这项工作是支持部分由美国国立卫生研究院R01EY012949，R21EY018885，T32 EY0707133赠款。我们感谢他的技术援助达尔文坑。

动物使用声明:动物照顾，在按照指南中描述的实验动物的照顾和使用的指引，使用由明尼苏达机构动物护理和使用委员会大学批准的协议。 1）准备的解决方案<ol><li>在进行电生理记录之前，细胞内，外，酶的解决方案需要做好准备。 </li><li>细胞外液:混合一瓶（8.8克）粉状艾姆斯"媒介与H 2 O和1升的碳酸氢钠1.9克（23毫米）（Sigma公司）。在所有点的细胞外液与95％O 2 / 5％CO 2混合气体饱和。将200毫升烧杯视网膜存储。在重力灌注的灌注系统容器将剩余的解决方案，一旦安装视网膜。 </li><li>细胞内的解决方案:细胞内的解决方案应该已经以前提出，然后分装成500-1000μL等份，并储存在-20 ° C细胞内的解决方案各不相同，取决于所需要的实验，电流钳这里显示的记录，我们使用（毫米）:125的K -葡萄糖酸钙，EGTA，10 HEPES，2 2 2 2 氯化钙 ，氯化镁2，10 NA 2 + - ATP， 0.5毫米NaGTP pH至7.2，用KOH 5 。一个荧光示踪剂，如10微米Alexafluor - 594肼（Invitrogen公司）可以加入到可视化实验过程中，和neurobiotin（0.3％）的树突也可包括细胞的解剖分析，以下记录。准备记录时，解冻细胞内的解决方案。 </li><li>酶液:结合83毫克到4.150毫升的细胞外液的透明质酸酶（沃辛顿化学品）的10000个单位胶原酶（沃辛顿化工）。在50μL分装储存在-20 ° C等分可用于几个星期。准备治疗视网膜时，稀释分装成500μL细胞外液起泡。 </li><li>磷酸盐缓冲液（PBS）:在800毫升的H 2 O，加8克氯化钠，氯化钾0.2克，1.4克的Na 2 HPO 4和0.24克的KH 2 PO 4 。把用NaOH的pH至7.4。调整音量至1 L。 </li><li> 0.2 M磷酸缓冲液:在800毫升的H 2 O添加21.8克的Na 2 HPO 4和6.4克的NaH 2 PO 4。带量至1 L。 </li><li> 4％多聚甲醛溶液:热50毫升H 2 O至60 ° C添加4G多聚甲醛，搅拌几分钟。 1M NaOH溶液中加入几滴（约5滴），不断搅拌，直至解决方案是明确的。添加50毫升到烧杯0.2 M磷酸缓冲液。检查pH值，使用pH值带（〜7.4）。过滤器和冷却。 </li><li>封闭液（10％山羊血清，0.5％TritonX100）:添加山羊血清和10μL，Triton X - 100的的0.2毫升，1.8毫升的PBS。 TritonX - 100服务permeabilise组织。 </li><li>链霉亲和素溶液（5％山羊血清，0.5％TritonX100 594链霉，1:500）:添加1.9毫升的PBS山羊血清0.1毫升，10μL，TRITON X - 100和4μL链霉594。 </li></ol> 2）从小鼠视网膜的分离解剖工具:1＃2钳，2＃5镊子，眼科剪刀<ol><li>安乐死鼠标按照批准的协议，在这种情况下，CO 2窒息和enucleate眼球，轻轻插入眼睛的角度＃2落后钳和起重眼球。 </li><li>菜放入35毫米的细胞外液的Petri的眼球。剪断视神经和任何附加的眼外肌或结缔组织，用眼科剪刀。用眼科剪剪去角膜和使用＃5镊子拉出镜头。撕裂巩膜使用一对＃5镊子和使用产钳切断视神经，视网膜和巩膜满足视盘。轻轻地揭下眼罩远离视网膜。提示:不要在两个视网膜之前，每一步移动到下一个步骤，以尽量减少时间。 </li><li>使用镊子去除附着在视网膜玻璃体。这可以由上述透明的玻璃体视网膜与钳"抢夺"。玻璃体，如果顺利取出，应作为一个明确的凝胶状物质，镊子可见。提示:切一半以下的玻璃体切除视网膜将最大限度地提高可用组织的数量，并在会议厅内安装容易。 </li><li>保持室温在黑暗的环境下以最小的环境光线的含氧细胞外液的视网膜，直到他们准备被消化。视网膜可以存储在这种方式〜6小时。 </li></ol> 3）治疗视网膜与酶的混合物，并安装在录音室<ol><li>胶原酶/透明质酸等份稀释500μL，细胞外液，在一个35毫米的培养皿的地方解决方案和转移的视网膜。 </li><li>保持视网膜包含ING培养皿中，覆盖在黑暗中，在95％O2 / 5％的CO 2环境中轻轻摇动15分钟。这一步艾滋病在消化任何剩余的玻璃体，并允许更容易进入神经节细胞层。提示:减少玻璃体或不利影响，如果是观察年轻的动物视网膜，时间可缩短至〜5分钟。 </li><li>视网膜含酶的培养基中删除，并在细胞外液洗广泛</li><li>转移到视网膜的感光层朝下录音室6。威克走剩余的液体，用吸管或组织，并推出视网膜两侧扁平化组织正在仔细只接触边缘的视网膜，特别是避免接触与神经节细胞层。铂金戒指放在视网膜尼龙网，锚的组织，并立即填写与细胞外液室。 </li><li>山显微镜舞台上的录音室，重视管灌注重力和真空吸力和附加接地线。 </li></ol> 4）本地化的EGFP表达神经节细胞光刺激<ol><li>重力在细胞外液1毫升/分或更快的速率灌注视网膜。提示:如果灌注速度太慢，该组织将不会有足够的含氧和突触的信号可能会中断。 </li><li>与细胞内的解决方案填充录音吸管插入吸管持有人。提示:在3至5MΩ电阻移液器提供最好的结果。 </li><li>根据红外微分干涉对比（DIC）在低倍率（10倍的目标，NA 0.3）本地化录音吸管的光学可视化视网膜。红外灯是用来减少视网膜暴露在可见光和减少光适应。移动到更高的放大倍率（40X客观的，不适用0.8），并把电极只是上述组织。 </li><li>在高放大倍率（40X目标），照亮epifluorescence（480纳米EGFP的）的视网膜，并选择使用磁带（图1A）一个荧光的神经节细胞和电脑显示器上的标志。 </li><li> DIC和红外线照射下，记录电极位置的附近有针对性的神经节细胞（图1A）。立即清理面积覆盖应用轻柔的正压记录电极通过一个12cc的塑料注射器用聚乙烯管材连接吸管持有人的细胞。放大器上，任何电压偏移为零。选定的神经节细胞，直到上放置电极酒窝出现在细胞表面。 </li><li>随着在电压钳模式放大器，适用于测试脉冲和监控通过吸液管的电流通过。应用负压创建电极和细胞之间的密封。释放负面的压力时，细胞开始密封和等待，直到电流是一个GΩ的细胞的细胞膜和吸管之间的电气密封。应用负压温柔脉冲，直到电容瞬态出现，然后释放任何压力的应用。提示:如果访问（系列）电阻高（&gt;30MΩ），这往往可以通过额外的，很温柔，负压脉冲减轻。 </li><li>一旦已达到一个稳定的密封采用负压脉冲。获得全细胞模式后，使细胞暗适应5分钟。纪录，在电流钳模式，响应全领域，白色的光刺激（图1B）。 </li><li> Alexafluor - 594录音之后，将有扩散的树突。示踪剂可根据epifluorescence显示，在594纳米，用于确定是否神经节细胞上，断，或epifluorescence和DIC红外光学 5之间切换双向分层。 </li><li>如果neurobiotin移液器和视网膜，免疫组织化学处理，然后必须将吸管轻轻地缩回，以提供最小的膜破坏。 </li></ol> 5）记录神经节细胞的解剖分析 neurobiotin灌装，允许更详细的神经节细胞的形态分析，下面的记录。提示:如果一个记录的神经节细胞和详细的形态之间的具体相关的需要，它可能是最好的记录和填写只有每一个研资局的准备。 <ol><li>然后放置在多聚甲醛溶液，如果neurobiotin是在细胞内的解决方案包括视网膜和修复一夜之间在4 ° C。 </li><li>在PBS冲洗，至少2个小时，至少有6变化。视网膜转移到板含有封闭液。留在阻断2个小时的解决方案，对室温​​摇床。 </li><li>更换与链霉亲和解决方案的封闭液。孵育2天，于4℃。上摇床彗星。在PBS冲洗的视网膜，6漂洗，每次20分钟。 </li><li>安装在玻片上的视网膜与Vectashield。必须放在视网膜神经节细胞。执行共聚焦显微镜（图1C）。 </li></ol>代表性的成果如果视网膜是健​​康的，夹层是成功的，个别的神经节细胞，应根据DIC的可见，在高放大倍率（图1A）。不健康的视网膜将有大粒细胞核，并应被丢弃。如果一个给定的神经节细胞突触的反应是完好的，那么细胞反应轻发病或抵消动作电位，一个内在的感光神经节细胞（图1B），持续的去极化的情况下。 <img src="/files/ftp_upload/2367/2367fig1.jpg" alt="图1" /> 图1。本地化，录制，和一种荧光标记的视网膜神经节细胞染色（A）GFP阳性研资局的可视化啶照明下40X放大倍率（左图）和DIC的照明下（右图）。 （二）M2的本质荧光标记的研资局在全细胞电流钳模式记录的感光视网膜神经节细胞突触的光响应。 （三）货币供应量M1内在感光视网膜神经节细胞的鉴定下epifluorescence，有针对性的全细胞记录，充满neurobiotin，然后进行免疫组化处理。

<ol>
	<li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li><li>Schmidt, T. M., Taniguchi, K., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+extrinsic+light+responses+in+melanopsin-expressing+ganglion+cells+during+mouse+development.">Intrinsic and extrinsic light responses in melanopsin-expressing ganglion cells during mouse development.</a> J Neurophysiol. 100, 371-384 (2008).</li><li>Huberman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+identification+of+an+On-Off+direction-selective+retinal+ganglion+cell+subtype+reveals+a+layer-specific+subcortical+map+of+posterior+motion.">Genetic identification of an On-Off direction-selective retinal ganglion cell subtype reveals a layer-specific subcortical map of posterior motion.</a> Neuron. 62, 327-334 (2009).</li><li>Siegert, S. <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>Schmidt, T. M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+differences+among+intrinsically+photosensitive+retinal+ganglion+cells.">Functional and morphological differences among intrinsically photosensitive retinal ganglion cells.</a> J Neurosci. 29, 476-482 (2009).</li><li>Newman, E. A., Bartosch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+eyecup+preparation+for+the+rat+and+mouse.">An eyecup preparation for the rat and mouse.</a> J Neurosci Methods. 93, 169-175 (1999).</li><li>Haverkamp, S., Inta, D., Monyer, H., Wassle, 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>Zhang, D. Q., Zhou, T. R., 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=Functional+heterogeneity+of+retinal+dopaminergic+neurons+underlying+their+multiple+roles+in+vision.">Functional heterogeneity of retinal dopaminergic neurons underlying their multiple roles in vision.</a> J Neurosci. 27, 692-699 (2007).</li><li>Hu, E. H., Dacheux, R. F., Bloomfield, 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=A+flattened+retina-eyecup+preparation+suitable+for+electrophysiological+studies+of+neurons+visualized+with+trans-scleral+infrared+illumination.">A flattened retina-eyecup preparation suitable for electrophysiological studies of neurons visualized with trans-scleral infrared illumination.</a> J Neurosci Methods. 103, 209-216 (2000).</li><li>Wu, S. M., Gao, F., Pang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+circuitry+mediating+light-evoked+signals+in+dark-adapted+mouse+retina.">Synaptic circuitry mediating light-evoked signals in dark-adapted mouse retina.</a> Vision Res. 44, 3277-3288 (2004).</li></ol>

这种技术适用于任何一个孤立的视网膜视网膜神经节细胞的录音。虽然在本质上感光上面使用鼠标线，黑视素的表达RGC的标记与EGFP 2，5，这个协议很容易转移到其他荧光标记的研资局线或可广义纪录，以及从随机选取的视网膜神经节细胞。这种技术是特别有价值的，因为每个研资局和其各自的输入神经元的树突状乔木留下完全完好无损。录音可以很容易地在电流或电压钳模式下，细胞贴?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ames&#x2019; Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase</td>
<td>&nbsp;</td>
<td>Whorthington Biochemical</td>
<td>LS005273</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Whorthington Biochemical</td>
<td>LS002592</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>AlexaFluor 594 Hydrazyde</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A10442</td>
<td>&nbsp;</td>
<tr>
<td>Neurobiotin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>SP1120</td>
<td>&nbsp;</td>
<tr>
<td>Electrodes</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>BF120-69-10</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>&nbsp;</td>
<tr>
<td>Ophthalmologic Scissor</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin 594</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S32356</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>&nbsp;</td>
<tr>
<td>Goat Serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>005-000-001</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>19210</td>
<td>&nbsp;</td>		</tbody>
		</table>
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an isolated retinal preparation to record light response from genetically labeled retinal ganglion cells

在脊椎动物视觉第一步骤，当光线刺激视网膜 1杆和锥光感受器。然后将这些信息分割成ON和OFF通路。光感受器信号光信息的双极细胞（BCS），其中光照强度的增加（BCS）的或减少（关BCS）去极化响应。这种光信息的隔离是保持在视网膜神经节细胞（RGC的），其中有分层内网状层（IPL），他们收到来自关闭的BC直接兴奋输入关sublamina的树突的水平，或分层在sublamina的彩光，他们收到直接的兴奋性输入在边界条件。这隔离的关于增加或减少照明（开启和关闭途径）的信息是守恒的，并暗示大脑平行。 RGC的是输出的视网膜细胞，因而是一个重要的细胞，以研究，以了解如何在大脑中的视觉核信号光信息。在过去几十年的小鼠遗传学的进展，导致各种具体的研资局的人群是一种荧光蛋白标记，以便研资局亚型 2 3 4和电生理记录针对特定的识别荧光记者鼠标线。在这里，我们提出了一种用于记录从一个完整的，孤立的视网膜准备荧光标记的神经节细胞的光反应的方法。这个与世隔绝的视网膜准备允许来自视网膜神经节细胞树突状乔木是否完好，并保留在整个研资局的树突状乔木输入录音。这种方法适用于跨多种神经节细胞亚型，是适合各种各样的单细胞的生理技术。

Watch this Scientific Journal Video about An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells at JoVE.com

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

本文提供了一个描述如何从孤立在小鼠视网膜准备的解剖和记录。特别是，我们介绍了如何从一个荧光标记的神经节细胞的人口记录对光反应和随后查明并分析其形态。

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 ...

an-isolated-retinal-preparation-to-record-light-response-from

Research

JoVE Journal

Neuroscience

16.7K 次观看.  University of Minnesota. 本文提供了一个描述如何从孤立在小鼠视网膜准备的解剖和记录。特别是，我们介绍了如何从一个荧光标记的神经节细胞的人口记录对光反应和随后查明并分析其形态。

视频: An Isolated Retinal Preparation to Record Light Response from Genetically Labeled 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

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 ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

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

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

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 ...

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 ...

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 ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

体内 大型动物视网膜神经节细胞及视神经功能及结构的评估方法

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...