R01AA016837（JCB）和P20AA017828（AGD和JCB）：这些研究是由美国国立卫生研究院酒精中毒和酒精滥用国家研究院​​的资助。

步骤1.在分析之前执行的日<ol><li>挑L4阶段蠕虫新鲜线虫生长培养基（NGM）板接种OP50 E.的草坪大肠杆菌 ，并对其进行培养，在20 CO / N。每个实验条件要求10蠕虫;挑过量蠕虫允许O / N损失的蠕虫。 <ol><li>只有实验动物，是第一天的成年人;许多突变体生长以较慢的速度比野生型。调整拾取对于具有发育迟缓，使所有测试的动物是第一天的成人菌株的定时。 </li></ol></li></ol>执行对检测的第2天步骤<ol><li>对于实验准备： <ol><li>在标准的非种子60×15毫米NGM板进行测定。干燥所有的板的情况下使用，在37℃，2小时后，用盖子关闭。每个实验试验，采用四干NGM板;这些将是0 20mM和400mM的乙醇测定板及其相应的驯化板。 注意：使用NGM板是很重要的位置;在该板的成分的差异，特别是它们的渗透压，可以强烈地影响对行为乙醇的剂量反应，这是由于，至少部分，以变化的乙醇由动物20中累积的量。 NGM琼脂160毫渗量。 </li><li>干燥后，称量各未播种NGM板的在测定中使用，并记下重量。确定的媒体中基于空盘的重量在板的体积。以近似重量的琼脂至体积的转化，假设介质的重量为相同的等体积水。 注：我们最一致的结果，已发现与已脱水足够的原始10毫升卷有8.3-8.9毫升之间的干燥后体积NGM媒体。一种替代的2小时的干燥时间是干燥直至板达到这个体积范围来解释在孵化的差异。 </li><li>熔体4铜环（1内径。6厘米）插入每个板的表面上以充当畜栏对于不同基因型或worms.Grasp每个环用钳子和热的治疗组中的火焰（来自本生灯强烈的火焰效果很好）约3秒。立即将戒指放在盘子里，而仍持有环钳，以防止“跳跃”。 <ol><li>确保该环靠在平贴在琼脂表面通过按下轻轻用钳子以环上的几个点。当放置戒指，要小心留有余地，另外三个圈。 </li><li>融化三个附加铜环进入板的表面上，同时注意把它们作为并拢尽可能使所有4将在视摄像机的视场。 注意：在制作与琼脂良好的密封是至关重要的，以保持在所述环中的蠕虫的测定期间。环即跳过围绕上盘不可能进行正确的密封与琼脂和可能疤痕琼脂，这使得蠕虫洞穴和干扰可视化的蠕虫。 </li><li>对于每个测定板制备一个伴随“驯化”板，它应被干燥和未接种的和将得不到乙醇。放在这些板块4铜环。 </li></ol></li><li>标签板的底部旁边的每个环与蜗杆应变环中所用的，并注意在视环本身的领域不写。匹配试验板的标签，其伴随的驯化板。 </li><li>计算的100％乙醇的量添加到每个测定板，以使最终浓度为0毫米或400毫米的乙醇（重量比体积）。对于每个实验（N = 1），将有1 0毫和一个400毫乙醇板;驯化板没有收到乙醇。添加乙醇，吹打它围绕板的表面上。用实验室膜以密封所述板，并允许它以平衡地置于工作台顶部2小时。 </li><li>当1.5小时过去后，开始检测，步2.2的驯化一步。 </li></ol></li><li>进行运动检测：测定板<ol><li>小心转移10蠕虫每个实验组，以在驯化板铜环的中心。删除任何食物，通过与蠕虫挑轻轻刮琼脂在这一点上可见。变化都带上跨实验性试验的板，其中所述实验组，使得相同的菌株不能把在平板上在各试验相同的顺序的顺序。 注意：我们的目标是将动物用食物数量最小转移到板，因为如果在驯化板食物被转移到测定板中的蠕虫会聚集周围的食物和运动的药物的作用将被遮蔽。 </li><li>要小心，不要打破挑的琼脂表面，因为这将允许蠕虫打洞，所以会影响检测。孵育蠕虫在室温30分钟。 </li><li>确保有各板的灌顶之间保持适当的间隔。一个有经验的实验者可以移动40动物，10 /环4环的&lt;1.5分，但任何间隔可达2分钟是可以接受的。标准测定具有电影记录在10-12分钟，并暴露于两者0 20mM和400mM的乙醇30-32分钟。 </li><li>启动驯化板为0毫曝光和驯化板为400毫米曝光约2分钟和30秒的间隔，以允许用于保存前的第二电影必须开始的第一个电影文件。 </li><li>在30分钟驯化期后，从驯化板蠕虫转移到测定板中。转移的相同顺序，它们被添加到驯化板，跟踪各板的完井之间的定时的蠕虫到测定板（0毫米或400毫米乙醇）。与实验室薄膜密封板，以减少乙醇损失汽化。 </li><li> ü瑟用细边扁平虫挑收集蠕虫的扁平挑顶部铲动作。在不使用的细菌，其通常在转印蠕虫使用，因为它有助于胶蠕虫拾取的执行蠕虫从非种子驯化板转移至测定板。 注：在该动物在这个步传送速度是非常重要的，因为在板的第一蠕虫将暴露于乙醇比加入到板的最后蠕虫更长，并且较早的时间点可能表现出一些时间依赖性影响。这是用于旋转该菌株被横跨实验重复放置在平板上第一的主要原因。 </li></ol></li><li>执行步态分析：拍摄<ol><li>用显微镜/摄像机组合，其允许所有四个环中的视场（约42x42毫米2平方），的同时成像诸如0.5倍的显微镜物镜，0.8倍的放大倍率和一个照相机与2048×2048 7.481;万像素CCD。 </li><li>使用，即使在整个视场，这在强度阈步骤有助于在物体识别照度（见下文）。 A 3“X3”背光效果很好。 </li><li>像一盘定位媒体朝上的蠕虫（盖下），产生对比正在使用与传输光源的传统显微镜相比，背光时丢失。 </li><li>准备的图像分析软件来捕获的移动蠕虫时间推移电影。设置软件来捕获12位灰度图像每1秒，如2分钟（120帧）的电影。以减小输出文件的大小，而同时仍保留足够的图像分辨率，使用2x2融合模式拍摄1024×1024像素的图像。 </li><li>录制的电影。开始记录第一板（0毫乙醇）的最后一个蠕虫后10分钟被放置在该板的一个120帧的电影。保存的电影文件。 </li><li>记录所述第二板（400毫乙醇）。重复此过程两个板开始最后的蠕虫30分钟后，被放置在第一板（0毫乙醇）来捕获用于每个曝光的30-32分钟的时间点。 注：允许有足够的时间开始下盘记录进行分析之前，每个捕获会话后保存图像文件。 </li><li>为适应未来发展的存档电影，以允许打开和其他公共领域的开源图像处理软件程序，如ImageJ的28分析了这些电影，每部电影的拷贝转换成8位256级灰度TIFF文件。 注：这里所描述的图像分析软件使用一种专有的文件格式。 </li></ol></li><li>分析使用ImagePro软件电影。 <ol><li>一旦捕获，分析使用的Image-Pro Plus软件或等值的电影。 注意：各种其他对象跟踪软件是可用的已被成功地用于跟踪多个C.线虫一次29和这种软件可以合理替代的是，这里所描述的对象识别步骤基于类似的原理。描述的方法涉及到的Image-Pro Plus软件版本6.0-6.3和7.0，新版本的的Image-Pro Plus软件可能有细微的差别。 </li><li>在2分钟（120架）段分析电影。首先，将过滤器应用于图像变平的背景和增强蠕虫的对象的对比度。选择过滤器，如下所示：菜单&gt;过程&gt;过滤器&gt;增强&gt;拼合：参数：背景=黑暗;特征宽度= 20 Pix的。 </li><li>重新保存电影的过滤步骤后，但始终保持在其未经过滤的形式原始影片。 <ol><li>在每个环带的感兴趣区域（ROI）的圆形区域被放置和尺寸与铜环重叠分别分析动物的运动。识别和跟踪的菜单&gt;办法蠕虫&gt;跟踪物体...命令;这带来了跟踪ðATA表窗口。跟踪选项按钮允许特定的曲目被排除和限制任何实验文物。 </li></ol></li><li>根据自动跟踪选项卡，使用下列参数：轨道参数：速度极限（搜索半径）= 400微米/帧，加速度限制=自动，最小总轨道长度= 400微米，主要运动型=混乱;在轨道参数的对象：允许部分曲目= YES，最小长度= 21帧，跟踪预测深度= 1架。 </li><li>要启动跟踪过程中，单击查找所有曲目自动功能按钮，弹出计算/尺寸选项对话框和跟踪对话框。选择手动选项，在计算/尺寸对话框，它提供了一个使用的蠕虫像素的灰度强度，突出分析蠕虫对象，忽视了更轻的背景像素的重要门槛一步的强度范围选择。 <ol><li>调整强度阈值SLI德尔斯（一到设定的上限，一到设定下限）创建一个具有包容性的范围，突出全暗的对象。中0-1,500的规模为0-4,095一系列是一个很好的起点，更精细的调整。 </li><li>适用的尺寸过滤器，以排除对象是比单个蠕虫对象更大和更小，如铜环和在平板上的任何碎屑。设置两个尺寸参数做到这一点，包括大小个别蠕虫在各种姿势与计量&gt;的计算/尺寸选项对话框中选择测量菜单项。 （面积范围：28,000-120,000微米2和周边范围：600-2,500微米）。 </li><li>如果一个突变株是显著小于或大于其它动物更大的板加以分析，扩大的过滤器设置的范围为对象的识别，以容纳不同尺寸的菌株的第一追踪动物之前，使设置不需要改变中期分析。 </li></ol> &lt;/ li&gt; <li>单击继续在跟踪对话框中完成了跟踪过程。直观地比较输出音轨与影片中的每一个蠕虫的进展，以确保每一个蠕虫表示，除非有基于自动过滤器设置一个合理的理由来排除它。手动删除由满足大小的过滤标准确定非蠕虫物体的存在被生产的轨道。 </li><li>使用软件来计算每个帧之间的每个蠕虫的速度（行驶距离为每帧之间1秒的对象的质心），并显示在所有磁迹的平均流速为每个轨道和平均速度在蠕虫的人口铜圈。考虑这最终平均为 n = 1的实验性试验条件。将数据导出到的统计分析和数据归档的电子表格程序。 </li><li>一旦数据已被记录为在板的第一环中，将ROI的到下一个环和重复跟踪处理，而无需改变任何参数。 </li><li>计算运动通过相对速度： </li><li>相对速度（％）=处理（400毫米）的速度/未处理（0毫米）的速度×100。 注：不同的遗传操作往往改变动物的基础运动速度。为了确定乙醇对动物的运动速度的影响，并能够比较不同基因型或条件下，这些效果，计算出相对速度。 </li></ol></li></ol>

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T., 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=Ethanol+metabolism+and+osmolarity+modify+behavioral+responses+to+ethanol+in+C.+elegans.">Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans.</a> Alcohol. Clin. Exp. Res. 36, 1840-1850 (2012).</li><li>Morgan, P. G., Sedensky, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+sensitivity+to+ethanol+in+the+nematode,+Caenorhabditis+elegans.">Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans.</a> Alcohol. Clin. Exp. Res. 19, 1423-1429 (1995).</li><li>Mitchell, P. H., 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+concentration-dependent+effects+of+ethanol+on+Caenorhabditis+elegans+behaviour.">The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.</a> Pharmacogenomics J. 7, 411-417 (2007).</li><li>Vidal-Gadea, 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=Caenorhabditis+elegans+selects+distinct+crawling+and+swimming+gaits+via+dopamine+and+serotonin.">Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin.</a> Proc. Natl. Acad. Sci. U S A. 108, 17504-17509 (2011).</li><li>Bettinger, J. C., Leung, K., Bolling, M. H., Goldsmith, A. D., Davies, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+environment+modulates+the+development+of+acute+tolerance+to+ethanol+in+Caenorhabditis+elegans.">Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.</a> PLoS ONE. 7, e35129 (2012).</li><li>Davies, A. G., 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=Different+genes+influence+toluene-+and+ethanol-induced+locomotor+impairment+in+C.+elegans.">Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans.</a> Drug Alcohol Depend. 122, 47-54 (2012).</li><li>Newlin, D., Thomson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+challenge+with+sons+of+alcoholics:+a+critical+review+and+analysis.">Alcohol challenge with sons of alcoholics: a critical review and analysis.</a> Psychol. Bull. 108, 383-402 (1990).</li><li>Ponomarev, I., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

简单的神经生物学和遗传学工具C.提供线虫使蠕虫病毒在其中研究乙醇对行为影响的分子基础的极好的模型。在这里，我们描述了已被用于识别急性行为反应的几种分子和环境介质为乙醇8,10,20,24,25的测定。这种方法允许分化和同时检查两个不同乙醇响应行为表型，初始灵敏度和急性官能耐受的发展，它们一起模拟响应在哺乳动物的水平的复合型。相同的测定，可以很容易地?...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 mm Petri plates, triple vented</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>Other plate brands will suffice.</td>
		</tr>
		<tr>
			<td>NGM agar</td>
			<td>Various</td>
			<td></td>
			<td>NaCl (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td>Various</td>
			<td></td>
			<td>For drying agar</td>
		</tr>
		<tr>
			<td>Digital balance</td>
			<td>Various</td>
			<td></td>
			<td>For determining plate weights and agar volume</td>
		</tr>
		<tr>
			<td>Copper rings</td>
			<td>Plumbmaster</td>
			<td>STK#35583 (48 cap thread gasket)</td>
			<td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>QImaging</td>
			<td>RET-4000R-F-M-12</td>
			<td>This camera has a large field of view.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with C-mount and 0.5X objective</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Discontinued model, M60 is current equivalent.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Schott</td>
			<td>A08923</td>
			<td>3&#8221;x3&#8221;&#160; backlight for even illumination across the field of view</td>
		</tr>
		<tr>
			<td>Imaging and tracking software</td>
			<td>Media Cybernetics</td>
			<td>ImagePro-Plus v6.0-6.3</td>
			<td>Newer versions of the software have tracking functions.</td>
		</tr>
	</tbody>
</table>

an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

从几个不同的基因型和它们的配对控制代表数据（ 图1）呈现8,24;数据特别选择在检测动物亮点的差异。的效果，在暴露10分钟的程度被认为是一个菌株，其上示出在图1B-G的左侧轴的初始灵敏性。突变菌株与相对速度比在10分钟的控制较大被认为是乙醇抗性（ 图1F，G），而突变菌株具有相对速度小于被认为是过敏乙醇（ 图1D的控制，...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

测量乙醇的影响上的步态速度的测定<em&gt;秀丽隐杆线虫</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

an-assay-for-measuring-effects-ethanol-on-locomotion-speed

Research

JoVE Journal

Neuroscience

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

8.5K 次观看.  Virginia Commonwealth University. C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay. 

视频: An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

科研

神经科学

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

的斑马鱼胚胎神经上皮通透性的测定

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

方法研究抗精神病药物的作用机制在<em&gt;秀丽隐杆线虫</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受<em&gt;果蝇</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

新颖的指标来表征线虫胚胎伸长率<em&gt;秀丽隐杆线虫</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

一个<em秀丽隐杆线虫</em基于营养状态的铜反转测定

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...