>
			<td>Ground Glass Diffuser Plate</td>
			<td>Edmund Optics</td>
			<td>custom</td>
			<td>Diffuses light, 25 cm x 30 cm</td>
		</tr>
		<tr>
			<td>US Std. No. 100</td>
			<td>Fischer Scientific</td>
			<td>04-881X</td>
			<td>Sieve with nominal opening of 150 &#956;m</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Lighting Option 1</td>
		</tr>
		<tr>
			<td>LED backlight 660 nm (20 cm x 20 cm)</td>
			<td>Spectra West</td>
			<td>BL47192</td>
			<td>a simpler but more expensive lighting option.</td>
		</tr>
		<tr>
			<td>Power Supply for LED Backlight</td>
			<td>Spectra West</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Lighting Option 2</td>
		</tr>
		<tr>
			<td>660 nm LEDs</td>
			<td>Superbrightleds</td>
			<td>RL5R1330</td>
			<td>Wavelength 660 nm (approximately 7 x 7 LED array for a 14.7 inch x 9.75 inch panel)</td>
		</tr>
		<tr>
			<td>Linear DC Power Supply</td>
			<td>GW Instek</td>
			<td>GPS-1830D</td>
			<td>Power supply for LED Panel</td>
		</tr>
		<tr>
			<td>Solderless Breadboard</td>
			<td>Digikey</td>
			<td>922354-ND</td>
			<td>Breadboard for LEDs</td>
		</tr>
	</tbody>
</table>

a single-fly assay for foraging behavior in drosophila

对于许多动物，饥饿促进了有利于寻找适当的食物来源的方式变化的嗅觉系统。在这个视频文章中，我们描述了一个自动检测测量饥饿或过饱的嗅觉依赖食物搜索行为的影响在成年果蝇果蝇 。在一个不透光的盒子由红灯是看不见的果蝇照明，链接到自定义数据采集软件的摄像机同时监控6苍蝇的位置。每个飞局限于走在包含在该中心食物的气味个别领域。测试领域休息多孔层的功能，以防止气味的积累。延迟来定位气味源，它反映在不同的生理状态嗅觉灵敏度的度量，是通过软件分析确定。在这里，我们讨论运行此行为范式的关键力学和涉及有关飞LOADI具体问题纳克，气味污染，检测温度，数据质量和统计分析。

数据分析软件和布局，其中的一个例子可在​​图1中可以看出，是根据一组数据的标准使用其10分钟试验期间，以评估每个蝇的效能。下列标准用于确定是否从每个苍蝇的数据将被用于数据分析，其目的是消除这些苍蝇是无法执行食品搜索任务因受伤，疾病，压力，或缺乏动力。 苍蝇不活动的时间超过300秒被认为是“无效”，并从数据集将被拒绝，除非它们一）...

Watch this Scientific Journal Video about A Single-fly Assay for Foraging Behavior in Drosophila at JoVE.com

A Single-fly Assay for Foraging Behavior in Drosophila

在这个视频文章中，我们描述了一个自动检测测量饥饿或过饱的嗅觉依赖食物搜索行为的影响在成年果蝇果蝇。

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

a-single-fly-assay-for-foraging-behavior-in-drosophila

Research

JoVE Journal

Neuroscience

A Single-fly Assay for Foraging Behavior in Drosophila

13.1K 次观看.  University of California-San Diego. 在这个视频文章中，我们描述了一个自动检测测量饥饿或过饱的嗅觉依赖食物搜索行为的影响在成年果蝇果蝇。 

视频: A Single-fly Assay for Foraging Behavior in Drosophila

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 ...

科研

神经科学

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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

染色昆虫行为检测：麻醉的交配行为<em&gt;果蝇</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

觅食路径长度协议<em&gt;果蝇</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

果蝇美容行为的量化

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

果蝇岛试验中运动行为的高通量分析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

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

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

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

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

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 ...

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. ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

果蝇的快速食物偏好分析

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...