这项工作是由德意志研究联合会（WE849/16 1 / 2，KD和RW）的支持。我们感谢托马斯欧拉（Töbingen德国）软件量子点的光刺激。

以下说明假设实验者有一个基本的了解视网膜结构，膜片钳记录和双光子显微镜。建立和运行一个设置膜片钳和双光子成像系统的有用信息，请参阅文献[7-12]。 1。动物和组织准备<ol><li>保持鼠标暗适应至少3小时。在此期间，准备1-2升胞组成的解决方案（毫米）125 2.5氯化钠，氯化钾，氯化钙，1.6 MgCl 2的，25日10碳酸氢钠3，D -葡萄糖和平衡pH值7.4 carbogen毒气（5％CO 2在O 2）在室温下。 </li>一<li> 二氧化碳过量颈椎脱位，安乐死在气密腔的鼠标。此过程，并应在黑暗中进行以下步骤。使用昏暗的长波长的光照（我们阻止短的波长由690纳米longpas的冷光源的过滤器），以支持个人愿景，同时保持动物的眼睛暗适应（小鼠只有在光谱的红色部分视力不佳）。在完全黑暗的使用红外线照明（&gt; 800 nm的）的工作，并佩戴夜视镜。 </li><li> Enucleate眼用一双弯曲的虹膜剪刀和他们转移到一碟放置在解剖显微镜下的细胞外液。 </li><li>开放与对春天的剪刀（我们第一次使用柳叶刀刺穿切割为起点）沿锯齿缘（视网膜及睫状体之间的边界）的眼球中取出的角膜和睫状体。取出镜头，仔细分离的视网膜色素上皮细胞。如果视网膜的方向是对你的学习至关重要，表明它像文献[12]。切断视神经和视网膜色素上皮之间，视网膜除去眼罩。注意视网膜表面的方向:卷曲的视网膜内的gangl离子电池的一面;外感光方。 </li><li>删除玻璃体视网膜内层表面轻轻拉出一个木制牙签援助。为了这个目的，使用了细胞外液的体积小，只是涵盖的视网膜。视网膜玻璃体支牙签，可以拖动离心关闭。然后，应用短切口，沿视网膜的周边，以方便扁平化的组织。 </li><li>转移到录音室视网膜感光的一面朝下，宣扬出去的玻璃底部（我们使用细刷）和固定的不锈钢尼龙串成帧。第二视网膜准备以同样的方式，并保持它在carboxygenated供日后使用外液暗适应。 </li></ol> 2。录音<ol><li>一个堂堂正正的激光扫描显微镜下安装在黑暗的录音室，并不断superfuse视网膜准备（不低于5毫升/公里n与细胞外液carboxygenated）加热到35 ° C。显微镜（还配备一个红外敏感CCD相机）坐落在一个吸震内的电子屏蔽法拉第笼的空气表。与非透明幕墙笼盖，继续在黑暗中准备。红色透明薄膜，我们还从电脑显示器屏幕关闭。 </li><li> 850-870 nm或更长的波长调谐红外激光，切换到锁模条件，并使用双光子激发可视化表达GFP的细胞。衰减激光的输出控制激光扫描软件在一定程度上的荧光细胞清楚地认识到，这只是足够使用中性密度过滤器</li><li>对于膜片钳记录，电流钳模式使用玻璃微量（我们用硼硅玻璃管材外径1.5毫米和0.225毫米壁厚）洋溢着细胞内组成的解决方案（毫米）125 K -葡萄糖酸，氯化钾10 0.5 EGT，10 HEPES，与氢氧化钾（给人一种吸管约5MΩ电阻）滴定至pH 7.4。请注意电压钳记录等其他实验条件下，需要不同的解决方案。如果需要注射染料，加入荧光探针（我们使用10毫米的Alexa Fluor 594）或示踪分子（3％Neurobiotin）。微管插入持有人，确保参比电极（氯化银线）与细胞外液在录音室接触。 </li><li>施加压力的微量和目标的GFP表达细胞（我们用40倍水浸泡的目的; NA 1.25）。坐落在神经节细胞层（直接在视网膜的电流方向表面之下）在内核层的近端部分（编制内深约55-75微米），以及无长突细胞组织。微量的靶细胞，内界膜（胶质endfeet鞘）在视网膜接触前表面有被击穿。是公认的成功渗透赶出从微管尖和分离从底层的视网膜组织内界膜红外线传输IR - CCD相机拍摄的图像上，可以观察细胞内的解决方案。 </li><li>办法从双光子图像进行比较SOMA的位置与图片的IR - CCD相机，记录微量的位置所需的细胞。请注意，这一步是不容易实现，需要一定的练习，因为绿色荧光蛋白表达细胞在确认以直接的微量红外线传输图像。正确的定位是实现时，微管尖端导致细胞表面可以看到在双光子图像的凹陷。替代此过程中，使用荧光染料在细胞内的解决方案（例如的Alexa Fluor 594，100微米），以揭示在双光子图像的微量位置。红外双光子激发允许足够的这种染料的荧光微量明显。为此，调整激光扫描软件，还包括红色荧光的检测范围。 </li><li>从微量释放的压力，并获得一个全细胞膜片钳配置在电流钳模式。一个可用的录音应该给的-50至-55 mV的膜电位和持续至少20分钟。 </li><li>目前创建一个刺激软件（我们使用，德国蒂宾根大学的托马斯欧拉，量子点），在计算机显示器上11的视觉刺激。调整要记录的细胞刺激的空间位置是:马克显示器上显示的IR - CCD相机和中心到它的一个斑点状的刺激传输图像的细胞位置。中性密度​​滤光片插入光路中的调整刺激强度。校准显示器的频谱和强度，见参考文献[14]。选择一个prolongeð刺激间隔（如15秒），以避免适应。包括一个光电二极管保持在光束路径上的刺激定时记录。 </li><li>启动刺激的协议，并记录光响应（我们使用与非尖峰细胞过滤5 kHz时为10 kHz的采样率;穗录音建议像更高的采样率20千赫）。使用光刺激软件触发录音软件。 </li><li>染料充填让前仔细回缩荧光剂扩散到30-45分钟的记录细胞从细胞的微管。小心不拉细胞体。如果使用非荧光示踪Neurobiotin，它需要通过有约束力的可视化链霉亲和共轭一个荧光团（我们在夜间使用链霉亲和素- Cy3标记，1:400 4 ° C，经过20分钟的视网膜多聚甲醛固定;染料耦合研究链霉亲和孵化，可以延长至3天）。另外，注射剂也可以进行与尖锐的电极（&gt; 100MΩ）当染料驱动iontophoretically刺穿细胞（矩形脉冲:0.25-1 NA，100毫秒，间隔100毫秒，总时间3-6分钟）。 </li></ol> 3。代表性的成果: 以下结果来自对鼠标的研究表达绿色荧光蛋白（GFP）启动子下，儿茶酚胺合成的限速步骤（TH:GFP的小鼠）的酶催化酪氨酸羟化酶 13,14 。基于GFP信号的两个不同的细胞群的亮度分辨（图2A）。细胞中表达的GFP水平较高，拥有位于内核层（INL，图2A），或在神经节细胞层（保利协鑫，图2B）流离失所的胞体和内网状层中的分层（图2C，彩光） 。他们被确定为2型细胞14-16的形态（图3）可以系统地进行研究，并选举rical活动（图4），即使它的人口密度仅为250细胞/毫米2。 <img alt="图1" src="/files/ftp_upload/3457/3457fig1.jpg" /> 图1。实验装置示意图。双光子激发（红色点缀光路）荧光团，表达完整的视网膜细胞，使视觉由微管（绿色发射光路）的目标。通过冷凝器显微镜（黄色光路）预计的光刺激，视网膜细胞对光反应记录。 <img alt="图2" src="/files/ftp_upload/3457/3457fig2.jpg" /> 图2。绿色荧光蛋白表达细胞在视网膜的一个TH flatmount:GFP的鼠标。可以区分两类人群的GFP信号的亮度:1型细胞（DA细胞）位于显示弱荧光（A，见箭头）的INL和强烈的标记2型细胞INL（A，见箭头）或在保利协鑫（B）和树突状分层阶层的IPL S3（三）在流离失所的细胞体。比例尺，50微米。 <img alt="图3" src="/files/ftp_upload/3457/3457fig3.jpg" /> 图3 2型细胞注射示踪Neurobiotin的形态。示踪剂后来可视化链亲和素- Cy3标记的绑定（洋红色）。的显微照片，这也是描绘的GFP信号（绿色），是一个覆盖GCL和IPL的形象栈的投影。比例尺，50微米。 <img alt="图4" src="/files/ftp_upload/3457/3457fig4.jpg" /> 图4。位于保利协鑫2型细胞对光反应。在暗的范围内增加强度的照明白光满场的响应模式。刺激强度是每秒每杆（RH * /棒/ S）photoisomerizations。用于更好的不同的是一个3秒的长时间刺激离子刺激发病响应元件（ON响应）和偏移量（OFF响应）。

<ol>
	<li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat. Neurosci. 4, 877-886 (2001).</li><li>Baccus, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+computation+in+inner+retinal+circuitry.">Timing and computation in inner retinal circuitry.</a> Annu. Rev. Physiol. 69, 271-290 (2007).</li><li>Haverkamp, S., Inta, D., Monyer, H., Wässle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+analysis+of+green+fluorescent+protein+in+retinal+neurons+of+four+transgenic+mouse+lines.">Expression analysis of green fluorescent protein in retinal neurons of four transgenic mouse lines.</a> Neuroscience. 160, 126-139 (2009).</li><li>Siegert, S., Gross-Scherf, B., Del Punta, K., Didkovsky, N., Heintz, N., Roska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+address+book+for+retinal+cell+types.">Genetic address book for retinal cell types.</a> Nat. Neurosci. 12, 1197-1204 (2009).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Euler, T., Detwiler, P. W., Denk, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directionally+selective+calcium+signals+in+dendrites+of+starburst+amacrine+cells.">Directionally selective calcium signals in dendrites of starburst amacrine cells.</a> Nature. 418, 845-852 (2002).</li><li>Sakmann, B., Neher, 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> Single-Channel Recording. , (1995).</li><li>Jackson, M. B., Gerfen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+voltage+clamp+recording.">Whole-cell voltage clamp recording.</a> Current Protocols in Neuroscience. , (1997).</li><li>Majewska, A., Yiu, G., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom-made+two-photon+microscope+and+deconvolution+system.">A custom-made two-photon microscope and deconvolution system.</a> Pf&#252;gers Arch. - Eur. J. Physiol. 441, 398-408 (2000).</li><li>Yuste, R., Konnerth, 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> maging in Neuroscience and Development: A Laboratory Manual. , (2005).</li><li>Euler, T., Hausselt, S. E., Margolis, D. J., Breuninger, T., Castell, X., Detwiler, P. B., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyecup+scope+-+optical+recordings+of+light+stimulus-evoked+fluorescence+signals+in+the+retina.">Eyecup scope - optical recordings of light stimulus-evoked fluorescence signals in the retina.</a> Pfl&#252;gers Arch. - Eur. J. Physiol. 457, 1393-1414 (2009).</li><li>Wei, W., Elstrott, J., Feller, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+targeted+recording+of+GFP-expressing+neurons+for+light+responses+and+live-cell+imaging+in+the+mouse+retina.">Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina.</a> Nat. Protoc. 5, 1347-1352 (2010).</li><li>Matsushita, N., Okada, H., Yasoshima, Y., Takahashi, N., Kiuchi, K., Kobayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+tyrosine+hydroxylase+promoter+activity+during+midbrain+dopaminergic+neuron+development.">Dynamics of tyrosine hydroxylase promoter activity during midbrain dopaminergic neuron development.</a> J. Neurochem. 82, 295-304 (2002).</li><li>Knop, G. C., Feigenspan, A., Weiler, R., Dedek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inputs+underlying+the+ON-OFF+light+responses+of+type+2+wide-field+amacrine+cells+in+TH-GFP+mice.">Inputs underlying the ON-OFF light responses of type 2 wide-field amacrine cells in TH-GFP mice.</a> J. Neurosci. 31, 4780-4791 (2011).</li><li>Zhang, D. Q., Stone, J. F., Zhou, T., Ohta, H., McMahon, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+genetically+labeled+catecholamine+neurons+in+the+mouse+retina.">Characterization of genetically labeled catecholamine neurons in the mouse retina.</a> Neuroreport. 15, 1761-1765 (2004).</li><li>Contini, M., Lin, B., Kobayashi, K., Okano, H., Masland, R. H., Raviola, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+input+to+ON-biploar+cells+onto+the+dopaminergic+neurons+of+the+mouse+retina.">Synaptic input to ON-biploar cells onto the dopaminergic neurons of the mouse retina.</a> J. Comp. Neurol. 518, 2035-2050 (2010).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat. Methods. 2, 932-940 (2005).</li><li>Dedek, K., Breuninger, T., de Sevilla M&#252;ller, L. P., Maxeiner, S., Schultz, K., Janssen-Bienhold, U., Willecke, K., Euler, T., Weiler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+type+of+interplexiform+amacrine+cell+in+the+mouse+retina.">A novel type of interplexiform amacrine cell in the mouse retina.</a> Eur. J. Neurosci. 30, 217-228 (2009).</li><li>Denk, W., Detwiler, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+light-evoked+calcium+signals+in+the+functionally+intact+retina.">Optical recording of light-evoked calcium signals in the functionally intact retina.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 7035-7040 (1999).</li></ol>

小鼠进行处理，并按照指引，对动物福利机构，由德国政府颁发的动物实验的法律安乐死。

这种方法提供了可能性研究的完整的视网膜视觉引导下的具体神经元的电性能，不影响视网膜adaptational条件。它特别适合于细胞，目前不佳而由于人口密度低，像大多数人口的无长突细胞研究的表征。双光子激发允许高分辨率和高对比度的影像，甚至较深部位的组织17，一个准确的定位的前提和成功的膜片钳细胞，特别是在其细胞体的高密度的INL。 孤立的小鼠视网膜是可行的实验条件下3-4小时。如果第二个视网膜下连续carbogen毒气完全黑暗中，它保留紫色的原色感光色素，可用于后整理与第一视网膜的实验。在开始的时候，针对选择与在视网膜wholemount微量细胞是一个挑战，需要一些练习，尤其是当在黑暗中工作。包括在微荧光染料，可以简化程序，因为细胞和微管是在同一时间的双光子激发下可见。然而，将组件添加到细胞内的解决方案可能阻碍gigaseal形成或导致录音的质量遭受。一旦成功地实现了，一个好的记录可以持续约1小时而红外激发光本身仅仅是视网膜感光细胞吸收不足，兴奋表达GFP的细胞发出的光在可见光谱部分。然而，荧光团很高兴，只有在一个小焦量，是不会改变视网膜adaptational条件。所有的多，激发只需要为目标，录制的光反应时，可以切换。 该usefulnESS是已经证明这种方法通过研究特定人群的无长突细胞14,18从电生理记录，否则会一直只能由事故12。最后，这个强大的技术，可以进一步扩展，包括药理学方法 14， 钙离子成像 19或使用注射的细胞免疫研究或电子显微镜。通过这种方式，一个特定类型的细胞在视网膜的电路的功能定位可以揭开。

<table border="1"><tbody><tr><th>试剂/设备</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>夜视镜</td><td> Gutzeit有限公司，Warthausen，德国</td><td> Xtron的F1 </td><td>包括一个800纳米光源</td></tr><tr><td>光学过滤器</td><td>肖特，美因茨，德国</td><td> RG9 </td><td>在690纳米，长通与截止过滤</td></tr><tr><td>虹膜剪刀</td><td>精细科学工具</td><td> 14061-09 </td><td>与22毫米刀片弯曲</td></tr><tr><td>春季剪刀</td><td>精细科学工具</td><td> 15000-00 </td><td>直3 mm的螺丝刀</td></tr><tr><td>录音室</td><td> Luigs诺依曼有限公司，德国拉廷， </td><td> 200-100 500 0180 A型（TC） </td><td>底部安装玻璃盖玻片聚四氟乙烯室（约0.15毫米; 200-100 500 0182号） </td></tr><tr><td>流量热水器</td><td>多声道系统，德国罗伊特林根， </td><td> PH01，TC01 </td><td>与温度小号可加热灌注导管恩索尔和温度控制器</td></tr><tr><td>激光扫描显微镜</td><td>徕卡显微系统</td><td>马克的LFS </td><td>徕卡共聚焦软件控制</td></tr><tr><td>空气球台</td><td>新港</td><td> VH 3036W - OPT </td><td></td></tr><tr><td>激光</td><td>光谱物理</td><td></td><td>海啸锁模钛蓝宝石激光</td></tr><tr><td> CCD相机</td><td> PCO公司，德国，Kelheim </td><td> PixelFly QE </td><td>包括控制软件</td></tr><tr><td>微管玻璃毛细管</td><td> Hilgenberg，Malsfeld，德国</td><td> 1408411 </td><td></td></tr><tr><td>微管拉拔</td><td>萨特仪器</td><td> P - 97 </td><td></td></tr><tr><td>的Alexa Fluor 594 </td><td> Invitrogen公司</td><td> A - 20004 </td><td>荧光染料</td></tr><tr><td> Neurobiotin </td><td> Axxora </td><td> VC - SP - 1120 - M050 </td><td>非荧光示踪</td></tr><tr><td>链霉菌- Cy3标记</td><td> Dianova </td><td> 016-160-084 </td><td></td></tr><tr><td>麦克风romanipulator </td><td> Luigs诺依曼有限公司，德国拉廷， </td><td> 210-100 000 0010 </td><td>电动Mini25操盘单元与显示SM - 5 </td></tr><tr><td>膜片钳放大器</td><td> NPI电子有限公司，德国，塔姆</td><td> SEC - 05LX NPI </td><td></td></tr><tr><td>数字化仪</td><td>美国国家仪器公司</td><td> BNC - 2090 </td><td></td></tr><tr><td>数据采集​​软件WinWCP </td><td>约翰登普斯特，苏格兰，英国格拉斯哥大学</td><td></td><td> http://spider.science.strath。 ac.uk / sipbs / software.htm </td></tr><tr><td>视觉刺激发生器量子点</td><td>托马斯欧拉，德国蒂宾根大学</td><td></td><td>要在一个单独的控制2台显示器（用户界面，刺激显示器）的计算机操作</td></tr><tr><td>中性密度​​滤光片</td><td> ITOS，美因茨，德国</td><td></td><td></td></tr></tbody></table>

electrophysiological characterization of gfp-expressing cell populations in the intact retina

学习的生理特性和完整的组织中的特定神经元的突触连接，这些细胞缺乏突出的形态特征，或显示一个人口密度低，是一种挑战。这尤其适用于视网膜无长突细胞，interneurons，包括大约30亚型在哺乳动物1分外多种形式类。虽然被塑造视网膜输出2的视觉处理的重要组成部分，这些亚型没有研究到现在功能方面，因为遇到这些细胞与记录电极是一种罕见的事件。 最近，众多的转基因小鼠线是可用的表达像促销员的控制下的绿色荧光蛋白（GFP）的膜受体或酶，是具体 ​​的，只有在一个给定的组织3,4神经元的一个子集的荧光标记。这些预标记细胞，因此accessiblE定向微电极定位的微观控制下，允许他们在原地的生理特性的系统的研究。然而，激发的荧光标记物是伴随着风险的光毒性的活组织。在视网膜上，这种做法是另外的问题阻碍，激发光引起的光感受器适当的刺激，从而造成感光色素，漂白和光适应条件转移到视网膜电路。克服了这些缺点是使用一个锁模飞秒短脉冲激光红外激发交付。双光子激发提供的能量足够激发荧光基团，并在同一时间限制激发到一个小的组织体积减少光损伤5危害。此外，叶以来未能photopigments 6吸收红外光（850纳米）的视觉刺激反应的视网膜。 达到原位录音电从绿色荧光蛋白表达细胞在视觉上有针对性的双光子激发。视网膜是在黑暗中准备和维护，并可以通过显微镜的冷凝器（图1）预计的光刺激。膜片钳记录的光反应，可与染料填充揭示的形态和检查耦合到邻近的细胞间隙连接介导的染料，使靶细胞可以通过不同的实验水平上研究。

Watch this Scientific Journal Video about Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina at JoVE.com

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

本文描绘出单个细胞荧光标记完好的小鼠视网膜神经人口的记录。通过使用双光子红外激发transgenetically标记的细胞是有针对性的膜片钳记录，研究他们的光反应，感受野特性和形态。

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

electrophysiological-characterization-gfp-expressing-cell-populations

Research

JoVE Journal

Neuroscience

13.4K 次观看.  University of Oldenburg. 本文描绘出单个细胞荧光标记完好的小鼠视网膜神经人口的记录。通过使用双光子红外激发transgenetically标记的细胞是有针对性的膜片钳记录，研究他们的光反应，感受野特性和形态。

视频: Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

脑植入的微型器件及周围组织的完整的组织学特性研究

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

从开发小脑显微切割分离的不同的细胞群

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.