This work was supported by the Deutsche Forschungsgemeinschaft DFG FOR701 and FE464/11-1 to A.F. The authors want to acknowledge the excellent technical assistance of Andrea Nerz.

所有程序都根据用于由德国联邦共和国发出动物实验的准则进行。 注意:由于视网膜组织将在此过程中的延长部分是无氧，所有步骤应尽可能快地进行。小鼠应该暗适应解剖前至少3小时。以避免视网膜，制备的光适应和切片应在昏暗的红光下进行。 1.媒体和其他化学品的制备<ol><li> 准备艾姆斯介质6。解剖后，沉浸视网膜组织埃姆斯"在任何时候都网上平台。 <ol><li>溶解2.2克艾姆斯培养基和298毫克的HEPES在250ml蒸馏水中。 HEPES浓度为5毫米。轻轻搅拌，直到所有的颗粒溶解。 </li><li>气泡艾姆斯介质在室温下用卡波金（95％氧气，5％的车盂兰盆氧化钛）为至少5分钟。供给卡波金用玻璃巴斯德吸管到溶液中。经由适当的管道的巴斯德吸管连接至气体调节器的出口。 </li><li>添加475毫克钠 - 碳酸氢盐溶液中。碳酸氢盐的起泡后加入防止碳酸钙的沉淀。 </li></ol></li><li> 制备低凝胶温度琼脂糖。琼脂糖需要vibratome切片期间视网膜组织包埋。 <ol><li>溶解于9ml埃姆斯"媒介180毫克琼脂糖和1ml蒸馏水。使用微波，直至所有固体溶解缓慢加热该琼脂糖溶液。热火在5 - 10秒为增量，以避免溢出。溶液应澄清透明。 </li><li>商店在水浴琼脂糖溶解在37℃的过程的持续时间。 </li></ol></li></ol> 2.设置Vibratome 注:所有切片已用进行在材料表vibratome上市。给定的设置指这类只vibratome的。有关神经组织vibratome切片进一步详情，请参阅参考资料7。 <ol><li>将vibratome喷油器刀片插入刀座。 </li><li>测量用"vibrocheck"装置中的叶片的垂直偏转。使用的刀片保持器的调节螺钉来倾斜叶片，直到它平行于切割平面。叶片的精确定位是必要的，以防止部分表面上百叶影响，并产生一个平坦部分，它包含在其表面上活细胞。 </li><li>设置vibratome以下设置:振幅，1.00;速度，0.20;厚度，180 - 200微米。 </li></ol> 3.鼠标视网膜解剖<ol><li>由约1ml异氟烷气化在气密容器麻醉成年C57BL / 6小鼠。当麻醉完成后，由颈椎脱位安乐死的动物。该鼠标不应早于3个月，否则片的质量将变差。 </li><li>通过放置一个弯钳眼睛下方取出眼球。轻轻抬起眼球会露出视神经。切用弹簧剪刀视神经和眼球转移到含2ml埃姆斯&#39;培养基在35毫米培养皿中。 </li><li>开展立体显微镜的阶段的所有后续步骤。调整放大倍数值使得下面的过程（步骤3.4 - 3.9）最佳的视觉控制。 </li><li>穿刺角膜在过渡到与尖镊子巩膜或20G的针。在此步骤中，稳眼球与另一个钳。要小心，不要损坏穿刺装置视网膜。 </li><li>将弹簧剪刀到在步骤3.3所做的小孔，减少角膜周围一圈。此外，稳健的眼球用钳子切割时。保持切口非常接近表面，以便不至cUT斯达康到视网膜。 </li><li>小心取出角膜，晶状体和玻璃体钳。玻璃体包括在眼睛后部仅一薄透明层。但是，它是用于vibratome切片的玻璃体完全除去必要的。 </li><li>在陪替氏培养皿，在某种程度上一个看起来通过孔向下到视网膜上的位置眼罩。开口的周缘应仅由巩膜组织。如果该圆形切口（步骤3.4）已进行了太靠近眼罩的赤道，视网膜组织也将存在接近切口。千万要注意了，不要破坏步骤3.8视网膜。 </li><li>抓住用钳子巩膜，仔细撕裂组织。不要在此步骤破裂视网膜至关重要。如果需要的话，通过用钳子稳住组织的巩膜部分，并仔细剥离与其他镊子视网膜脱离从锯齿缘视网膜。保持镊子关闭;从来没有办理再蒂娜与自组织钳很柔软，这样会马上给。 注:在这一步骤中，在视网膜和眼罩的其余部分将只在视神经头相连。 </li><li>切断附着位点在视神经乳头与弹簧剪刀。视网膜现在应该自由浮动在艾姆斯中单件的组织。 </li><li>通过举办视网膜随着钳的边缘，它操纵到位的使用弯曲手术刀片约23毫米6小矩形 - 切视网膜成4。避免夹持矩形内视网膜组织。取从视网膜，而不是远周边的中央部分件。 </li><li>转移视网膜件具有大直径的塑料巴斯德吸管35毫米的塑料陪替氏培养皿，其底部的已被取代的具有网格尺寸小于1毫米的网格。固定烧杯或锥形瓶中的上三分之一内培养皿并在埃姆斯&#39;中蒲式耳淹没视网膜件bbled与卡波。 </li></ol> 4.嵌入视网膜饮片琼脂糖<ol><li>小心地转移2 - 3视网膜片放入35毫米的塑料培养皿连同一些艾姆斯媒介。它是最方便使用的大直径的塑料巴斯德吸管转让在该步骤中，视网膜片的确切取向并不重要。 </li><li>用玻璃巴斯德吸管除去尽可能剩余艾姆斯培养基。一旦流体已被删除与步骤4.3进行。不要让视网膜组织干的。 </li><li>立即覆盖视网膜片用2ml琼脂糖溶解（37℃）。定在步骤3.10中提到的尺寸，片不会在琼脂糖内卷曲。 </li><li> 内的液体琼脂糖，轻轻地推视网膜件培养皿的底部。神经节细胞层应朝下。使用立体显微镜的精确位置的判断。为定位，处理视网膜PI欧洲经委会用小铲。视网膜定位为快速，精确地因为琼脂糖会很快凝固。 <ol><li>安排平行于玻璃盘底部视网膜件;否则部分将切或斜。单件不应该接触对方，但也不宜相距甚远也。尝试把视网膜碎片上同一水平更多或更少。 </li></ol></li><li>把玻璃培养皿嵌入视网膜片放入冰箱（4 - 6℃）至少2分钟。这将迅速变硬，以便进一步处理的琼脂糖。一旦琼脂糖聚合下一步骤继续进行。 </li></ol> 5.安装视网膜饮片<ol><li>小心取出含有从培养皿视网膜个琼脂糖块。用刮刀或类似设备，以解除从玻璃琼脂糖块。由于粘合力，这可以证明是相当困难的。但是，避免折断琼脂糖块。进行在室温下这和所有后续步骤。 </li><li>放置在玻璃盖玻片琼脂糖块，并用手术刀刀片调整其大小。留下1 - 在视网膜件每一边琼脂糖2毫米。块的最终尺寸应约为1066毫米（10毫米指块，6mm至其边缘的长度的高度）。 </li><li>用胶水瞬间胶粘剂琼脂糖块到vibratome的试样架。视网膜件应该位于10毫米高的块的顶部和取向平行于它的表面，而底部固定在支架上。 </li><li>通过倾倒先前鼓泡艾姆斯中等入vibratome的缓冲盘，立即覆盖块。 </li></ol> 6.准备水平Vibratome部分<ol><li>开始切片。首先，设置手动切片的厚度直至视网膜组织的片段出现在该切片。然后将设置更改为在步骤2.3。由于小鼠视网膜仅约200微米厚，就会有不超过2 - 每琼脂糖块3片。 </li><li>从用玻璃棒缓冲纸盘中取出切片，并将它们在35毫米培养皿转移至2ml艾姆斯培养基。 </li><li>商店切片立即在37℃（5％二氧化碳/ 55％O 2）的培养箱。周围部分中的琼脂糖会留在培养箱固体和可用于处理的部分，而不会损坏视网膜。相比氧化艾姆斯在RT中的各自气氛中，在37℃下储存时，会获得更好的片质量。部分可以立即使用或可被存储到4小时中具有良好的生理效果的孵化器。 </li><li>嵌入在琼脂糖和重复第4下一视网膜片 - 6。 </li></ol>

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The authors have nothing to disclose.

在对比垂直切片，如水平细胞和无长突细胞径向定向神经元的树突形态保存完好。相比于整个视网膜的准备工作，这些细胞类型是微电极方便且可由两个电生理和成像技术进行研究。在这里，我们专注于视网膜外层;然而，类似的方法已经报道尤其用于内视网膜10的无长突细胞。 水平切片准备的属性有功能性研究的几个问题。锥感光细胞和突触后水平细胞之间的突触联系的数量从完整的视网膜几乎没有区别。突触接触是功能性的，显示出在黑暗中神经递质的释放进补。突触可以通过内源的刺激像光/暗转换或由外部双极刺激被激活。 这里提出的技术大多局限于水平定向神经元。因此，不可能从感光细胞和完整双极细胞记录在水平片段中，由于双极细胞轴突将被截断。另外，也不可能完全控制切片的级别。一个单一的视网膜一块米飞行从而在不希望的水平被削减，对于光感受器的外表面和内区段之间实例。增加在一块至少切片的一个正确平面的琼脂糖增加机会的一个块内的视网膜的件数。 的水平片段的准备是非常适合于学习杆和视锥感光细胞的带状突触的独特性能。到目前为止，我们已经使用的动物基因敲除系统来调查突触前蛋白质complexin 3和锥感光细胞和水平细胞11之间的突触传递complexin 4的作用。另外，也可以研究突触后神经递质受体的特性，如前面9所示。 未来的应用将包括基于与电生理和成像技术的转基因小鼠系突触蛋白的研究。它也将是可能的调查生物物理亲水平细胞之间的缝隙连接的perties。由于光响应可以被测量，该技术也将被用于在视觉系统的第一突触到研究内源性刺激的编码。它是可以进行在黑暗的制备，以防止组织的光适应，否则光响应将在很大程度上锥驱动的。 总之，在水平切片准备代表一种新颖的方法，以增加个体突触的辅助，以在哺乳动物视网膜12研究它们的功能性质的先决条件。

视觉信息被编码为感光体活化的动态空间 - 时间模式，及光感受器和二阶神经元之间的色带突触确定的视觉信息的下行传输。哺乳动物视网膜的垂直切片制备是高度有价值的工具来研究在垂直通路的信号处理， 即感光细胞和双极细胞之间，和之间双极细胞和某些类型的无长突细胞1，2，3，4。 然而，垂直切片总是导致许多视网膜神经元的树突领域的严重截断，导致突触联系的相当大的损失。特别是在外侧视网膜，水平细胞由于它们的横向扩展的扩展树突字段和轴突终端系统5的影响。在垂直因而切片制备，数百锥感光体终端到水平细胞的树突的突触输入被降低到几稀疏联系人。这可能足以研究单个突触的性质，但是它确实绝非表示感光体色带突触的同步激活基础的信号处理能力。 因此，我们开发了一个横向切片准备，这让几乎所有的锥感光的突触输入水平细胞树突的完整和功能。平行于视网膜表面切片运行的平面上，通过水平细胞和无长突细胞之间的内核层理想地切割。因此，外视网膜的水平片段创建含有上突触前部位，用内外段以及突触终端，水平细胞和双极细胞上的突触后现场光感受器。这种准备是非常适合ŤØ通过其树突领域内的所有锥感光细胞研究协调突触的投入水平细胞树突。它因此可能证明是有用的，以便更好地理解在感光色带突触，这是视觉信息的传送从感光体活化的基质成突触后响应关键的运作。

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; Medium</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A1420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbogen</td>
			<td style="width:125px;">Air Liquide</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A9045</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">35-mm petri dish (plastic)</td>
			<td style="width:125px;">Nunc Thermofisher</td>
			<td style="width:119px;">150318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass petri dish</td>
			<td style="width:125px;">Chemoline</td>
			<td style="width:119px;">50-1470-40</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Isoflurane</td>
			<td style="width:125px;">Dispensary university hospital</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome injector blade</td>
			<td style="width:125px;">Biosystems</td>
			<td style="width:119px;">39053250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibratome</td>
			<td style="width:125px;">Leica Microsystems</td>
			<td style="width:119px;">VT1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibrocheck</td>
			<td style="width:125px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microwave</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20-gauge needles</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spring scissors</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Curved scalpel blades</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic Pasteur pipette</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Pasteur pipette</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of horizontal slices of adult mouse retina for electrophysiological studies

垂直切片准备工作已经非常成熟，研究电路和信号传输在成年哺乳动物的视网膜。这些制剂切片的平面垂直于视网膜表面，使其成为理想的像光感受器和双极细胞径向定向神经元的研究。然而，水平细胞，宽视场的无长突细胞和神经节细胞的大树突乔木大多截短，留下显着降低突触活动在这些细胞中。而神经节细胞和无长突移位细胞可以在视网膜的一个整体安装的制剂进行研究，水平细胞和位于内核层无长突细胞是只很差在整个视网膜组织电极访问。 为了实现最方便的访问和突触的完整性，我们开发了小鼠视网膜的水平切片准备，并就读于光感受器和水平之间的突触信号传递细胞。横切片允许（1）容易和明确为电极定位水平细胞体的视觉识别，和（2）保存在扩展水平细胞树突的字段，作为完整且功能锥体突触输入到水平细胞树突的先决条件。 从水平切片水平细胞表现出补品突触活动在黑暗中，他们回答了介绍光闪烁以减少内向电流和减弱突触的活动。免疫细胞化学证据表明，水平细胞的树突领域内几乎所有的锥体建立与周边树突突触。因此，水平片段制备是公适合研究水平延伸视网膜神经元的跨越选定突触生理特性以及传感信号传输和整合。

<html>在小鼠视网膜的水平片段，水平细胞体可以很容易地根据它们与几个主要树突从多边形细胞体（ 图1A）的新兴独特的形态识别。位于切片的表面附近水平细胞机构是对膜片钳电极例行访问。期间记录，将细胞填充有荧光染料，揭示其复杂树突分支，这非常类似于完整小鼠视网膜8（ 图1B）的轴突承载水平细胞的形态。用荧光素偶联花生凝集素锥体椎弓根的标记显示锥体椎弓根和水平细胞树突（ 图1C）之间的密切的空间关系。除了少数例外，水平细胞的树突领域内的所有锥蒂被树枝状突起联系。 ENT"FO:保together.within页="1"&gt;为了证明锥和水平细胞之间的接触是功能性的，膜片钳记录从水平细胞机构开展在暗适应条件下，目前的反应是。其特征在于由一个大内向电流伴有中度至高频突触活性（ 图2A）。从基线和偏差的兴奋性突触后电流被谷氨酸在黑暗中恒定的释放引起的，激活突触后AMPA-和红藻氨酸盐型谷氨酸盐受体9。由一个单相波形或更复杂的电流轨迹（ 图2B）。补品突触后活动的存在表明，谷氨酸在图1C所示锥感光细胞和水平细胞树突之间的突触联系释放个别突触事件进行了表征。 Horizo记录ntal细胞响应于光强度的变化，这表明在锥体光感受器的信号转导级联也完整且功能性的水平切片制备。由于递质释放的减少，补虚向内暗适应水平细胞的电流抑制，突触活动满场光刺激（1300 瓦 /平方厘米，470 40纳米）的存在（ 图中出现了明显减少图3A）。同样地，一个开关以从光适应状态下的黑暗引起内向电流和增加的突触活性（ 图3B）。另外，简短电脉冲（1-15伏，1毫秒）通过铂 - 铱双极刺激电极被施加到去极化所有锥光感受器在水平细胞体的附近。谷氨酸的刺激诱导的释放在记录水平细胞（ 图3C）诱发大振幅兴奋性突触后电流。以summary，这些发现令人信服表明锥感光细胞和水平细胞间的突触可通过任一的内源性刺激物的光或由突触前元件的受控去极化实验操纵。 <img alt="图1" src="/files/ftp_upload/55173/55173fig1.jpg" /> 图1:形态和水平切片准备水平细胞的突触联系。位于切片的表面附近的一个水平细胞（箭头）与Dodt对比度光学可视化。比例尺= 10微米（A）。用于成像实验中，水平细胞经贴剂电极，它仍然连接到该单元填充有俄勒冈绿488 BAPTA-1（100μM）。比例尺=10μm的（B）中。的染料注入水平细胞和用荧光素标记偶联花生凝集素锥体椎弓根的阵列的重叠揭示推定的突触联系。<html>比例尺=10μm的（C）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55173/55173fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55173/55173fig2.jpg" /> 图2: 从一个水平细胞体记录突触活动。在-60 mV的钳制电压全细胞电压钳记录显示高频补品突触活动（A）。在较高的时间分辨率（B）中，突触事件显示单相波形（箭头）或更复杂的电流轨迹（箭头）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55173/55173fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> g3.jpg"/&gt; 图3:由光与电刺激诱发水平的细胞体的电流响应。在-60毫伏记录保持电位的暗适应水平细胞体的光响应。全视场照明抑制补品内向电流，从而降低突触活动（A）。在同等条件下录制的光适应水平细胞体显示以下的黑暗（B）期间伴随增加突触活动深色闪光内向电流。在A和B水平线代表经济刺激的时机。通过短暂电脉冲的细胞外刺激诱导大振幅的兴奋性突触后电流由于谷氨酸从锥光感受器的同步释放（℃）。保持电位:-60 mV的。箭头指示的刺激脉冲的定时。在跟踪开始截断电流代表的刺激文物。.COM /文件/ ftp_upload / 55173 / 55173fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies at JoVE.com

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

我们开发了成年哺乳动物视网膜的水平切片准备具有平行切片于视网膜表面的平面。非径向导向的视网膜神经元树突领域并没有截断，使信号处理与膜片钳和成像技术研究。

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 ...

preparation-horizontal-slices-adult-mouse-retina-for

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.0K 次观看.  Friedrich-Alexander-University (FAU) Erlangen-Nürnberg. 我们开发了成年哺乳动物视网膜的水平切片准备具有平行切片于视网膜表面的平面。非径向导向的视网膜神经元树突领域并没有截断，使信号处理与膜片钳和成像技术研究。 

视频: Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

器官型培养的全厚成年猪视网膜

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

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 ...

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

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

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 ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the clear majority have been performed on dissociated primary cells. To improve our understanding of how teleost pituitary cells, behave in a more biologically relevant environment, this protocol shows how to prepare viable brain-pituitary slices using the small freshwater fish medaka (Oryzias latipes). Making the brain-pituitary slices, pH and osmolality of all solutions were adjusted to values found in body fluids of freshwater fish living at 25 to 28 &#176;C. Following slice preparation, the protocol demonstrates how to conduct electrophysiological recordings using the perforated whole-cell patch-clamp technique. The patch-clamp technique is a powerful tool with unprecedented temporal resolution and sensitivity, allowing investigation of electrical properties from intact whole cells down to single ion channels. Perforated patch is unique in that it keeps the intracellular environment intact preventing regulatory elements in the cytosol from being diluted by the patch pipette electrode solution. In contrast, when performing traditional whole-cell recordings, it was observed that medaka pituitary cells quickly lose their ability to fire action potentials. Among the various perforation techniques available, this protocol demonstrates how to achieve perforation of the patched membrane using the fungicide Amphotericin B.

The article describes an optimized protocol for making viable brain-pituitary tissue slices, using the teleost fish medaka (Oryzias latipes), followed by electrophysiological recordings of pituitary cells using the patch-clamp technique with the perforated patch configuration.

健康脑-垂体切片对硬骨鱼类鱼垂体细胞电生理的研究

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...