We thank the staff in the Ecology Branch, Toxicology Branch and Environmental Chemistry Branch of the U.S. Geological Survey, Columbia Environmental Research Center for technical and analytical assistance. Funding provided in part by Teck American Incorporated through an agreement with the US Environmental Protection Agency (USEPA) Region 10 with funds provided by USEPA to US Geological Survey (USGS) through the Department of Interior Central Hazmat Fund.

1.设置稀释器幼虫鲟鱼暴露于水铜含量<ol><li>采用流通式系统，如改性山和Brungs 12稀释体系以下通过ASTM国际13,14,15准则设置曝光。 </li><li>选择25微克/升的基础上，绕着4-6微克/升观察疗效以前的测试结果高铜浓度。使用试剂级铜II五水合物（纯度&gt; 98％），并搭配了一个测试原液。 注:示例浓度系列的风险敞口25，12.5，6.25，3.125，1.0625和0微克/升。开始使用25微克/升的高浓度50％的连续稀释将针对一系列观察到导致行为障碍的浓度。 </li><li> 48小时之前准备在容量瓶中试原液到曝光的启动和设置为投放使用穗稀释器混合室自动注射器饮水机。 </li><li>使用电子表格模板，以确定要加入到1升的去离子水，这将导致在25微克/升的铜浓度，当稀释混合室用1ml试料液中掺入化学的重量。 注意: 图1描绘了用于化学备料用于稀释毒性试验的电子表格模板的实例。 </li><li>称出195毫克硫酸试剂级铜二五水合物使用分析天平，倒入1升容量瓶中，并用1L去离子水混合的储备浓度的48.65毫克/升。 </li><li>从自动注射器饮水机进入测试原液放置​​进气管，并设置秒杀量至1ml，并打开电源开关打开稀释并让周期为48小时使其达到平衡之前备货相应的铜浓度鲟。 </li><li>附加一个直列4路分流器<s高达&gt; 16到每个交付线，以水流量划分到每个在稀释水浴四个重复曝光室。重力进料水。循环时的电源稀释接通通过稀释器的水和电磁阀被打开，允许水流到稀释槽。设定制冷装置至15℃，并开启水泵在稀释水浴循环水。 注:此过程是通过使用一种自动计时器的调节。 </li><li>使用自动计时器稀释器设定为周期每30分钟，并以带来2.50 ml的试验水的每个周期，从而导致每天12卷增补每个复制试验室中。 </li><li>基于所述鲟鱼的大小选择暴露试验室在任何给定的时间保持在可接受的负荷率是&lt;10克鱼湿重/ L的任何腔室。例如，要进行30天的后舱口（DPH）白色鲟（平均重量以克0.17克）使用12×曝光21.5厘米2玻璃瓶与侧面4厘米的洞。覆盖该侧带有30微米网孔不锈钢筛网通过测试水的，以允许流动。试验水在曝光罐子体积为1升</li><li>使用50毫升的塑料注射器采取两种复制50ml水洗涤样品在每个浓度为总共12个样品和分配测试水入100毫升的玻璃烧杯中并测量溶解氧（DO），温度，导电率，pH值，碱度，硬度，总氨，主要阳离子，阴离子主要使用标准商用设备，并按照生产商的说明溶解有机碳。 注:样品应在开始和曝光的结束作出。 </li><li>收集子样品进行化学分析，使用25毫升塑料注射器，使用附连到注射器而不是针一吸管吸管曝光室制定约24 ml的试验水的。 </li><li>从SY取出吸管稻草RINGE并放置一个聚丙烯过滤器滤芯外壳的塑料注射器一个0.45μm孔径，聚醚砜膜。 </li><li>推4毫升试验水通过过滤器和处理。 </li><li>通过过滤器分配所述剩余的20 ml的试验水的成酸清洗聚乙烯瓶中并酸化至1％体积/高纯度卷，16进制硝酸的长达3个月的存储。 注:化学分析样品应在开始，中间和曝光的结束被带到确认铜的含量。 </li><li>使用下面的美国环境保护署方法6020A 17电感耦合等离子体质谱法进行化学分析</li><li>把所有水样和稀释的自行车，股票10（随机，随意）鲟鱼在每个复制后试验室。收集从他们使用的是小网眼非研磨网住的培养罐中的鲟鱼。将鲟鱼在一个小bucke吨，养殖水体。总共240鱼需要开始曝光。 </li><li>在曝光过程中不要喂鱼。 注:请参考图1的稀释布局的可视化设置。 </li><li>读取测试每天曝光和记录鱼死亡的持续时间和监测游泳行为。 注:其它终点，寻找包括昏睡，失去平衡，呼吸的变化，色素沉着，位置鱼在水柱，隐藏活动，并能直观地识别任何其他异常。 注:在同一时间阅读测试的每一天的一致性。 </li><li>测量和量化游泳活动（时间鱼花费移动，速度和移动的距离）用市售的数字跟踪软件程序。 </li></ol> 2.观测和计算死亡率在曝光<ol><li>目测每个测试通道琥珀和记死亡率和96小时暴露在日常使用的数据表一个行为清单（ 表1）大约在同一时间，最好是在早晨的异常行为的观察。 注:如果引人注目的是，一反常态，主观上，从管制质的差别被认为是不正常的行为。最理想的观察者不知道的治疗。 注:平衡的损失被定义为鱼无法维持水柱内的直立位置和不动性被定义为鱼无法移动或游泳除非戳。如在水柱嗜睡，多动，增加或减少呼吸，颜色的变化，震颤，痉挛，腹部臃肿，位置和其他任何不寻常的游泳方式等异常也应记录在数据表上。 注:请参阅视频1异常的例子行为。 </li><li>记录和日常去除死鲟鱼。 </li><li>使用带有探头举行溶解氧（DO）计，一只手测量每个接触浓度并记录在数据表上的两个重复现场和记录水温溶解氧。 </li></ol> 3.录像制度游泳活动<ol><li>使用手持式摄像机捕获的视频数据的样本安装到设置在测试腔室的直接开销来记录行为异常三脚架。 </li><li>量化游泳活动，切下一块的聚氯乙烯管13厘米，直径13厘米高的作为测试竞技场（ 图1）使用。放置PVC管中的每个对应的铜浓度曝光罐之内的稀释。 PVC管作为测试竞技场内使用的区域，因为这是足够大的鲟鱼约自由游动。 </li><li>在96小时的曝光结束时，随机子采样5从各铜concentratio存活鲟n至衡量游泳活动，并将其放置在使用小网眼网测试的舞台。 注意:在更高的测试浓度，其中鲟死亡率为盛行，任何剩余的存活鲟应该被用于测量游泳活动，并在某些情况下，可能小于5。 </li><li>放置鱼中对测试竞技场后，让鱼适应一段30分钟。 注:成功，视频的无差错分析需要的鱼对具有最小的结构，可能会掩盖或隐藏鱼的图像的背景的高对比度图像。鱼的形象必须是在良好的焦点，并且必须是自由表面眩光的或免费的，由于流动的水因此稀释系统必须关闭的扭曲。 </li><li> 30分钟后，打开摄像机，并设置在REC录制的游泳活动，为期2分钟。 </li><li>安乐死鱼取录像，以确定是后havior。 </li><li>放置鲟在三卡因甲磺酸酯（MS222）的水，至少10分钟，浓缩溶液，以允许鳃运动停止。 注:推荐至少250毫克/升的浓度，并可能对某些种类的高得多。 </li><li>放置在冷冻机进行处置的塑料拉链锁袋并置于安乐死鲟在稍后的时间。关闭摄像机，并用数字跟踪软件的所有视频文件传输到电脑进行后期处理。 </li></ol>从视频播放游泳活动的4措施<ol><li>定位的数据表和视频数据文件进行分析实验。视频文件转换成兼容的格式数字分析软件可以处理。 </li><li>上传的所有文件被加工成软件。通过点击图标打开跟踪软件。点击"新建默认实验"下的"创建新实验的"option主屏幕上。 </li><li>对于实验在屏幕上出现的"新实验"对话框中输入名称。选择定位试验文件被保存。点击"确定"。选择在"视频源"下的"设置"。选择"从视频文件"，"实验设置"选项。 </li><li>选择"1"为"赛场数"。选择"3"为"每个舞台科目数"。选择在"跟踪功能"，"中心点检测"。选择需要的单位。 </li><li>选择"设置"下的"审判名单"选项。点击"添加视频"在屏幕的顶部。选择"字母顺序"下的"排序顺序"选项，在该屏幕上出现的"添加视频"对话框。点击"浏览"。导航到文件夹中的视频文件的位置。 </li><li>突出显示所有视频文件。点击"打开"。点击"添加变量"在屏幕的顶部。进入"集中"到"标签"中输入"微克/升"到"说明"框中铜浓度。 </li><li>选择"数值"，从下拉列表中的"类型"。点击"预定义值"对话框。选择"定义个人价值观"，在出现的"预先定义数字值"对话框中的选项。 </li><li>输入"0"，"3"，"6"，"13"，"25"，和"50"中的"预定值"的空间。点击"添加&gt;&gt;"每个号码添加之间。取消"允许其他值"选项。点击"确定"。 </li><li>选择"试用"从"范围"框下拉菜单。输入适当的浓度在提供箱，每箱的审判。点击"添加变量"在屏幕的顶部。输入"复制"到"标签"框中。输入"复制号码"到"说明"框中。选择"数值"，从下拉列表中的"类型"。 </li><li>点击"P重新定义值"框中，选择""，在选择"预先定义数字值定义个人价值观"，"2"，""和"4"中的"1 3预定义值"对话框，输入"，"空间，点击"添加&gt;&gt; "每个号码添加之间。取消选中"允许其他值"option.Click"OK"。 </li><li>选择"试用"从"范围"框下拉菜单。在提供框中每次试验输入相应号码重复。选择"设置"选项卡下的"竞技场设置"在屏幕的顶部。名头设置"试用1"。点击从"竞技场设置（试行1）"对话框的"抢背景图片"选项。 </li><li>点击"浏览"上的"抢背景图片"对话框。找到视频文件，试行1，然后单击"打开"。视频出现后，点击"抢背景图片"对话框中"抢"选项。点击屏幕附近的UND顶部的白色圆形图标呃"竞技场设置"。 </li><li>操作出现，使整个游泳区被封闭在圈圈。影线标记出现在舞台区域被定义在哪里。点击附近的"竞技场设置"下的屏幕上方"校准比例"图标。留在舞台上的一个边缘点击。按住并拖动鼠标到舞台的另一端。松开左​​键。 </li><li>输入"10.5"，在出现的"校准距离"对话框中"现实世界距离"框。点击"确定"。如果需要，调整校准线，使得它跨越了圆形竞技场的整个直径。 </li><li>点击"设置竞技场（试行1）"对话框"验证竞技场设置"选项。解决任何问题，如果不进行验证设置。右键点击下的"设置"选项"竞技场设置"，从"实验资源管理器"工具栏上的屏幕左侧，从菜单中选择"新建"。 </li><li>重复步骤4.11-4.15，直到舞台设置h。AVE已每次试验的创建。一定要选择每次试验适当的视频文件。选择下从"资源管理器实验"工具栏中的"设置"选项，"检测设置"上的屏幕左侧。 </li><li>选择"动态减法"，从下拉菜单下的"方法"中的":检测设置1检测设置"对话"检测设置检测设置1"对话，在appears.Choose的"主题标识"下的每个主题不同的填充颜色框框。 </li><li>选择"选择视频"，并在找到的"视频"下的"采样速率"框试行1.Click"打开"。选择"5.9941"视频"检测设置:检测设置1"对话框。点击"设置"下"检测"中的"参考图像"选项"检测设置:检测设置1"对话框。 </li><li>点击"开始学习（C）4;选项​​，在"参考图像"对话box.Wait方案学习参考图像。曾经在"参考图像"对话框中的图像出现没有动物，单击对话框中的"使用动态参考图像"下的"采集设置"。 </li><li>点击"关闭"。从下拉菜单中选择为"浓"，"主题是"下的"检测"，在"检测设置:检测设置1"对话框。设置较小的数字为"33"或更大号为"153"下"检测""检测设置"明暗对比":检测设置1"对话框。 </li><li>点击"保存更改"在右下方的"检测设置:检测设置1"对话框。点击"播放控制"对话框中的播放按钮并确认软件成功追踪动物，而不是阴影或碎片。调整号码"黑暗反政府t"的必要。 </li><li>一旦跟踪为宜，点击右下角的"保存更改""检测设置:检测设置1"对话框。选择下从"资源管理器实验"工具栏中的"设置"选项"收购"的屏幕左侧。 </li><li>在"采集设置"对话框中点击"追踪下一个计划试"。确认正确的审判，视频和舞台设定在"采集设置"对话框中的"设置"显示。 </li><li>检查"采集控制"对话框中的"确定检测速度"选项。点击与封闭在一个白色方形的绿色圆圈按钮开始采集过程。重复步骤4.22-4.23，直到所有的试验已经跟踪。 </li><li>从"实验资源管理器"工具栏上点击下的"分析"选项"数据配置文件"左screen.Choose"时代"的"嵌套"在option在"组件"工具栏。调整"到""零时02分00秒"，在"选择曲目的时间间隔"中的"时间"对话框标题下。点击"确定"。 </li><li>在屏幕的右侧拖动"数据配置文件"区域中的"开始"框和"结果1"框的"鸟巢"框。点击下从"实验资源管理器"工具栏上的"分析"选项"分析档案"上的屏幕左侧。点击下的"距离和时间"，在出现的"因变量"工具栏标题"速度"。 </li><li>点击"速度"对话框中的"添加"。点击"移动距离"下的"距离和时间"中的"因变量"工具栏标题。点击"移动距离"对话框中的"添加"。点击"个人行为"，在"因变量"标题T下面的"运动"oolbar。 </li><li>调整"离群过滤器"下的"平均间隔"到"1"中的"运动"对话框的标题。调整"启动速度"到"2.00"和"停止速度"到"1.75"下的"门槛"中的"运动"对话框的标题。 </li><li>两个复选框为"动"与"不动"下的"计算统计信息"中的"运动"对话框的标题。点击"添加"，在"运动"对话框的底部。在"实验资源管理器"工具栏上点击下的"结果"选项"分析输出"上的屏幕左侧。点击"计算"在屏幕的顶部。 </li><li>一旦因变量进行计算，然后单击"导出"在屏幕的顶部。选择"导出分析输出"对话框目标文件夹。从"文件类型"下拉菜单中的"导出分析Outpu选择"创先争优"T"对话框，单击"确定"。 </li><li>点击"保存实验"的"文件"选项卡下在屏幕的顶部。关闭数字跟踪软件。将数据导入到电子表格文件，并使用商业统计分析软件包分析。 </li></ol>

<ol>
	<li>Gerhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+behavioral+ecotoxicology-prospects+and+limitations.">Aquatic behavioral ecotoxicology-prospects and limitations.</a> Hum Ecol Risk Assess: An International Journal. 13 (3), 481-491 (2007).</li><li>Beitinger, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+reactions+for+the+assessment+of+stress+in+fishes.">Behavioral reactions for the assessment of stress in fishes.</a> J Great Lakes Res. 16, 495-528 (1990).</li><li>Beitinger, T. L., McCauley, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-animal+and+physiological+processes+for+the+assessment+of+stress+in+fishes.">Whole-animal and physiological processes for the assessment of stress in fishes.</a> J Great Lakes Res. 16, 542-575 (1990).</li><li>Dell'Omo, 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> Behavioural Ecotoxicology. , (2002).</li><li>Little, E. E., Finger, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+behavior+as+an+indicator+of+sublethal+toxicity+in+fish.">Swimming behavior as an indicator of sublethal toxicity in fish.</a> Environ Toxicol Chem. 9, 13-19 (1990).</li><li>Rand, G. M., Rand, G. M., Petrocelli, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> Fundamentals of Aquatic Toxicology: Methods and Applications. , 221-256 (1985).</li><li>Calfee, R. 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=Acute+sensitivity+of+white+sturgeon+(Acipenser+transmontanus)+and+rainbow+trout+(Onchorhynchus+mykiss)+to+copper,+cadmium,+or+zinc+in+water-only+laboratory+exposures.">Acute sensitivity of white sturgeon (Acipenser transmontanus) and rainbow trout (Onchorhynchus mykiss) to copper, cadmium, or zinc in water-only laboratory exposures.</a> Environ Toxicol Chem. 33 (10), 2259-2272 (2014).</li><li>Little, E. E., Calfee, R. D., Linder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+smelter+slag-contaminated+sediments+from+Upper+Lake+Roosevelt+and+associated+metals+to+early+life+stage+White+Sturgeon+(Acipenser+transmontanus+Richardson,+1836).">Toxicity of smelter slag-contaminated sediments from Upper Lake Roosevelt and associated metals to early life stage White Sturgeon (Acipenser transmontanus Richardson, 1836).</a> J Appl Ichthyol. , 1-11 (2014).</li><li>Wang, 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=Chronic+sensitivity+of+white+sturgeon+(Acipenser+transmontanus)+and+rainbow+trout+(Onchorhynchus+mykiss)+to+cadmium,+copper,+lead+or+zinc+in+water-only+laboratory+exposures.">Chronic sensitivity of white sturgeon (Acipenser transmontanus) and rainbow trout (Onchorhynchus mykiss) to cadmium, copper, lead or zinc in water-only laboratory exposures.</a> Environ Toxicol Chem. 33 (10), 2246-2258 (2014).</li><li>Little, E. E., Dell'Omo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+measures+of+injuries+to+fish+and+aquatic+organisms:+regulatory+considerations.">Behavioral measures of injuries to fish and aquatic organisms: regulatory considerations.</a> Behavioural Ecotoxicology. , 411-431 (2002).</li><li>Little, E. E., Brewer, S. K., Schlenk, D., Benson, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioral+toxicity+in+fish.">Neurobehavioral toxicity in fish.</a> Target Organ Toxicity in Marine and Freshwater Teleosts New Perspectives: Toxicology and the Environment. Volume 2. , 139-174 (2001).</li><li>Mount, D. I., Brungs, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+dosing+apparatus+for+fish+toxicological+studies.">A simplified dosing apparatus for fish toxicological studies.</a> Water Res. 1, 21-29 (1967).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+guide+for+performing+early+life-stage+toxicity+tests+with+fishes.">Standard guide for performing early life-stage toxicity tests with fishes.</a> Annual.Book of ASTM International Standards. Volume 11.06. , 1241-1305 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+guide+for+measurement+of+behavior+during+fish+toxicity+tests.">Standard guide for measurement of behavior during fish toxicity tests.</a> Annual.Book of ASTM Standards. Volume 11.06. , 1711 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+guide+for+conducting+acute+toxicity+tests+on+test+materials+with+fishes,+macroinvertebrates,+and+amphibians.">Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians.</a> Annual.Book of ASTM Standards. Volume 11.06. , 729-796 (2014).</li><li>Brunson, , 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=Assessing+bioaccumulation+of+contaminants+from+sediments+from+the+upper+Mississippi+River+using+field-collected+oligochaetes+and+laboratory-exposed+Lumbriculus+variegatus.">Assessing bioaccumulation of contaminants from sediments from the upper Mississippi River using field-collected oligochaetes and laboratory-exposed Lumbriculus variegatus.</a> Arch Environ ConTox. 5, 191-201 (1998).</li><li>Brumbaugh, W. G., May, T. W., Besser, J. M., Allert, A. L., Schmitt, 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=Assessment+of+elemental+concentrations+in+streams+of+the+New+Lead+Belt+in+southeastern+Missouri,+2002-05.">Assessment of elemental concentrations in streams of the New Lead Belt in southeastern Missouri, 2002-05.</a> U.S. Geological Survey Scientific Investigations Report 2007-5057. , (2007).</li><li>Kane, A. S., Salierno, J. D., Gipson, G. T., Molteno, T. C. A., Hunter, 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+video-based+movement+analysis+system+to+quantify+behavioral+stress+responses+of+fish.">A video-based movement analysis system to quantify behavioral stress responses of fish.</a> Water Res. 38, 3993-4001 (2004).</li><li>Sprague, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoidance+of+Copper-Zinc+Solutions+by+Young+Salmon+in+the+Laboratory.">Avoidance of Copper-Zinc Solutions by Young Salmon in the Laboratory.</a> JWater Pollut Control Fed. 36 (8), 990-1004 (1964).</li><li>Saunders, R. L., Sprague, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+copper-zinc+mining+pollution+on+a+spawning+migration+of+Atlantic+salmon.">Effects of copper-zinc mining pollution on a spawning migration of Atlantic salmon.</a> Water Res. 1 (6), 419-432 (1967).</li><li>Barron, M. G., Dell'Omo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+contaminants+altering+behavior.">Environmental contaminants altering behavior.</a> Behavioural Ecotoxicology. , 167-186 (2002).</li><li>Ellgaard, E. G., Guillot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+the+swimming+behavior+of+bluegill+sunfish,+Lepomis+macrochirus+rafinesque,+exposed+to+copper:+hypoactivity+induced+by+sublethal+concentrations.">Kinetic analysis of the swimming behavior of bluegill sunfish, Lepomis macrochirus rafinesque, exposed to copper: hypoactivity induced by sublethal concentrations.</a> J Fish Biol. 33, 601-608 (1998).</li><li>Drummond, R. A., Spoor, W. A., Olson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+short-term+indicators+of+sublethal+effects+of+copper+on+brook+trout,+Salvelinus+fontinalis.">Some short-term indicators of sublethal effects of copper on brook trout, Salvelinus fontinalis.</a> J Fish Res Board Can. 30, 698-701 (1973).</li><li>Hansen, J. A., Rose, J. D., Jenkins, R. A., Gerow, K. G., Bergman, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chinook+salmon+(Oncorhynchus+tshawytscha)+and+rainbow+trout+(Oncorhynchus+mykiss)+exposed+to+copper:+neurophysiological+and+histological+effects+on+the+olfactory+system.">Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss) exposed to copper: neurophysiological and histological effects on the olfactory system.</a> Environl Toxicol Chem. 18, 1979-1991 (1999).</li><li>Sandahl, J. F., Baldwin, D. H., Jenkins, J. J., Scholz, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensory+system+at+the+interface+between+urban+stormwater+runoff+and+salmon+survival.">A sensory system at the interface between urban stormwater runoff and salmon survival.</a> Environ Sci Technol. 41 (8), 2998-3004 (2007).</li><li>McIntyre, J. K., Baldwin, D. H., Beauchamp, D. A., Scholz, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+copper+exposures+increase+visibility+and+vulnerability+of+juvenile+coho+salmon+to+cutthroat+trout+predators.">Low-level copper exposures increase visibility and vulnerability of juvenile coho salmon to cutthroat trout predators.</a> Ecol Appl. 22 (5), 1460-1471 (2012).</li><li>Green, W. W., Mirza, R. S., Wood, C. M., Pyle, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+binding+dynamics+and+olfactory+impairment+in+fathead+minnows+(Pimephales+promelas).">Copper binding dynamics and olfactory impairment in fathead minnows (Pimephales promelas).</a> Environ Sci Technol. 44 (4), 1431-1437 (2010).</li></ol>

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在行为由于暴露于污染物的变化常常被用来作为用于亚致死毒性的端点，但可能难以测量。通常，行为响应由目视观测和人工数据分析这需要大量的时间来处理测量。然而与发展的技术，用于量化游泳活性的方法集中在使用录像18和运动分析或数字跟踪软件，它减少了处理和分析时间。在视频的分析捕获的数据，量化游泳变量手动将是非常耗时所以使用视频数据记录，并提供用于分析鲟鱼游泳行为更加有效和高效的方式鱼类跟踪软件。虽然过程突出游泳的鱼的行为，定制其他生物，如两栖类和水生无脊椎动物需要简单的修改。根据什么行为endpoinTS正在解决，实验设计和摄像头的系统可以与几乎所有市售的跟踪软件包的使用进行开发。 该方法是使用溶解铜表现出来，但也适用于其他的水性污染物或特征，如温度或氧含量。本文开发和提出的协议利用一个简单的数字摄像机为录音设备。数字文件被容易地传输到计算机，并在给运动分析软件上传。该方法在不断进行修改和完善，以简化量化过程。当务之急是视频质量在高清晰度，以便用于分析软件来识别用于跟踪每个单独的鱼。试图处理数据文件时不与鱼对比任何背景会引起问题。与二维视频跟踪另一个常见的​​问题是识别个体当游泳路径交叉。这可以手动路径穿越期间识别每条鱼和衔接软件中的路径段被校正。可替代地，总活性可以从每个重复室为一组平均来确定。与每一个含有一个鱼几个单独腔室可在同一视场进行拍摄来计算个人的鱼的运动。 目前我们已经升级到使用一系列的开销监控摄像机上面的链接到高清晰度数字视频记录设备（HD-DVR）的曝光室。然而，使用任何摄像系统，可以录制高清晰度的MPEG-4视频会工作。用HD-DVR可以在指定的时间被设置为记录并设置最多7天。自动化的方法这种放手允许的几部影片拍摄的同时保持一致性，同时尽量减少可能危害鱼的行为外部干扰。在HD-DVR SYSTE毫秒被连接到内部网络，以便文件的传输是相对简单的。而自动相机系统是用于量化游泳行为大大改进的技术，它仍然是有利的，进行视觉观察以作为附加的支持信息用于在毒性试验记录行为损伤。 还有就是从接触到金属追溯到20世纪60年代初19,20,21导致文学文档化改变鱼的行为很长的历史。铜已被证明导致活动水平的变化，例如在翻车22活动减退（ 蓝鳃太阳鱼rafinesque），并在运动和变化河鳟鱼23的摄食活性（ 红点 鲑 ）。至少有一些幼鱼依靠嗅觉来探测和躲避天敌，和铜引起的化学感应剥夺可能会影响相关检测报警化学品24,25,26行为</sup&gt;。嗅觉上皮因铜曝光从而影响，可能会造成定向障碍，行为回避，摄食减少和由嗅觉27引导任何其他行为感觉机制受损。这些改变的行为均与曝光过程中观察到的是一致的。 白鲟的游泳行为亚致死暴露于含水铜的浓度。这些结果说明行为是如何在铜的亚致死浓度的影响，并且可以作为有毒应力的指示期间，大大影响。视频为基础的分析证明是有效的量化游泳行为，还担任过接触到铜鲟鱼严重影响的定性视觉文档。该分析软件也能够量化等各种行为端点。请参考表1中的列表。曝光系统可以修改，以处理每个端点中实时模式和可以用来量化暴露于关注的各种污染物相关联的行为差异。 在水产毒理学研究中使用行为的端点是越来越多的利用和评估污染物的效果时，因为自适应行为功能是在环境损伤9的确定关键应予以考虑。环境污染物鱼行为的影响常常是主观的并用亚致死端点在没有标准方法处理时挑战尤其.. 游泳活动通过使用这些方法，可严格监测作为定量，是无损最小应力对生物体和可重复。游泳行为是应该在测试协议被掺入到扩大的标准毒性试验5的灵敏度亚致死毒性的有效的和一致的索引。

了解污染物暴露会如何影响行为有时是非常具有挑战性的和主观的。行为通常被定义为一系列的通过中枢神经系统操作和使生物体生存，生长，繁殖公开的，可观察，全身活动。在行为由于暴露于一个毒物变化环境胁迫倍常常10-100之间更敏感的最敏感的指标之一相比存活1时。这些研究大部分的游泳年审活动，通风和觅食的鱼2,3,4的行为。游泳活动是在确定响应于污染物在毒性试验5一行为改变中最常用的亚致死端点。游泳变量包括在水柱的频率和行进的移动，速度和距离的持续时间，频率和匝数角度，位置和游泳的图案。游泳活动评估的基础上由兰德6毒理学中的基础书第九章中提出的标准毒物敏感性，当游泳行为的有效措施。 本文提出了一种毒理研究作为如何铜的早期生命阶段的白鲟在早期开发的各个阶段中的毒性（ 鲟transmontanus）只有水的敞口相对于鲟鱼游泳的行为进行评价的例子，说明了量化游泳方法行为。 在以前的研究中，不良的行为反应急性和慢性接触铜的开始几天是早发型明显，逐渐变得过度曝光时间和浓度7,8,9更加严重。幅度和这些行为反应的发作时间可能足以限制长期生存，因此是令人关注的给定为中心人才招聘的影响tment失败10。为了准确地解释这种敏感性金属曝光方法和程序的意义被用于定量相对于铜的浓度的行为障碍的时间过程和程度的发展。 对行为功能和开发测试协议被曝光室分析鲟鱼的开销视频样本建立。视频样本提供铜处理间的发育和功能的定性评估整个曝光周期。暴露于水铜的含量​​在行为和庇护寻求活性评估，以确定时间效应和表征损害的时间顺序，影响寻求庇护，嗜睡，游泳协调，平衡和呼吸。此外，视频记录作了鲟从各子采样的复制用于定量记录自发游泳ACTI的目的VITY。这些终点包括持续时间的措施或所花费的时间移动，游泳用市售的数字图象分析软件包的速度和行驶距离5,11。这个软件定义各图像的轮廓的视场之内，并从这个，定义了每个图像的质心。然后，该软件可以通过帧连续跟踪每个质心的一帧中的位置，以确定运动的路径。 这项研究与动物福利法的规定（9 CFR）的最终规则所有适用的部分，并与测试生物的文化和实验过程中人性化的对待所有机构准则规定。在研究结束时，所有的鱼都按照批准的机构动物护理和使用委员会的美国地质调查局，哥伦比亚大学环境研究中心适当的指导方针安乐死。

<table border="0" cellpadding="0" cellspacing="0" width="885">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">copper II sulfate pentahydrate</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">contaminant of concern</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">syringe dispenser</td>
			<td style="width:285px;">Hamilton MicroLab 600 Series</td>
			<td style="width:119px;"></td>
			<td>apparatus to spike chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">2-L volumetric flask</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td>container for holding stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">24-1.5 L glass jars</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td>test chamber for 2 dph sturgeon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">video camera</td>
			<td style="width:285px;">Sony Handycam HDR-CX550V</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">digital tracking software</td>
			<td style="width:285px;">Noldus Ethovision</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">3-17&#34; flat screen monitors</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">24 surveillance cameras</td>
			<td style="width:285px;">Model CL101</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">3-16 channel digital recording devices</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">DO meter</td>
			<td style="width:285px;">YSI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">pH meter</td>
			<td style="width:285px;">Orion 940</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ph probe</td>
			<td style="width:285px;">Orion&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ammonia meter</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">ammonia probe</td>
			<td style="width:285px;">Orion</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">chiller unit</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">recirculating water pump</td>
			<td style="width:285px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying fish swimming behavior in response to acute exposure of aqueous copper using computer assisted video and digital image analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

手动处理所述视觉观测数据表明百分之异常带2天后的孵化（DPH）鲟（ 图2）启动只是72小时暴露后，用铜浓度的增加而增加。视频样本记​​录铜暴露对鲟鱼的游泳行为极端冲击（视频2），并在限定的行为损伤，结果辅助。在另一实例中，鲟鱼，在30 DPH基于只死亡证明40.3微克/升用的96小时半致死效应浓度（LC50）铜暴露敏感。然而，当平衡与固定损失的行为致死端点与死亡率都被包含大约96小时平均浓度的效果（EC50）为2.4至5.0微克/升的敏感性增加。行为异常的视频文件拍摄的这些亚致死效应，并进一步验证人类观察记录在曝光期间。采用数字化追踪软件分析游泳活动时减少显著后处理时间。鱼游泳速度，花费的时间运动，而且行驶的距离都显著下降（ 图3）铜浓度的增加。游泳路径也与铜浓度的增加（ 图4）减小。 <img alt="图1" src="/files/ftp_upload/53477/53477fig1.jpg" /> 图1:。用来检测试验原液浓度的稀释毒性试验使用基于目标浓度的电子表格模板，确定化学备料电子表格模板的电脑屏幕截图 <a href="https://www.jove.com/files/ftp_upload/53477/53477fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> 图2:稀释器的设置和实验设计白色鲟的各种生命阶段暴露于铜。根据鱼的大小被选择用于曝光稀释的大小。 （ 一 ）早期生活阶段鲟用一个小稀释设置和（b）较旧的生命阶段鲟使用大型稀释设置暴露露出。 <img alt="图3" src="/files/ftp_upload/53477/53477fig3.jpg" /> 图3:游泳行为导致从早期生命阶段白鲟（ 鲟transmontanus）72 小时铜曝光（开始三十天孵化后[DPH]鱼 ）。游泳活动端点（ 一 ）中30 DPH白鲟运动的持续时间; （ 二 ）游泳速度; （ 三 </移动STRONG&gt;）距离谢绝中暴露96小时白鲟增加铜浓度。星号表示与对照显著差异，误差棒代表标准偏差。 <img alt="图4" src="/files/ftp_upload/53477/53477fig4.jpg" /> 图4:从早期生命阶段白鲟（鲟transmontanus）96小时铜暴露的结果（从2天孵化后[DPH]鱼）在2 DPH幸存的白鲟后的72小时表现出平衡和固定的损失。 96小时暴露随铜浓度。星号表示与对照显著差异，误差棒代表标准偏差。 <img alt="图5" src="/files/ftp_upload/53477/53477fig5.jpg" /> 图5:从早期生命阶段白鲟为例游泳路径的结果（鲟transmontanus）96小时铜曝光（三十天孵化后[DPH]鱼）凝视。鲟鱼的游泳路径，使用数字跟踪软件从（ 一 ）控制重复（N = 5鱼）和（b）从高处理（50数字化的96小时暴露后微克/升）的复制（n = 3的鱼）。注意游泳路径的数量并不代表室目前鱼的数量，因为一些鱼都是无效的。 <a href="https://www.jove.com/files/ftp_upload/53477/53477fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 量化的参数 </td><td> 观测参数 </td></tr><tr><td>速度</td><td>嗜睡/多动症</td></tr><tr><td>移动距离</td><td>失去平衡</td></tr><tr><td>的持续时间在区花</td><td>痉挛/震颤/上攻下</td></tr><tr><td>次机体移动选定区域之间的过渡区号码</td><td>在水柱位置</td></tr><tr><td>前往点计算对兴趣点动物的路径的偏差</td><td>呼吸（快/慢） </td></tr><tr><td>标题计算所选体点的标题</td><td>着色</td></tr><tr><td>转角度差在两个样本之间标题</td><td>藏</td></tr><tr><td>角速度计算由采样间隔除以转角</td><td></td></tr><tr><td>除以移动的距离的转弯角度蜿蜒计算。用于比较转动物以不同的速度移动</td><td></td></tr><tr><td>花费的时间移动</td><td></td></tr><tr><td>移动性状态，计算其检测为动物正在改变完整的区域的持续时间，即使分呃点保持不变</td><td></td></tr><tr><td>当所选择的身体点具有360°的累积转角度旋转一次旋转被完成。轮流在小于阈值被忽略相反的方向。 </td><td></td></tr><tr><td>流动性的连续计算移动性的所检测动物的完整区域的比例，即使中心点保持相同。 </td><td></td></tr><tr><td>之间的距离科目，计算所有演员和选定的接收器之间的距离</td><td></td></tr><tr><td>近程计算的量，演员是或不是在接近接收器中的持续时间</td><td></td></tr><tr><td>相对运动</td><td></td></tr><tr><td>净加权运动-的演员的至（正）的运动，并从（负）的接收器，通过它们之间的距离的加权</td><td></td></tr><tr><td>加权移动距离，MoveMe的<html>来自接收机的演员，通过它们之间的距离加权的核苷酸。 </td><td></td></tr><tr><td>加权运动于─演员到接收器的移动，通过它们之间的距离的加权</td><td></td></tr><tr><td>试用控制元件的两个事件，或者一个元素中的试用控制语句期</td><td></td></tr><tr><td>发生在其中一个元素中的事件您在试用控制定义试验控制事件的时刻。 </td><td></td></tr></tbody></table> 表1:通过数字跟踪软件定量行为端点这些端点可以个人或团体使用，也可以作为在曝光过程中行为发生减值的视觉观察检查清单。 <img alt="视频1" src="/files/ftp_upload/53477/53477movie1.jpg" /> /www.jove.com/files/ftp_upload/53477/53477video1.avi"&gt; 视频1:受影响白鲟表现出异常行为定义可视化（右键点击下载）使用每日观察检查表来记录异常的。平衡和固定的损失是风险敞口。从卡尔菲等修改过程中观察到的最常见的异常。7 <img alt="视频2" src="/files/ftp_upload/53477/53477movie2.jpg" /> <a href="https://www.jove.com/files/ftp_upload/53477/53477video2.avi">视频2:视觉文档凸显白鲟游泳行为的一个例子。 （点击下载），</a>白鲟鱼游泳活动大大暴露还原为铜浓度的增加。在此视频中描绘的鲟是从一个控制，中低，并且在96小时的水性铜的端部的高处理曝光。 虽然白鲟还活着这是显而易见的鱼相比，控制的治疗受到极大损害。从卡尔菲等进行修改。7

Watch this Scientific Journal Video about Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis at JoVE.com

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

使用计算机辅助视频和数字图像分析量化鱼游泳行为应对水铜的急性接触

测量鱼的行为环境污染物的影响往往是主观的和亚致死打交道时特别具有挑战性。我们描述方法，包括中，后96小时急性暴露于各种浓度的铜的视频技术来量化早期生命阶段白鲟（ 鲟transmontanus）的游泳行为。

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

quantifying-fish-swimming-behavior-response-to-acute-exposure-aqueous

Research

JoVE Journal

Neuroscience

11.1K 次观看.  US Geological Survey. 测量鱼的行为环境污染物的影响往往是主观的和亚致死打交道时特别具有挑战性。我们描述方法，包括中，后96小时急性暴露于各种浓度的铜的视频技术来量化早期生命阶段白鲟（ 鲟transmontanus）的游泳行为。 

视频: Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

科研

神经科学

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

计算机辅助多电极膜片钳系统

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

自由游泳弱电鱼的长期行为跟踪

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

的自动分析<em&gt;℃。线虫</em&gt;游泳行为使用天书软件

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

试验以检测紫外线反射结构，并使用茉莉花鳉确定其重要性伴侣偏好<em&gt;孔雀鱼latipinna</em

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

体外刺激对龟脑神经的运动测量

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

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 ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

麻醉大鼠中枢神经系统操作对肾交感神经活动的急性变化量化

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...