We thank members of the UNC Zebrafish Aquaculture facility for maintenance of the zebrafish. We would also like to thank Diagnosys, LLC for assistance with the setup of the ERG apparatus. Additional thanks go to Dr. Portia McCoy and the laboratory of Dr. Ben Philpot for assistance with electrophysiological methods. We also wish to thank Lizzy Griffiths for her illustration of a larval zebrafish. This work was supported by National Institutes of Health awards F32 EY022279 (to J.D.C) and R21 EY019758 (to E.R.W).

动物保养和实验方案由北卡罗莱纳大学教堂山分校的机构动物护理和使用委员会批准，并符合实验动物福利NIH办公室和协会的评估和实验动物保健国际资格认证的所有要求。 注：要获得幼虫ERG分析，公布的协议标准的斑马鱼的饲养和维护被雇用18。幼虫通过自然繁殖获得，住在一个14小时光照/ 10小时黑暗周期。该协议已优化幼虫5-7天受精后（DPF），但也最好能在老鱼的小修改的程序进行。在这里，使用野生型斑马鱼幼虫的TL应变5 DPF。 1，微管生产<ol><li>用拉1.5×0.86毫米（外径由内径）若干微量火抛光硼硅玻璃毛细管丝（熔融温度821°C）和P-97火焰山/布朗微量拉马装有箱热灯丝。使用该程序用于制作表1所述微量。 </li><li>请与相应的刻度标尺，在显微镜下每个微管，以确保提示是10-15微米，直径有一个平滑的顶部开口（ 即无锯齿边缘）。 </li><li>小心保存微量防止尖损伤和接触灰尘。存储选项包括培养皿实验室胶带，泡棉内衬盒，或市售的微量储存容器。 注意：其他微量拉拔器及玻璃毛细管可以使用，只要微量的正确直径和高品质的尖端得以实现。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 压力 </td><td> 热 </td><td> 拉 </td><td> 速度 </tD&gt; <td> 时间 </td></tr><tr><td> 500 </td><td> 560 </td><td> - </td><td>三十</td><td> 200 </td></tr><tr><td> 500 </td><td> 450 </td><td> - </td><td>三十</td><td> 200 </td></tr><tr><td> 500 </td><td> 410 </td><td> 55 </td><td> 40 </td><td> 200 </td></tr></tbody></table> 表1：程序用于使用P-97燃烧的/棕色微量拉马微量的制备配有框热灯丝微量移液器使用1.5×1.0 平方毫米（外径由内径）火抛光的硼硅玻璃毛细管长丝制成。 （熔融温度，821℃）。 2.缓冲准备<ol><li>使用过滤，充氧金鱼林格氏缓冲器19中的微电极的毛细管，并以饱和的聚乙烯醇（PVA）海绵在其上的幼虫被放置于实验。此外，使用E3<html>胚胎培养基或Hank氏平衡盐溶液。 </li><li>制备10倍金鱼林格氏溶液，如表2中所述，调整至pH 7.8，并消毒用0.22微米的过滤器，并存储10倍的库存在4℃。 </li><li>通过稀释10倍林格氏液为1x用去离子蒸馏水创建在实验当天的工作溶液。过滤器采用0.22微米的过滤系统。含氧化合物通过用95％氧气鼓泡4/10％CO 2的气体10分钟。帽紧紧事后以确保该溶液保持氧化。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>氯化钠</td><td>的1.25M </td></tr><tr><td>氯化钾</td><td> 26毫米</td></tr><tr><td> 氯化钙 </td><td> 25毫米</td></tr><tr><td> 氯化镁 </td><td> 10毫</td></tr><tr><td>葡萄糖</td><td> 100毫米</td></tr><tr><td> HEPES </td><td> 100毫米</td></tr></tbody></table>jove_content“&gt; 表2：10X金鱼林格氏溶液的制备。 3.平台电图<ol><li>执行ERG实验上的防振台法拉第笼内部，以改善信噪比。安装一个定制的钢平台使用六角螺母的防振动台。放置一个可移动的塑料平台，在桌子上的光源下，粘弹性聚氨酯聚合物吸震底部。 </li><li>定位相机与磁化的立场，在可移动的塑料平台，旨在下来。定位显微（其将保持在记录微电极）与第二磁化支架于可动塑料平台的右侧。确保相机和显微不会被其他设备的运动受到干扰，而它们不会阻挡从所述光源照明。 </li><li>将相机连接到视频监控和定位它来查看的眼睛幼虫用于放置电极，在适当的位置。 </li><li>确保安装正确地与铜线接地。检查噪声，放置在记录微电极的参考电极和尖端在35毫米培养皿中填充有林格氏液。检查设置的电噪声水平用示波器或ERG的装置的内置功能。噪音水平应不超过±10μV从基线。 </li></ol> 4.海绵准备<ol><li>切一小矩形的干PVA海绵将紧贴在35毫米培养皿。海绵的厚度不应大于盘的深度。使用一个工具刀，用干净的刀片进行切割。 </li><li>进行额外切成海绵容纳参比电极（无论是一个浅切口长度在海绵上的底部或竖直切过的小端1蝶式）。 </li><li>使用耐化学性的标记物标记上的海绵（其中幼虫将被放置），可以被用于定位所述相机的小圆点。 </li><li>浸泡PVA海绵林格氏液，直至饱和。取出并迅速涂抹在纸巾上的2-3倍。放置海绵在一个干净的35mm平皿。 </li><li>定位含有该塑料平台上的海绵，使得该标记可被相机可视培养皿。 </li></ol> 5.电极的制备注：斑马鱼设置包括在与林格氏溶液饱和PVA海绵和与角膜接触的记录电极接触的参考电极。参考电极由一个银/氯化银颗粒。记录电极是一个拉玻璃微量吸管由含Ag丝微电极保持器填充有林格氏溶液和保持。 <ol><li>通过5分钟（记录MICR浸泡在6-9％次氯酸钠（漂白剂），氯化电极oelectrode线）或15分钟（参考电极）。空气干燥上的Kimwipe 5分钟。 <ol><li>根据剪裁的样式在步骤4.2制成，将银/氯化银沉淀的参考电极的成（用于垂直蝶形切）或下（对于浅切纵向在底部）的海绵。附加参比电极引线到记录系统。 </li><li>或者，如果设置ERG具有空间限制或有从Ag / AgCl电极特别强光伏工件，通过琼脂盐桥连接的基准电极到海绵移动电极离开光路。 </li></ol></li><li>附上〜40厘米适当尺寸的管道，以5ml的非鲁尔锁注射器。充满林格氏液的注射器。微持有人持有的压力端口通常附带的适配器，以适应管与1/16内径“3/32”，1/8“或5/32”。 </li><li>填写1毫升非鲁尔锁注射器林格氏溶液和，使用Micro-FIL，小心地填充微电极保持器。防止气泡的形成。 </li><li>附上5毫升注射器与管的微电极保持器的压力端口，并用它来确保微电极保持器充满林格氏液。使用微FIL和1-ml注射器填充有林格氏液，从尖端填充微量的玻璃，并确保没有气泡存在。 </li><li>装上玻璃微到微电极夹，小心翼翼地保持电极丝直。一旦保护，​​使用5号毫升注射器的仔细强制通过微林格氏液，直至溶液小钱是尖端可见。的压力的注射器（当没有施加到角膜）偶发的应用将防止形成气泡，以及遮挡由于灰尘或盐的积累，在微量移液器尖端。 <ol><li>如果解决方案出来作为流，更换克姑娘微量，由于顶端开口太大或损坏。 </li></ol></li><li>小心地在显微记录微电极，并连接引线到记录系统。 </li></ol> 6.电图分析注：由于幼虫视网膜锥支配地位，高品质的ERG结果可以当根据间接白光低水平执行的准备用于记录获得的（&lt;1勒克斯）或具有短周期更高强度（&lt;1分钟）（ ≤250勒克司）工作灯。暗适应短时间的前记录仍然是必需的（见步骤6.7）。然而，实验可以在昏暗的红色或红外光使用红外感光摄像头进行。所有实验均在过滤灭菌（0.22微米）系统水从所述UNC斑马鱼养殖设施进行，但可使用的替代胚胎介质。 <ol><li>切纸巾方格测量大约1厘米2。 </li><li>如果测量孤立锥体质量受体电位，孵育3-5幼虫与500微米（±）-2-氨基-4- phosphonobutyric酸（APB）系统水5分钟。 注意：虽然APB是活动（L）的外消旋混合物和非活性（R） - 形式的AP4的，这是因为有效的为L-AP4和更便宜。 </li><li>麻醉3-5幼虫在系统水用0.02％（重量/体积）三卡因直至反应迟钝，约1-2分钟。 </li><li>使用巴斯德吸管和吸管泵在使用最小照度（≤250勒克司&lt;1分钟）解剖体视个人仔细的幼虫转移到纸巾广场。检查每个幼虫的位置，并选择一个候选者是背侧与未被遮挡的眼睛。 <ol><li>延长录音（&gt; 30分钟），由玻璃体内达到，但用细驼毛刷刷不包括头3％甲基纤维素保持幼虫湿润。 </li></ol></li><li>使用镊子，转移纸巾SQUARË与幼虫到潮湿的PVA海绵。 <ol><li>扩展记录（&gt; 30分钟），通过在含有蒸馏水的侧臂烧瓶一个airstone鼓泡气体申请水饱和的100％ 的 O 2气体的连续流过的幼虫。定位管道离开烧瓶的侧臂，传达附近幼虫的头部的加湿氧气。 注意：步骤6.4.1和6.5.1步骤将延长鱼16的寿命。 </li></ol></li><li>下最小照度，使用显微和照相机在鼻和眼的尾端部之间的中点来定位微电极前端轻轻压在角膜的背极限。 注意：电极末端到角膜的远末端区域的错位可导致极性相反ERG波形。 </li><li>允许幼虫到暗适应5-10分钟。 </li><li>使用阿瓦伊记录测试闪光响应从LED光源或光刺激提供的光拉布勒刺激和录音设备。调整协议参数，如闪光强度，闪长，闪颜色，背景亮度和颜色过滤器设置，以适应实验。 </li><li>当与试验结束时，根据AVMA / IACUC准则安乐死幼虫。 </li></ol>

<ol>
	<li>Dowling, J. E. <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 retina: an approachable part of the brain. , (1987).</li><li>Makhankov, Y. V., Rinner, O., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inexpensive+device+for+non-invasive+electroretinography+in+small+aquatic+vertebrates.">An inexpensive device for non-invasive electroretinography in small aquatic vertebrates.</a> J Neurosci. Methods. 135, 205-210 (2004).</li><li>Wu, J., Peachey, N. S., Marmorstein, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-evoked+responses+of+the+mouse+retinal+pigment+epithelium.">Light-evoked responses of the mouse retinal pigment epithelium.</a> J Neurophysiol. 91, 1134-1142 (2004).</li><li>Heckenlively, J. R., Arden, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practice of Clinical Electrophysiology of Vision. , (2006).</li><li>Perlman, I., Kolb, H., Nelson, R., Fernandez, E., Jones, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Webvision: The Organization of the Retina and Visual System. , (1995).</li><li>Fadool, J. M., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+eye+genetics.">Zebrafish: a model system for the study of eye genetics.</a> ProgRetin. Eye Res. 27, 89-110 (2008).</li><li>Doerre, G., Malicki, 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+analysis+of+photoreceptor+cell+development+in+the+zebrafish+retina.">Genetic analysis of photoreceptor cell development in the zebrafish retina.</a> Mech. Dev. 110, 125-138 (2002).</li><li>Brockerhoff, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+stimulates+a+transducin-independent+increase+of+cytoplasmic+Ca2++and+suppression+of+current+in+cones+from+the+zebrafish+mutant+nof.">Light stimulates a transducin-independent increase of cytoplasmic Ca2+ and suppression of current in cones from the zebrafish mutant nof.</a> J Neurosci. 23, 470-480 (2003).</li><li>Rinner, O., Makhankov, Y. V., Biehlmaier, O., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+cone-specific+kinase+GRK7+in+larval+zebrafish+leads+to+impaired+cone+response+recovery+and+delayed+dark+adaptation.">Knockdown of cone-specific kinase GRK7 in larval zebrafish leads to impaired cone response recovery and delayed dark adaptation.</a> Neuron. 47, 231-242 (2005).</li><li>Harada, T., Harada, C., Parada, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+visual+system+development:+more+than+meets+the+eye.">Molecular regulation of visual system development: more than meets the eye.</a> Genes Dev. 21, 367-378 (2007).</li><li>Branchek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+photoreceptors+in+the+zebrafish,+brachydaniorerio.+II.+Function.">The development of photoreceptors in the zebrafish, brachydaniorerio. II. Function.</a> J Comp Neurol. 224, 116-122 (1984).</li><li>Schmitt, E. A., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+retinal+development+in+the+zebrafish,+Daniorerio:+light+and+electron+microscopic+analyses.">Early retinal development in the zebrafish, Daniorerio: light and electron microscopic analyses.</a> J Comp Neurol. 404, 515-536 (1999).</li><li>Bilotta, J., Saszik, S., Sutherland, 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=Rod+contributions+to+the+electroretinogram+of+the+dark-adapted+developing+zebrafish.">Rod contributions to the electroretinogram of the dark-adapted developing zebrafish.</a> Dev Dyn. 222, 564-570 (2001).</li><li>Wong, K. Y., Adolph, A. R., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+bipolar+cell+input+mechanisms+in+giant+danio.">Retinal bipolar cell input mechanisms in giant danio.</a> I. Electroretinographic analysis. J Neurophysiol. 93, 84-93 (2005).</li><li>Nelson, R. F., Singla, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spectral+model+for+signal+elements+isolated+from+zebrafish+photopicelectroretinogram.">A spectral model for signal elements isolated from zebrafish photopicelectroretinogram.</a> Vis Neurosci. 26, 349-363 (2009).</li><li>Korenbrot, J. I., Mehta, M., Tserentsoodol, N., Postlethwait, J. H., Rebrik, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EML1+(CNG-modulin)+controls+light+sensitivity+in+darkness+and+under+continuous+illumination+in+zebrafish+retinal+cone+photoreceptors.">EML1 (CNG-modulin) controls light sensitivity in darkness and under continuous illumination in zebrafish retinal cone photoreceptors.</a> J Neurosci. 33, 17763-17776 (2013).</li><li>Gurevich, L., Slaughter, 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=Comparison+of+the+waveforms+of+the+ON+bipolar+neuron+and+the+b-wave+of+the+electroretinogram.">Comparison of the waveforms of the ON bipolar neuron and the b-wave of the electroretinogram.</a> Vision Res. 33, 2431-2435 (1993).</li><li>Westerfield, M. <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 Zebrafish Book: A guide for the laboratory use of zebrafish (Daniorerio). , (2007).</li><li>Kim, D. Y., Jung, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction+contributions+to+the+goldfish+electroretinogram+at+the+photopic+illumination+level.">Gap junction contributions to the goldfish electroretinogram at the photopic illumination level.</a> Korean J PhysiolPharmacol. 16, 219-224 (2012).</li><li>Brockerhoff, S. E., Dowling, J. E., Hurley, 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=Zebrafish+retinal+mutants.">Zebrafish retinal mutants.</a> Vision Res. 38, 1335-1339 (1998).</li><li>Naka, K. I., Rushton, 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=S-potentials+from+colour+units+in+the+retina+of+fish+(Cyprinidae).">S-potentials from colour units in the retina of fish (Cyprinidae).</a> J Physiol. 185, 536-555 (1966).</li><li>Naka, K. I., Rushton, 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=S-potentials+from+luminosity+units+in+the+retina+of+fish+(Cyprinidae).">S-potentials from luminosity units in the retina of fish (Cyprinidae).</a> J Physiol. 185, 587-599 (1966).</li><li>Shao, X. M., Feldman, 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=Micro-agar+salt+bridge+in+patch-clamp+electrode+holder+stabilizes+electrode+potentials.">Micro-agar salt bridge in patch-clamp electrode holder stabilizes electrode potentials.</a> J Neurosci. Methods. 159, 108-115 (2007).</li><li>Brockerhoff, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+behavioral+screen+for+isolating+zebrafish+mutants+with+visual+system+defects.">A behavioral screen for isolating zebrafish mutants with visual system defects.</a> ProcNatlAcadSci. U S A. 92, 10545-10549 (1995).</li><li>Fleisch, V. C., Jametti, T., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroretinogram+(ERG)+Measurements+in+Larval+Zebrafish.">Electroretinogram (ERG) Measurements in Larval Zebrafish.</a> CSH protocols. , (2008).</li><li>Seeliger, M. W., Rilk, A., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganzfeld+ERG+in+zebrafish+larvae.">Ganzfeld ERG in zebrafish larvae.</a> Doc Ophthalmol. 104, 57-68 (2002).</li><li>Kainz, P. M., Adolph, A. R., Wong, K. Y., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lazy+eyes+zebrafish+mutation+affects+M&#252;ller+glial+cells,+compromising+photoreceptor+function+and+causing+partial+blindness.">Lazy eyes zebrafish mutation affects M&#252;ller glial cells, compromising photoreceptor function and causing partial blindness.</a> J Comp Neurol. 463, 265-280 (2003).</li><li>Lewis, 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=Celsr3+is+required+for+normal+development+of+GABA+circuits+in+the+inner+retina.">Celsr3 is required for normal development of GABA circuits in the inner retina.</a> PLoS. genetics. 7, e1002239 (2011).</li></ol>

没有利益冲突的声明。

在这个协议中的幼虫斑马鱼的ERG记录一个简单的过程详细说明。这个程序允许视觉function.There的快速和全面的分析是整个应该牢记的程序几个关键步骤。斑马鱼幼虫应该是健康的实验前，以避免死亡时潜在的药物治疗，并确保在ERG录制长时间的生活。此外，重要的是，在实验中使用的幼虫是密切年龄匹配。这是由于视网膜（锥体亚型即 ，差动发展），同时也幼虫的整体形态和生理的快速发?...

电图（ERG）是已被广泛地应用于临床，用于确定在人类视网膜的功能的非侵入性的电生理学方法。响应于光激励的电活动是通过将记录电极对角膜的外表面测量的。刺激范例和响应波形的特性限定的视网膜神经细胞贡献的响应。该方法已被改编为与一些动物模型包括小鼠和斑马鱼中使用。典型的脊椎动物的ERG响应具有四个主要组成部分：一个波，这是从光感受器细胞活性衍生的角膜负电位; b-波，从接通双极细胞衍生的角膜正电位;的d波，角膜正电位解释为OFF双极细胞的活性;而C波，其中b波后出现几秒钟，反映了米勒的神经胶质细胞和RET活动伊纳勒色素上皮1-4。了解ERG分析的历史和原则在人类和动物模型的附加 ​​引用了网上购书，Webvision，犹他州和文本的大学，如原则和愿景4,5临床电生理实践。 斑马鱼（斑马鱼）长期以来被青睐的一种模式脊椎动物发展，由于其快速成熟和透明性，其允许器官系统，行为分析的非侵入性的形态学分析和正向和反向遗传筛选（对于综述，参见Fadool和道林6）。斑马鱼幼体是高度适合于遗传学和药理学处理，其中，加上它们的高繁殖力时，使它们的优异动物模型，用于高通量生物分析。锥体在幼虫斑马鱼棒较高比率 - 大约1：1的比小鼠（〜3％浓S） -使其成为锥功能7-9的研究特别有用。 在脊椎动物的视网膜，锥杆10前发展。有趣的是，斑马鱼锥体可操作早在4单丝旦，允许锥体的选择性电生理分析在该阶段6中，11,12。与此相反，在杆ERG响应11和21旦13之间出现。因此，斑马鱼幼体在4-7 DPF功能作为全视网膜锥。然而，4-7单丝旦幼虫天然明视视网膜电响应由b波为主。 （+） - - 2-氨基-4-膦酰基丁酸（L-AP4），激动剂为代谢型谷氨酸（mGluR6）受体由上表达双极细胞，有效地阻止了生成的药理学试剂，如L-应用b-波和揭示了分离的锥形质量受体电位，（以下简称“a波”），14-17。 在这里，我们描述了一个简单而reliabl使用市售ERG设备设计用于与已被改编为与斑马鱼幼虫用鼠标E法进行ERG分析。该系统可用于对不同的遗传背景，以及那些具有药理剂处理，以帮助研究人员在信号通路有助于视觉灵敏度和光适应16的识别的斑马鱼幼虫。在这个协议中所列的实验程序将引导侦查员在使用ERG分析来解答各种有关视觉的生物学问题，并表现出灵活的ERG安装施工。

<table border="0" cellpadding="0" cellspacing="0" width="2170">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;"><a name="RANGE!A1:D30">Name of the Material/Equipment</a></td>
			<td style="width:168px;">Company</td>
			<td style="width:165px;">Catalog Number</td>
			<td style="width:1504px;">Comments/ Description (optional)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Faraday cage</td>
			<td>80/20 Inc</td>
			<td>custom</td>
			<td>Custom designed aluminum &#34;Industrial Erector Set&#34; for Cage framework</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PVA sponge</td>
			<td>Amazon</td>
			<td>B000ZOWG1C</td>
			<td>Provides a soft, moist platform for placement of zebrafish larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150 ml Sterile Filter systems</td>
			<td>Corning</td>
			<td>431154</td>
			<td>Filtering solutions to prevent small articulates from blocking micropipettes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Espion E2</td>
			<td>Diagnosys, LLC</td>
			<td>contact</td>
			<td>Modular electrophysiology system capable of generating visual stimuli for any stimulator and digital recording and analysis of responses using propietary software, more information at http://www.diagnosysllc.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Colordome</td>
			<td>Diagnosys, LLC</td>
			<td>contact</td>
			<td>Light stimulator with RGB LED and Xenon light sources for Ganzfeld ERG, more information at http://www.diagnosysllc.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanipulator</td>
			<td>Drummond</td>
			<td>3-000-024-R</td>
			<td>Holding and positioning the recording microelectrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic ring stand</td>
			<td>Drummond</td>
			<td>3-000-025-MB</td>
			<td>Holding and positioning of the camera and refrence electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lead extensions</td>
			<td>Grass Technologies</td>
			<td>F-LX</td>
			<td>Spare female to male 1.5 mm lead cables for connecting electrodes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:333px;">Male Pin to Female SAFELEAD Adaptor</td>
			<td>Grass Technologies</td>
			<td>DF-215/10</td>
			<td>Connecting 2 mm pins to 1.5 headboard pins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Window screen frame (metal) and spline</td>
			<td>Lowes or Home Depot</td>
			<td>various</td>
			<td>For attaching copper mesh to Faraday cage framework</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Steriflip 50 ml filters</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td>Filtering solutions to prevent small articulates from blocking micropipettes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BNC adaptor</td>
			<td>Monoprice</td>
			<td>4127</td>
			<td>Connecting camera to BNC cable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BNC cable</td>
			<td>Monoprice</td>
			<td>626</td>
			<td>Connecting camera to video adaptor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Camera lens</td>
			<td>Navitar</td>
			<td>1582232</td>
			<td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Camera coupler</td>
			<td>Navitar</td>
			<td>1501149</td>
			<td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luna BNC to VGA + HDMI Converter</td>
			<td>Sewell</td>
			<td>SW-29297-PRO</td>
			<td>BNC to VGA adaptor allowing camera image to project on computer monitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APB</td>
			<td>Sigma</td>
			<td>A1910</td>
			<td>mGluR6 agonist, blocks b-wave allowing analysis of the isolated cone mass receptor potential</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosilicate glass</td>
			<td>Sutter</td>
			<td>BF-150-86-10</td>
			<td>Fire- polished borosilicate glass (metling temperature = 821&#176;C) with filament and dimensions of 1.5mm x 0.86 mm (outer diameter by inner diameter)&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P97 Flaming/Brown puller</td>
			<td>Sutter</td>
			<td>P97</td>
			<td>For pulling glass micropipettes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sorbothane sheet</td>
			<td>Thorlabs</td>
			<td>SB12A</td>
			<td>Synthetic viscoelastic urethane polymer, placed under Passive Isolation Mounts and ERG platform to absorb shock and prevent slipping, can be cut to size</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Breadboard</td>
			<td>Thorlabs</td>
			<td>B2436F</td>
			<td>Vibration isolation platfrom for ERG stimulator and zebrafish specimen</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Passive Isolation Mounts</td>
			<td>Thorlabs</td>
			<td>PWA074</td>
			<td>Provides vibration isolation to breadboard</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Copper mesh</td>
			<td>TWP</td>
			<td>022X022C0150W36T</td>
			<td>To line Faraday Cage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette pump</td>
			<td>VWR</td>
			<td>53502-233</td>
			<td>Used with Pasteur pipettes to carefully transfer zebrafish larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pasteur pipettes</td>
			<td>VWR</td>
			<td>14672-608</td>
			<td>Used with Pipette pump to carefully transfer zebrafish larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Camera</td>
			<td>Watec</td>
			<td>WAT-902B</td>
			<td>Visualizing the positioning of the recording microelectrode onto the larval cornea</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tricaine (MS-222)</td>
			<td>Western Chemical</td>
			<td>Tricaine-S</td>
			<td>Pharmaceutical-grade anesthetic,</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-fil</td>
			<td>WPI</td>
			<td>MF28G-5</td>
			<td>Filling microelectrode holder and microelectrode glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microelectrode holder</td>
			<td>WPI</td>
			<td>MEH2SW15</td>
			<td>Holds glass microelectrode, connects to ERG equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reference Electrode</td>
			<td>WPI</td>
			<td>DRIREF-5SH</td>
			<td>Carefully break off last centimeter of casing to drain electrolyte and expose sintered Ag/AgCl pellet electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reference Electrode (alternative)</td>
			<td>WPI</td>
			<td>EP1</td>
			<td>Alternative to DRIREF-5SH. Ag/AgCl electrode that must be wired/soldered to connecting lead</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low-noise cable for Microelectrode holder</td>
			<td>WPI</td>
			<td>13620</td>
			<td>Connecting recording microelctrode holder to adaptor/headboard</td>
		</tr>
	</tbody>
</table>

electroretinogram analysis of the visual response in zebrafish larvae

电图（ERG）是确定视网膜功能的无创性的方法电。通过放置在角膜的表面上的电极的方法，电活性响应于光而产生可以测量并且用于评估视网膜细胞在体内的活性。这个手稿描述了使用所述的ERG的测定视觉功能在斑马鱼。斑马鱼一直被用作模型脊椎动物发育由于易于基因抑制由吗啉代寡核苷酸和药理操纵。 5-10 DPF，只有视锥细胞的功能是在幼虫视网膜。因此，斑马鱼，不像其它动物，是对于锥形视觉功能的体内研究的强有力的模型系统。该协议使用标准麻醉，显微操作，并且是常见的执行斑马鱼的研究实验室立体显微镜协议。概述的方法使用标准的电EQuipment和低光相机的放置记录微电极引导到幼虫角膜。最后，我们将演示如何市售ERG刺激/录像机最初设计用于小鼠可以很容易地适合与斑马鱼中使用。幼虫斑马鱼的ERG提供在动物中测定锥体视觉功能的一个极好的方法，已经由吗啉代寡核苷酸注射改性以及诸如锌指核酸酶（ZFN）较新的基因组工程方法，转录因子样效应核酸酶（TALENS），和群集定期相互间隔短回文重复序列（CRISPR）/ Cas9，所有这些都大大地提高了效率和基因在斑马鱼的靶向效力。此外，我们采取的药理学试剂穿透斑马鱼幼体来评估向光响应的分子成分的能力的优势。这个协议概述了可以被修改并用来研究一个安装各种实验目标。

通常情况下，尔格记录从斑马鱼幼虫在5旦，由于大量的研究发表的ERG记录在这个阶段9，16,20。使用白色LED的光的20毫秒刺激没有背景照明的暗适应的条件下，测定幼虫的响应。我们利用选自由Ganzfeld光刺激器和计算机控制器/记录器的市售的E​​RG系统。刺激器采用了严格控制的专有脉冲宽度调制（PWM）系统，以控制两者的背景和闪光刺激的亮度。使用专有的全差动放大器与硬件抗混叠滤?...

Watch this Scientific Journal Video about Electroretinogram Analysis of the Visual Response in Zebrafish Larvae at JoVE.com

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

在斑马鱼幼虫的视觉反应的视网膜电图分析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

electroretinogram-analysis-of-the-visual-response-in-zebrafish-larvae

Research

JoVE Journal

Neuroscience

15.5K 次观看.  University of North Carolina at Chapel Hill. We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

视频: Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

科研

神经科学

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

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

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

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

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

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

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

Zebrafish are a proven model for vision research, however many of the earlier methods generally focused on larval fish or demonstrated a simple response. More recently adult visual behavior in zebrafish has become of interest, but methods to measure specific responses are new coming. To address this gap, we set out to develop a methodology to repeatedly and accurately utilize the optokinetic response (OKR) to measure visual acuity in adult zebrafish. Here we show that the adult zebrafish's visual acuity can be measured, including both binocular and monocular acuities. Because the fish is not harmed during the procedure, the visual acuity can be measured and compared over short or long periods of time. The visual acuity measurements described here can also be done quickly allowing for high throughput and for additional visual procedures if desired. This type of analysis is conducive to drug intervention studies or investigations of disease progression.

Zebrafish have been a valuable tool for many research areas. Here we demonstrate a method to elicit a visual response and calculate a functional visual acuity in the adult zebrafish.

在视动反应在斑马鱼视力的定量测量

Zebrafish are a proven model for vision research, however many of the  earlier methods generally focused on larval fish or demonstrated a simple  ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage (ICH). Modelling ICH pre-clinically has proven difficult, and current rodent models poorly recapitulate the spontaneous nature of human ICH. Therefore, there is an urgent requirement for alternative pre-clinical methodologies for study of disease mechanisms in ICH and for potential drug discovery.
The use of zebrafish represents an increasingly popular approach for translational research, primarily due to a number of advantages they possess over mammalian models of disease, including prolific reproduction rates and larval transparency allowing for live imaging. Other groups have established that zebrafish larvae can exhibit spontaneous ICH following genetic or chemical disruption of cerebrovascular development. The aim of this methodology is to utilize such models to study the pathological consequences of brain hemorrhage, in the context of pre-clinical ICH research. By using live imaging and motility assays, brain damage, neuroinflammation and locomotor function following ICH can be assessed and quantified.
This study shows that key pathological consequences of brain hemorrhage in humans are conserved in zebrafish larvae highlighting the model organism as a valuable in vivo&#160;system for pre-clinical investigation of ICH. The aim of this methodology is to enable the pre-clinical stroke community to employ the zebrafish larval model as an alternative complementary model system to rodents.

Here we present a protocol to quantify brain injury, locomotor deficits and neuroinflammation following bleeding in the brain in zebrafish larvae, in the context of human intracerebral hemorrhage (ICH).

斑马鱼幼虫研究出血性脑卒中的病理后果

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

在体内成像完全活跃的脑组织在清醒斑马鱼幼虫和青少年由骷髅和皮肤去除

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...