这项工作是支持的澳大利亚研究理事会（ARC DP0988227）和生物医学研究计划资助的生物医学科学学科，悉尼大学。设备膜片钳放大器是由EPC 8的启动资金从悉尼大学生物医学科学学科。从Rebecca L.库珀从悉尼大学生物医学科学学科的基础和启动基金的资金购买设备InstruTech产品立8 +8的数据采集系统。我们想感谢匿名审稿人，他们有见地的建议和意见。

1。一般设置和组织准备<ol><li>按住鼠标在黑暗中30分钟（旨在减少其压力水平）。在等待时，准备外液1升。首先Milli-Q水半升碳酸氢钠1.9克溶解。通过鼓入95％O 2和5％CO 2，它的pH值保持在7.4。五分钟后，溶解8.8克艾姆斯中等Milli-Q水100毫升，加入碳酸氢钠溶液，Milli-Q水1升，拌匀。保持本方案的carboxygenated实验的其余部分。 </li><li>艾姆斯中carboxygenated 250毫升，并成立了流体灌注系统在电设置。保持解决方案carboxygenated。 </li><li>均匀地涂抹一层薄薄的真空脂灌注室的底部上。密封盖玻片，并确保它是不透气的。取少量润滑脂（〜2毫米的直径），并将其定位在顶部和底端室。这些球将举行电网（铂框架串成的牙线）到位，并防止它在视网膜上按太硬。修复室的平台上，并对齐两个开口。 </li><li>第一异氟醚麻醉，持续2分钟，然后颈椎脱臼处死动物。快速消除眼睛用一双小剪刀（弯叶片），彻底清洗与carboxygenated细胞外液小烧杯中，然后放置在一个充满与carboxygenated细胞外液的培养皿。 </li><li>解剖显微镜下，用19号针头在角膜上的插通孔（〜2 mm从边缘）。重复另一只眼睛。这一重要步骤，应该是迅速允许双方视网膜氧合。删除一个角膜虹膜剪一双小平行切割边缘。接下来，取出一双细镊子的光圈和镜头。重复另一只眼睛。一只眼杯转移到烧杯中含有细胞外液carboxygenated。 </li><li>用手术刀刀片，切到一半，一只眼杯能够扁平化的整个安装，并获得两个全坐骑每视网膜。小心地分离视网膜色素上皮细胞，用钝的解剖探头轻轻拉动它。一旦脱离，视网膜是相当透明的。请注意的是凸面侧的光感受器。的凹面是神经节细胞侧，它可能会粘在粘玻璃体液的存在下。 </li><li>一双细镊子，保持分离的视网膜组织接近的边缘锯齿缘，抢锯齿缘，另一对细镊子，在视网膜的中心向外拉出。这是一个微妙的过程，可能需要几分钟的尝试。如果成功的话，锯齿缘，睫状体和玻璃体液过程去除的，和在视网膜的曲率减小（ 即平坦）。 </li><li>修剪的边缘，然后将其转移到每神经节细胞融合室朝上。保持组织到位，通过放置在网格上的油脂球。将剩余的组织与其他眼杯以供以后使用。视网膜剥离中描述的类似程序，米德尔顿和16普罗蒂Pottek 等 17。 </li><li>录音室安装一个堂堂正正的显微镜下。立即成立的视网膜与carboxygenated细胞外液中（35.5℃）的速率为3 - 5毫升/分钟的连续灌注。确保接地电极地方。 </li><li>连接到显微镜是一种数码相机，允许可视化的组织。整套起来坐在上面的空气表（减震）和一个法拉第笼（块外部静态和非静态电场）内。 </li><li>打开光源（红外，850纳米）和40X的目标，将其集中到神经节细胞的胞体。寻找中心区的整个安装RECOR小丁。 </li><li>解冻冷冻小瓶中K +的细胞内液（300微升，pH值= 7.4）含有（以mM）K-葡糖酸：140，HEPES酸：10，MgCl 2的4.6，ATP-Na的+：4，GTP-钠+ ：0.4和EGTA：10。所有试剂购自Sigma-Aldrich公司。 </li></ol> 2。修补视网膜神经节细胞的胞体<ol><li>第一把火擦亮硼硅玻璃毛细管管的两端，然后拉他们两个加热步骤（电阻：5 - 8MΩ）用玻璃微电极拔轮器。 </li><li>加入3微升路西法黄河（终浓度为0.2％），细胞内的解决方案，拌匀，离心。接着，最高的85％的溶液转移到1 ml注射器，将它连接到0.22μm的f ILTER单元，然后到30 G的可重复使用的皮下注射针。冷藏。 </li><li>修复玻璃吸管，用于录音的那些类似的针尖大小，在其持有人，并在显微镜下移动使用micromanipula器。 40X的目标下，慢慢地降低吸移管，直到它使一个小凹坑内界膜的表面上。吸管小心地向前推进，直到它捕获的少量组织，然后将它向上和/或侧身撕了一个小洞，露出神经节细胞的胞体。孔越小，较高的程度的视网膜网络的完整性被保持。如果需要，磺基若丹明101（SR101 0.5 - 1μM）可以被添加到细胞外介质的标签损伤的神经元，通过使用荧光显微镜15。 </li><li>填补了一个干净的吸管8 - 细胞内液的10微升。把它插入到连接到放大器的头阶段的持有人，并确保氯化氯化银电极被淹没。丢弃，如果灰尘和/或气泡/本。 </li><li>打开所有的设备上。我们使用的EPC 8膜片钳放大器实现全细胞记录由PatchMaster控制电压钳CON成形。需要注意的是，也可以用其他的放大器，使在快速模式下的电流钳记录。应用内液和保持正压。选择一个单元格与胞体较大，所以最有可能的，它是一个，而不是一个流离失所的无长突细胞或神经胶质细胞，神经节细胞。慢慢地降下移液管尖，所以使膜的表面上的一个小的凹坑，停止，并释放压力。立即降低保持电位 - 60毫伏。一旦gigaseal（GΩ）和被实现它们之间，轻轻一吸，打破膜实现全细胞膜片钳。本程序是一个细致的步骤，如果过分施加负压，则连接变成漏水，如果太少，膜保持完整。 </li><li>随着时间的推移，荧光黄将填充单元格，允许可视化的细胞的形态。如果稳定的话，这种设置应持续30分钟或更多。 </li></ol> 3。神经节细胞的录音使用DYNAMIC型夹<ol><li>首先，在电流 - 电压测试（从 - 75到+35 mV的每10毫伏）使用PatchMaster执行。只有当大的Na +电流（&gt; 1 NA）是目前继续后面的测试。请注意修补流离失所的无长突细胞在罕见的场合，在这种情况下，不会回应列车的动作电位，随后注入电流。 </li><li>打开LabVIEW和执行自定义的书面Neuroakuma程序，然后加载各种电导波形。设置重复次数为8（6 - 8作统计用途）。兴奋性和抑制电导的可逆电位设置在0 - 75毫伏。选择一个配对的兴奋和抑制的电导测试。兴奋电导跟踪（绿色）和抑制电导曲线（红色）将出现在下部面板。 </li><li>转到回PatchMaster，选择电流钳模式，并立即切换放大器“CC +通讯FAST”电流钳模式。此模式允许符合urately遵循快速的膜电位变化。如果保持良好的密封膜之间和吸管，小/无补偿是必要的，否则，记录将不会持续很长时间。在Neuroakuma，按下“记录”按钮。细胞反应（通常是阶段性突发的动作电位， 图1A）将出现在面板上。进入细胞内注入电流低缩放电导第一，如果很少/没有反应观察，以较小的增量增加，直到它产生了强烈的反响。过大的电流注入可以杀死细胞。一旦测试完成后，选择一个新的对的电导波形，重复上述所有对生理和人工电导。注射实时的生理电流（I）的基础上： I（T）= G EXC（T）×（V M（T） - V EXC）+ G INH（T）×（V M（T） - V INH） </li></ol>可能是SY电导（G在NS）naptic或人工。兴奋性和抑制性突触传导波形采集从以前的实验进行普罗蒂，马哥孛罗迪，黄，Vonhoff，阮和所罗门（结果未显示）在控制条件下，以不同的视觉刺激和存在河豚毒素（TTX，1微米） 。人工导波形为蓝本使用阿尔法函数。 V M（毫伏）是记录膜电位。 ĝEXC和G INH分别表示兴奋和抑制电导，而V EXC和V INH代表逆转兴奋性和抑制电导潜力。时间吨毫秒。采样率是40千赫。 I S = 0（T /α）E-αT 上面的方程为α的函数（I 0 =最大电流，1 /α=峰值时间（秒-1）的电流）。的上升时间和衰减时间的突触电流，取决于α4。兴奋性电导抑制电导延时是不变的，而进行了修改，降低其延迟相励磁（ 图2D）的发病。 <ol start="4"><li>录制完成后，仔细收回移液器，所以胞体保持完好。立即固定在4％多聚甲醛固定30分钟，随后由3个10分钟，用0.​​1M磷酸缓冲液洗涤组织。冷藏及以下的一天或一个星期内进行抗体染色。 </li></ol> 4。对路西法黄河填充视网膜神经节细胞的抗体染色<ol><li>抗体染色对路西法黄河充满神经节细胞在室温下（主抗路西法黄河兔IgG（稀释= 1:10000）五天第六天，染色继发的羊抗兔IgG（1:500稀释过夜））。湿摩视网膜荧光保留媒体。盖玻片密封与钉子polish。 </li><li>径使用规格-II的Leica共聚焦显微镜（ 图1B）的共聚焦图像细胞形态的影响。轴突的存在下，使神经节细胞的确认。 </li></ol>

<ol>
	<li>Robinson, H. P. C., Kawai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+digitally+synthesized+synaptic+conductance+transients+to+measure+the+integrative+properties+of+neurons.">Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.</a> Journal of Neuroscience Methods. 49, 157-165 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp:+artificial+conductances+in+biological+neurons.">The dynamic clamp: artificial conductances in biological neurons.</a> Trends in Neuroscience. 16, 389-394 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clamp:+computer-generated+conductances+in+real+neurons.">Dynamic clamp: computer-generated conductances in real neurons.</a> Journal of Neurophysiology. 69, 992-995 (1993).</li><li>Johnson, D., Wu, S. M. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Foundations of cellular neurophysiology. , (1995).</li><li>Taylor, W. R., Vaney, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+synaptic+mechanisms+generate+direction+selectivity+in+the+rabbit+retina.">Diverse synaptic mechanisms generate direction selectivity in the rabbit retina.</a> Journal of Neuroscience. 22 (17), 7712-7720 (2002).</li><li>Jurkat-Rott, K., Lehmann-Horn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+patch+clamp+technique+in+ion+channel+research.">The patch clamp technique in ion channel research.</a> Current Pharmaceutical Biotechnology. 4, 221-238 (2003).</li><li>Prinz, A. A., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp+comes+of+age.">The dynamic clamp comes of age.</a> Trends in Neuroscience. 27, 218-224 (2004).</li><li>Williams, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+compartmentalization+and+functional+impact+of+conductance+in+pyramidal+neurons.">Spatial compartmentalization and functional impact of conductance in pyramidal neurons.</a> Nature Neuroscience. 7 (9), 961-967 (2004).</li><li>Perkins, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-attached+voltage-clamp+and+current-clamp+recording+and+stimulation+techniques+in+brain+slices.">Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices.</a> Journal of Neuroscience Methods. 154 (1-2), 1-18 (2006).</li><li>Feng, S. S., Jaeger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+SK+calcium-dependent+potassium+currents+in+regulating+the+activity+of+deep+cerebellar+nucleus+neurons:+a+dynamic+clamp+study.">The role of SK calcium-dependent potassium currents in regulating the activity of deep cerebellar nucleus neurons: a dynamic clamp study.</a> Cerebellum. 7 (4), 542-546 (2008).</li><li>Butera, R., Lin, R., Destexhe, A., Bal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+factors+for+improving+dynamic+clamp+performance.+In:+Dynamic-Clamp+From+Principles+to+Applications+Series.">Key factors for improving dynamic clamp performance. In: Dynamic-Clamp From Principles to Applications Series.</a> Dynamic-Clamp From Principles to Applications Series: Springer Series in Computational Neuroscience. 1, (2009).</li><li>Marco, S. D. i., Nguyen, V. A., Bisti, S., Protti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+functional+reorganization+of+retinal+circuits+induced+by+early+long-term+visual+deprivation.">Permanent functional reorganization of retinal circuits induced by early long-term visual deprivation.</a> The Journal of Neuroscience. 29 (43), 13691-13701 (2009).</li><li>Zhao, Y., Inayat, S., Dikin, D. A., Singer, J. H., Ruoff, R. S., Troy, 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=Patch+clamp+technique:+review+of+the+current+state+of+the+art+and+potential+contributions+from+nanoengineering.">Patch clamp technique: review of the current state of the art and potential contributions from nanoengineering.</a> Proceedings of the Institution of Mechanical Engineers, Part N: Journal of Nanoengineering and Nanosystems. 222 (1), 1-11 (2009).</li><li>Lobb, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+NMDA+receptor+conductance+in+rat+midbrain+dopaminergic+neurons+using+the+dynamic+clamp+technique.">Application of a NMDA receptor conductance in rat midbrain dopaminergic neurons using the dynamic clamp technique.</a> J. Vis. Exp. (46), e2275 (2010).</li><li>Briggman, K. L., Euler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+electroporation+and+population+calcium+imaging+in+the+adult+mammalian+retina.">Bulk electroporation and population calcium imaging in the adult mammalian retina.</a> Journal of Neurophysiology. 105 (5), 2601-2609 (2011).</li><li>Middleton, T. P., Protti, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+spontaneous+synaptic+activity+in+retinal+ganglion+cells.">Cannabinoids modulate spontaneous synaptic activity in retinal ganglion cells.</a> Visual Neuroscience. 28 (5), 393-402 (2011).</li><li>Pottek, M., Knop, G. C., 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=Electrophysiological+characterization+of+GFP-expressing+cell+populations+in+the+intact+retina.">Electrophysiological characterization of GFP-expressing cell populations in the intact retina.</a> J. Vis. Exp. (57), e3457 (2011).</li></ol>

所有的程序首先由悉尼大学动物护理伦理委员会批准，然后按照澳大利亚守则的护理和使用动物用于科学目的的指引（澳大利亚国家卫生和医学研究理事会进行）。

这里，我们表明的使用，动态钳位评估的兴奋和抑制视网膜神经节细胞的输出的比值和相对定时的影响。动态钳利用计算机模拟引入生理记录或人工活体神经元的突触传导到。这种方法提供了一个互动的工具，通过它可以修改和电导注入神经元计算其影响神经元的反应。能够得到电导波形的视觉刺激实验中，用于激活光感受器的控制条件和药理学试剂的存在下在特定的细胞或电路隔离的贡献。注?...

在视网膜后面的眼睛里，是一个近乎透明的神经组织。许多研究使用视网膜的模型，以探讨视觉处理和突触信号机制的第一步。由于视网膜网络中的整个安装准备解剖后保持不变的，它代表了一个理想的系统研究突触的相互作用，作为其生理反应非常相似， 在体内条件。因此，使用一个孤立的视网膜神经元的特性，可以研究采用膜片钳技术（技术的评论，看到6,9,13）。的识别的确切贡献的具体电路和神经递质的神经节细胞的反应，但是，通常阻碍药理学试剂作用于各种网站。 视网膜神经元的生理反应轻，自然的刺激，可以记录充满了细胞内液与玻璃吸管。使用补丁CL可以记录膜电位的波动（电流钳）或电流（电压钳）放大器技术，神经元对光刺激的反应。通过在不同的电压和实施后验电导分析膜电位，它是未能隔离抑制和兴奋性突触输入5,12。这种类型的实验，可以进行正常沐浴介质中，并在不同的药理剂的存在下，以隔离的贡献不同的神经递质和受体的神经元的反应。从许多实验室的研究特征在于丰富的尖峰输出和兴奋性和抑制输入刺激属性，例如大小，对比度，空间和时间的频率，方向，方位和其他刺激变量的依赖。虽然这些实验方法提供尖峰输出和突触输入功能刺激物业之间的关系的信息，解释特定的细胞类型和细胞的兴奋性突触输入的贡献并不简单。这是由于这样的事实，通常兴奋性和抑制输入随刺激属性，因此，它是无法评估的确切影响，这些输入的变化对神经元扣球。 另一种方法来规避这些限制是开展动态钳记录，这也让个别突触输入输出扣球的贡献的关键评价。动态钳技术允许电流直接注入到单元格，在一个给定的时间依赖于电流注入量的记录，可在那个时候1,2,3（综述见7,14）的膜电位。它是一个修改后的电流钳设置在一个实时记录和下方的单元格的装置，其特征在于，包括专门的硬件，软件之间的相互作用，快速反馈和一台计算机来实现。电流注入到细胞的量的计算。因此，这种方法的优点是可以刺激细胞与电导波形的不同组合，其响应将模仿激活的受体介导的突触输入。例如，注射兴奋性和抑制的一个小点，响应注射兴奋性的一个小点的电导电导响应相比，只提供抑制对细胞响应的影响的信息。同样地，其他的组合生理记录的电导可以共同注入揭示了如何依赖于刺激兴奋性和/或抑制电导的变化会影响尖峰输出。 在我们的研究中，动态钳技术用于演示的相对幅度和时序上的发射性能的视网膜神经节细胞的突触输入的影响。各种电导在控制的条件下或在药理学试剂的存在下得到的生理实验作为输入。此外，人工电导基于阿尔法功能也被用于为了研究如何集成神经元突触输入。因此，这是一个多功能的技术，可以使产生的各种类型的电导生理，药理或计算被注入到相同的神经节细胞，所以这些输入的响应的比较可以。

<table border="1">
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			<td>&nbsp;</td>
			<td>&nbsp;</td>
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			<td>Reagent</td>
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			<td>Isoflurane Inhalation Anaesthetic</td>
			<td>Pharmachem</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ames Medium with L-Glutamate (Powder)</td>
			<td>Sigma-Aldrich</td>
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		<tr>
			<td>Potassium Gluconate, Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>&nbsp;</td>
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			<td>HEPES Sodium salt</td>
			<td>Sigma-Aldrich</td>
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			<td>Magnesium chloride solution (4.9 mol/l)</td>
			<td>Sigma-Aldrich</td>
			<td>&nbsp;</td>
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			<td>Adenosine 5'-triphosphate (ATP) disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>&nbsp;</td>
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		</tr>
		<tr>
			<td>Guanosine 5'-triphosphate sodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Paraformaldehyde Powder, 95%</td>
			<td>Sigma-Aldrich</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Lucifer Yellow, Rabbit IgG Fraction (3 mg/ml)</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Goat Anti-Rabbit IgG (H+L) 2 mg/ml</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescent Preserving Media</td>
			<td>BioFX Laboratories Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Capillary Glass Tubing with flame polished ends (OD = 1.50 mm, ID = 0.86 mm, Length = 15 cm)</td>
			<td>Warner Instruments</td>
			<td>64-0794</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Single Stage Glass Microelectrode Puller</td>
			<td>Narishinge Japan</td>
			<td>Model PP-830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Minipuls 2</td>
			<td>Gilson</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Millex-GV 0.22 &mu;m Filter Unit</td>
			<td>Millipore Corporation</td>
			<td>SLGV004SL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luer Lock Reusable Hypodermic Needle: 30 G</td>
			<td>Smith &amp; Nephew (Australia)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Single Inline Solution Heater</td>
			<td>Warner Instruments</td>
			<td>Model SH-27B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual Automatic Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus Stereomicroscope SZ61</td>
			<td>Olympus Corporation</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus Microscope BX50WI: with 40X objective</td>
			<td>Olympus Corporation</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0-30 V 2.5 A DC Power Supply</td>
			<td>Dick Smith Electronics</td>
			<td>Q1770</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Digital Microscopic Camera ProgResMF cool</td>
			<td>Jenoptik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micromanipulator MP-225</td>
			<td>Sutter Instrument Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Patch Clamp Amplifier EPC 8</td>
			<td>HEKA Elektronik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8 Data Acquisition System</td>
			<td>HEKA Elektronik</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Computer: DELL</td>
			<td>Dell Corporation</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

implementing dynamic clamp with synaptic and artificial conductances in mouse retinal ganglion cells

神经节细胞输出的视网膜神经元，他们的活动体现了集成多个突触输入所产生的特定的神经回路。膜片钳技术，在电压钳和电流钳配置，常用来研究神经元的生理特性和表征其突触输入。虽然这些技术的应用是非常翔实，他们带来种种限制。例如，它是难以量化的兴奋性和抑制输入交互操作的精确确定响应输出。为了解决这个问题，我们使用了修改后的电流钳技术，动态钳，也称为电导钳1，2，3，检查的影响兴奋性和抑制性神经元兴奋性突触输入。这种技术需要电流注入到单元格，是依赖于在那个时候，其膜电位的实时反馈。 injecteD电流计算从预定的兴奋性和抑制性突触传导，其反转的潜力和细胞的瞬时膜电位。实验程序的详细信息，说明在这个视频文章膜片钳细胞的动力钳技术，实现了全细胞的结构和就业。这里，我们表明小鼠视网膜神经节细胞的反应的生理实验在控制的条件下或在药物的存在下得到的各种导波形。此外，我们使用人工使用alpha调查反应的细胞的功能产生的兴奋和抑制电导。

不同来源的神经节细胞的反应的抑制性输入的贡献表现通过应用各种电导波形。这些波形不同的亮度得到的刺激，在正常条件下和在TTX，电压门控Na +通道阻断剂的存在下，仅在一个子集，抑制视网膜中间块动作电位的产生。 图2A示出了有代表性的反应注射兴奋和抑制电导波形记录在刺激反应，在正常情况下在灰色背景上一个小黑点。当电导有一个黑色斑点的大小不同的波形?...

Watch this Scientific Journal Video about Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells at JoVE.com

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

此视频文章说明设置，程序，修补细胞组织，以及如何实现动态钳记录整个安装鼠标视网膜神经节细胞的。这种技术允许调查的兴奋性和抑制性突触输入精确的贡献，他们的相对幅度和时机神经扣球。

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

implementing-dynamic-clamp-with-synaptic-artificial-conductances

Research

JoVE Journal

Neuroscience

12.2K 次观看.  University of Sydney. 此视频文章说明设置，程序，修补细胞组织，以及如何实现动态钳记录整个安装鼠标视网膜神经节细胞的。这种技术允许调查的兴奋性和抑制性突触输入精确的贡献，他们的相对幅度和时机神经扣球。

视频: Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

全细胞膜片电生理方法对突触倍数的评价

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

用于研究视网膜神经节细胞的小鼠视网膜分离和甲醇处理

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (&#8722;20 &#176;C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (&#8722;20 &#176;C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

作者聚焦：用于研究视网膜神经节细胞的基于甲醇的整装制剂  

Methanol can be used as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage, which is useful for the investigation of retinal ganglion cells.

基于甲醇的全支架制备用于视网膜神经节细胞的研究

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...