This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (Werner Reichardt Centre for Integrative Neuroscience T&#252;bingen, EXC 307 to T.E. and T.S.; KFO 134 to B.W. and F.P.D.), the German Federal Ministry of Education and Research (BMBF) (BCCN T&#252;bingen, FKZ 01GQ1002 to T.E and T.B.), and the European Union (DRUGSFORD; HEALTH-F2-2012-304963 to M.K. and F.P.D).

所有动物的程序进行遵守的准则和法律对动物保护由德国联邦政府决定，并经图宾根大学的体制动物福利委员会。 1.动物模型<ol><li> HR2.1：TN-XL 钙的生物传感器鼠标<ol><li>使用转基因HR2.1：TN-XL鼠标线表达的Ca 2+生物传感器TN-XL在选择性光感受器锥体10。使用3 - 6周龄小鼠不论男女使用标准的12小时昼/夜的节奏上调。 </li></ol></li></ol> 2.视网膜解剖<ol><li>生理溶液<ol><li>制备新鲜的2L的胞外溶液，含有125 mM氯化钠，2.5mM的氯化钾，1mM的MgCl 2的，1.25毫的NaH 2 PO 4，26 mM的碳酸氢钠 ，0.5 2mM L-谷氨酰胺，和20mM（+）葡萄糖。混合直至所有成分溶解。 </li><li> “泡沫”细胞外液与carboxygen（95％O 2，5％的CO 2）5 - 10分钟。添加氯化钙 ，以达到2mM的浓度。 </li><li>冒泡细胞外液后，测量其pH值。的pH值应为7.4，否则很可能有错误的溶液的制备过程中已取得。保持冒泡率和溶液温度恒定在整个实验中，以确保恒定的pH值。 </li><li>股票分离成2烧瓶，一个用于视网膜剥离，一个用于记录设置（灌注）。 </li></ol></li><li>眼眼球摘除和视网膜的隔离（5 - 10分钟） <ol><li>保持眼球摘除工作区域暗淡的红光。使用LED具有650nm（或更长）的峰值波长，以避免在解剖锥体的漂白。 </li><li>通过把它的笼子到通风良好，不透光盒子，以确保锥充分认识在录音时暗适应鼠标2小时。 </li><li>麻醉鼠标下laborato用一个蒸发器和保持一个标准鼠笼气密容器Ry的罩用异氟烷（5％）。处理时，蒸发器小心地按照生产厂家的说明书。使用手套和口罩，以减少接触过敏原。 </li><li>使用双目显微镜用10 - 40倍放大倍率解剖。 </li><li>断头牺牲麻醉鼠标。 </li><li>对于定位，标记每只眼睛（=背）的顶部具有防水笔。继续使用是否左眼或右眼的轨道。 </li><li>通过切断视神经眼球的后面取出谨慎采用弯剪刀的眼睛。解剖，眼球转移到新鲜含外carboxygenated解决培养皿。对于转移眼球，由视神经残端持有它。 </li><li>皮尔斯在眼球沿角膜和巩膜（ 即，在锯齿缘 ）之间的边界用锋利的注射针的任何一点。 </li><li>持有电子在你们用钳子轻轻巩膜插入一个剪刀刀片成在上一步中所做的孔。沿着锯齿缘切以分离从后部（眼罩）眼睛（角膜，晶状体，玻璃体）的前部。径向切眼罩（朝向视盘）在笔标记的位置来指示在视网膜上背的位置。 注意：准备这样，眼罩可以保持在供以后使用不断carboxygenated溶液。 </li><li>打开眼罩，在培养皿中，使得背砍分远离自己。通过插入视网膜和巩膜之间的钳子提示抢在左侧和右侧的眼罩的具有一对各钳子的巩膜。 </li><li>轻轻倒转眼罩的巩膜部分分离视网膜。最后，使用微解剖剪刀以释放从巩膜视网膜切开视神经。 注意：这是一个关键步骤。避免损坏视网膜（ 例如，通过用钳子触摸并挤压）。 </li><li>持视网膜的边缘之一与一对镊子轻轻从使用的第二对钳子的视网膜表面上除去碎屑和玻璃体。当视网膜表面是干净的，用微解剖剪刀，使三个附加较短，径向切口（大约每90°）。这些切口允许人们小心弄平视网膜与感光面朝下在培养皿中（ 图1A）。 </li></ol></li><li>切片准备（10 - 15分钟） <ol><li>准备视网膜切片和玻璃盖，单硝酸纤维素事先过滤膜安装片，将它们转移到录音室。切断硝基纤维素过滤膜进入用剪刀约10×5mm的尺寸的矩形块。切盖玻片成使用一个玻璃刀约10×5mm的尺寸的矩形块。 </li><li>慢慢沉浸载玻片进入细胞外液接近组织。轻轻地，拉视网膜上的神经节细胞的一面朝上搏命通过使用一对镊子抓住它的边缘。此过程减少机械损伤和组织的折叠。 </li><li>选择相关于各个研究问题的视网膜的区域（也见讨论）。记住，切长在步骤2.2.9取得标志着背视网膜一半（ 图1A）。切的矩形，大约为1×2mm的大小的一块出使用弯曲手术刀刀片所选视网膜区域。擦去组织周围多余的解决方案。 </li><li>将滤膜上的片视网膜的，使得神经节细胞侧粘结到膜的顶部。立即，加一滴外介质的在膜上牢固附着的组织到膜上。 </li><li>传送膜安装视网膜组织到含有新鲜细胞外溶液中的切片室。 </li><li>切视网膜到200微米的垂直切片厚度（ 图1B）使用连接到组织断续器23一个新鲜刀片。对于每一块视网膜改变刀片。 注：刀片需要被完美的切片室底部的表面排列，使得整个膜同时分割，通过指示“咔嗒”切割时的声音 - 否则叶片可弯曲和损坏切片。 </li><li>通过施加高真空润滑脂向膜粘合的单个膜装片的玻璃盖玻片结束只（ 图1C）。保持视网膜下方的玻璃表面无油脂。 </li><li>保持覆盖一滴在保持室外溶液盖玻片装片-闭合并遮光的容器中， 例如，一个陪替氏培养皿覆盖有铝箔盖-在RT一个carboxygen气氛下进行。通过鼓泡小蓄水池，以保持气氛中保持引进carboxygen加湿室;这防止了片干燥。 </li><li>允许切片休息在保持室10 - 他们（逐个）移动到录音室前15分钟。片可以维持长达4-5小时在保持室中于室温（〜21℃）。 </li></ol></li></ol> 3.双光子钙成像<ol><li>双光子显微镜<ol><li>使用一个可移动目标显微镜（MOM）型双光子显微镜。无论MOM设计和成像程序前面24进行了描述，详情另见10,21,25（来源用于和公司， 见表1）。 注意：任何直立双光子显微镜，满足下列最低要求，可以用：它必须配备（a）一种脉冲激光调谐到〜860纳米，（b）一种至少有两个同时获得的荧光通道，（三）过滤器ECFP和黄水晶荧光，（D）的光刺激（对于可能的设计，见24,26），和（e）的软件，允许录制时间已过图像序列的帧频足以解决这个钙的兴趣2+信号。 </li><li>启动双光子成像系统由制造商所指示的。严格按照工厂的激光安全指引。启动激光和调整其〜860纳米。 </li><li>从保持室转移切片到录音室，并立即开始使用carboxygenated细胞外液灌注。保持2毫升/分钟的灌注流速和37℃的录音室中的温度。 </li><li>使用20X 0.95 NA水浸泡的目的。如果可用，则使用CCD照相机结合红外线记录 ​​室下方的LED定位的视网膜切片（ 图2）。否则，使用定位双光子成像（见3.1.5）切片。 </li><li>切换到双光子成像，以查看生物传感器的表达。打开两个检测通道ECFP和黄水晶的荧光成像。 </li><li>使用控制双光子显微镜进行扫描，并选择一排锥形终端，用于记录（ 图3A）的图像采集软件。集图像采集到128×16像素的图像（31.25 Hz）或类似的配置。限制扫描区域到锥形终端，以避免在外段光合色素的褪色。 注：对于TN-XL钙传感器（τ= 0.6〜秒， 钙离子结合，τ=〜0.2秒， 钙解除绑定;见16）〜8赫兹的最低帧率建议。 </li></ol></li><li>光刺激和记录<ol><li>使用一个子级全视野光刺激如别处所述10,21,26以执行以下步骤。 注：一个完整场光刺激一个简单的解决方案是使用两个带通滤波的LED（ 例如，“蓝”：360 BP 12，“绿色”：578 BP 10）匹配的小鼠锥体的波长灵敏度，但在同一时间不重叠与用于荧光检测的过滤器（参照3.2.4）。来自LED的光通过记录室（ 图2）的底部聚焦的冷凝器。有关该刺激的设计，其校准的细节，使用的光强度和诱发锥光异构化率，见10,21,26。 </li><li>打开激光器，并允许锥体，以适应在扫描激光器和刺激背景光（20 - 30秒，也看潜在缺陷）呈递的光刺激（见3.2.3）或施加药剂之前。 </li><li>开始呈现任意刺激（光如，闪烁， 如图3B，C）的。在步骤3.2.1中所述的刺激的情况下，通过调节这两个LED的强度随时间使用微处理器板由定制软件控制（生成的刺激详细信息，请参阅10,21,26）。 </li><li>开始记录在两个荧光通道（ 例如 ，在483毫微米为“蓝色”FRET的供体ECFP和535nm处为“黄”FRET的受体;对于规格见表1）使用各自的图像获取软件同时（也见3.1 0.6）。例如，在闪烁感（ 图3B，C），记录至少8时- 10刺激演示（试验）。 </li></ol></li><li> “在片” 钙校准<ol><li>记录的Ca在锥形端子2+信号（如在3.1.6和3.2.4中所述），并提取一组圆锥的基线的Ca 2+水平（如在3.4中描述）。 </li><li>切换到“ 钙离子游离”胞外培养基中为5μM离子霉素（溶于DMSO）和10mM EGTA（或，替代地，BAPTA）加入。记录锥体终端再次每次5分钟，以确定最小荧光比（R 分钟 ），&lt;EM&gt;即，在（近）无细胞内钙的比例2+。这可能需要之间的任何地方15 - 30分钟。 注：灌注系统是必需的，允许不同储层之间的切换。 </li><li>切换到细胞外介质与5μM的离子霉素（溶于DMSO）和2.5mM 钙离子。之后的5分钟的温育时间，记录锥体再次每隔5分钟以测量最大荧光比（R 最大 ）， 即，在饱和的细胞内钙的浓度2+的存在比例。以下离子霉素应用，它可能需要3至- 15分钟为的Ca 2+比变得稳定。 注意：揭露切片高的Ca 2+和离子霉素一段较长的时间将最终损伤细胞。仅包括细胞保留其正常形态的分析。浓度（ 即，EGTA，离子霉素）可能需要进行调整。有关钙校准详情协议，见13,17。 </li></ol></li><li>数据分析 注意：使用图像处理和数据分析软件包与各自的显微镜软件兼容，并能够运行定制分析脚本。 <ol><li>加载的成像数据文件并绘制的利息（投资回报）围绕每个锥端（ 图3A）的区域。对于每一帧，平均荧光强度的ROI中并减去背景荧光，计算FRET的受体（柠檬色）和施主（ECFP）荧光（R = F A / F D; 图3B，C）之间的比（R）之前。该比率通常用作用于相对内Ca 2+浓度的代理，但校准（见3.3）后，也绝对浓度可估计（见3.4.3）。 注意：椎弓根可以通过其位置在所述外丛状层被识别和它们的形态（有些扁平“斑点”小于锥胞体，位于所述锥轴突的末端）。 </li><li>平均刺激试验（ 图4A）。提取前光刺激，峰值幅度（R 放大器 ），和曲线（R A），作为响应的大小的措施下面积平均的痕迹（ 图4B），如Ca 2+基线水平（R 基本 ）响应参数。此外，从平均化的痕迹，如响应上升（叔上升 ）和衰变时间（t 衰变 ）提取反应动力学（在20％和80％R 放大器 =相应的时间间隔;见插图图4B）。有关如何确定这些参数的详细信息，请参阅第10。 </li><li>计算基于从步骤3.3.1-3.3.3测量绝对锥体Ca 2+浓度的估计。使用比率为最小和最大Ca 2+浓度，R 最小和R 最大分别在钙的供体荧光2+ </s向上&gt; -bound（F D，钙结合 ）和-unbound（F D，无钙 ）的状态，以及在体内解离常数（K D = 0.77，见16），用于TN-XL与下列式（模拟到27）： </li></ol></li></ol><img alt="式（1）" src="/files/ftp_upload/52588/52588eq1.jpg" />

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M., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+reporter+of+synaptic+activity+in+vivo.">A genetically encoded reporter of synaptic activity in vivo.</a> Nature Methods. 6, 883-889 (2009).</li><li>Mank, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophysical Journal. 90, 1790-1796 (2006).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+locus+control+region+adjacent+to+the+human+red+and+green+visual+pigment+genes.">A locus control region adjacent to the human red and green visual pigment genes.</a> Neuron. 9, 429-440 (1992).</li><li>Connaughton, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+retinal+slice+preparation.">Zebrafish retinal slice preparation.</a> Methods in Cell Science. 25, 49-58 (2003).</li><li>Baden, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tale+of+two+retinal+domains:+near-optimal+sampling+of+achromatic+contrasts+in+natural+scenes+through+asymmetric+photoreceptor+distribution.">A tale of two retinal domains: near-optimal sampling of achromatic contrasts in natural scenes through asymmetric photoreceptor distribution.</a> Neuron. 80, 1206-1217 (2013).</li><li>Kemmler, R., Schultz, k., Dedek, K., Euler, T., Schubert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+cone+calcium+signals+by+different+horizontal+cell+feedback+mechanisms+in+the+mouse+retina.">Differential regulation of cone calcium signals by different horizontal cell feedback mechanisms in the mouse retina.</a> The Journal of Neuroscience. 34, 11826-11843 (2014).</li><li>Werblin, F. 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Pflugers Archive.</a> European Journal of Physiology. 457, 1393-1414 (2009).</li><li>Baden, T., Berens, P., Bethge, M., 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=Spikes+in+mammalian+bipolar+cells+support+temporal+layering+of+the+inner+retina.">Spikes in mammalian bipolar cells support temporal layering of the inner retina.</a> Current Biology. 23, 48-52 (2013).</li><li>Breuninger, T., Puller, C., Haverkamp, S., 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=Chromatic+bipolar+cell+pathways+in+the+mouse+retina.">Chromatic bipolar cell pathways in the mouse retina.</a> The Journal of Neuroscience. 31, 6504-6517 (2011).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> The Journal of Biological Chemistry. 260, 3440-3450 (1985).</li><li>Thoreson, W. B., Mangel, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+interactions+in+the+outer+retina.">Lateral interactions in the outer retina.</a> Progress in Retinal and Eye Research. 31, 407-441 (2012).</li><li>Trifunovic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-dependent+cone+photoreceptor+degeneration+in+the+cpfl1+mouse+retina.">cGMP-dependent cone photoreceptor degeneration in the cpfl1 mouse retina.</a> The Journal of Comparative Neurology. 518, 3604-3617 (2010).</li><li>Sahaboglu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+pigmentosa:+rapid+neurodegeneration+is+governed+by+slow+cell+death+mechanisms.">Retinitis pigmentosa: rapid neurodegeneration is governed by slow cell death mechanisms.</a> Cell Death &amp; Disease. 4, e488 (2013).</li><li>Sahaboglu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP1+gene+knock-out+increases+resistance+to+retinal+degeneration+without+affecting+retinal+function.">PARP1 gene knock-out increases resistance to retinal degeneration without affecting retinal function.</a> PloS One. 5, e15495 (2010).</li></ol>

作者什么都没有透露。

使用电单细胞记录或Ca 2+成像用合成荧光指示剂预先存在的协议很难记录在小鼠锥感光细胞的钙离子动力学为一定数量的技术原因（见导言）。这里所描述的协议允许的Ca 2+信号，甚至绝对的Ca 2+水平的个体，确定鼠标锥体终端以有效和相对简单的方法测量。 该协议已经被成功地用于在三个研究在健康小鼠视网膜寻址锥体功能的不同方面。在第一项研究10，HR2.1：TN-XL小鼠用免疫组织化学，ERG录音，双光子钙成像和药理学特征，显示出钙离子的生物传感器的圆锥特异性表达不妨碍圆锥体解剖结构和功能。在第二项研究中21，色度和鼠标锥消色差响应性能进行了映射整个视网膜，展示了“绿色”视蛋白为主的背和“蓝色”视蛋白为主的腹小鼠视网膜之间的显着差异，锥的功能。在锥体属性这些区域差别匹配在自然环境中的微分对比度分布（ 即，天空与地面），这表明不同光谱类型的鼠标锥体提供消色差对比（近）最优采样的，因此，可以提供一进化优势。在第三项研究22的往复反馈锥轴突终端接收从水平细胞进行了研究。由于所有提出的水平细胞反馈机制上的电压门控的Ca 2+通道的锥形轴突末端28充当，锥形终端的Ca 2+可以作为水平细胞-锥体相互作用的代理。由KEMMLER和研究同事22支撑认为水平细胞使用，包括多种机制来控制光感受器谷氨酸释放一个复杂的反馈系统。 这些研究说明了所描述的协议的通用性，并表明，它可适应很宽范围的关于锥体功能和它的突触电路的问题。此外，该协议使学习当地的钙信号在不同的车厢锥，对更好地理解锥生理学。这种知​​识是重要的是理解在退化锥病理生理过程，以最终允许对潜在的治疗方法的合理开发，特别是用于影响锥退行性疾病。 在HR2.1：TN-XL鼠标线， 钙生物传感器被表示整个锥体，除所述外段的。这为钙离子的动态直接和比例的评估机会S在不同的车厢锥。由于改变的Ca 2+电流的外段都反映在通过膜电位和产生的电压-门控的Ca 2+通道的活化端子，在所述外段的过程可以间接观察到。 潜在的隐患： 视网膜剥离是一个关键的一步：在小鼠，视网膜从眼罩通常光感受器外段和色素上皮之间的分离。因此，分离的视网膜的光敏感的感光外段暴露和机械损伤极其敏感。极大必须小心不通过触摸感光侧与工具或通过侧向移动所述组织上的粘合面（ 例如，一个过滤膜）的损害。 高品质的视网膜切片在显微镜下，他们的清洁切割面识别和由一个组织严密的感光层有明确界定外节。切片质量功能评估可以通过闪烁明亮的光线刺激和响应确定的视锥细胞的百分比（ - 20锥例如，在视图10场）迅速完成。这里，响应质量应通过计算信噪比（S / N）（基线噪声的光刺激与光响应的振幅之前振幅）进行评估;一个S / 2 N - 3，应考虑的最低阈值。通常情况下，我们丢弃片，用小于50％的响应锥体。还切片与显示过度的自发扣球行为（参见图4中的10）应该被丢弃锥体。 符合上述解剖和功能标准录音室片显示1相一致的响应- 2小时（有关响应一致性的详细信息，请参见10）。由于片生存用于h我们在保持室，一个成功的实验可长达6小时。值得注意的是，有关于学习锥和水平细胞之间的长范围的空间相互作用的一些限制，如切片不可避免切断视网膜网络横向连接。然而，视网膜片的厚度增加至300微米的可改善这一问题22。 利用垂直视网膜切片由激发激光避免扫描感光锥体外段，因此，在很大程度上可以防止视蛋白漂白（对于广泛的讨论，见10,21）。然而，所记录的Ca 2+信号不仅取决于光刺激，而且还得到受由扫描激发激光产生的背景照明组件。事实上，在这样的双光子成像实验的有效背景照明是散射激光，荧光灯由记录策发射的组合LLS，以及任何LED背景刺激成分。因此，圆筒应允许适应至少20 - 30秒到激光扫描（与光刺激的背景成分接通）记录之前。 优势和应用： 虽然一些应用这种锥钙 -imaging协议上面已经描述了10,21,22，其他应用程序可以设想：在锥钙成像相结合药理研究可以确认的Ca 2+锥信号传导通路，可能是用于测试的疗效和钙中针对不同玩家的药物药性2+ -信号10。然而，一个关键的应用可以是研究影响锥功能疾病。许多视网膜变性小鼠模型模仿人类的​​疾病都可用。例如，锥光感受器损失1（CPFL 1）小鼠是伯锥体变性模型痛苦从PDE6C突变29。相反，该杆变性1（RD 1）小鼠遭受一个PDE6B突变。虽然这会导致初级视杆变性30，一次次 1杆损耗完毕，二次锥变性套1。杂交这些动物用HR2.1：TN-XL线将使学习和在初级和次级锥形变性比较钙动力学和可能在锥体细胞死亡提供有价值的见解的Ca 2+的作用。此外，药理学诱导锥变性-例如使用选择性抑制剂PDE6 -可能有助于查明锥变性10,29,31下游的机制。 综上所述，该协议描述这里允许在小鼠锥感光细胞亚细胞器测量的Ca 2+，并提出很好的机会来发现在广泛的生理锥生理和病理生理条件。此外，该协议可以用于旨在干扰锥体的Ca 2+ -信号，从而有助于建立锥体疾病的新疗法药剂的筛选。

视力开始光致活化的视网膜感光的光转导级联。视杆细胞允许在视力低光照水平，而锥感光细胞介导的​​色彩和高分辨率的视觉白天。许多感光体特异性基因易受突变导致这些细胞的变性。若干与感光体损失相关的分子标记物已被鉴定1，但迄今的详细的分子机制和事件的序列仍不清楚。改变的Ca 2+动态平衡被推测是光感受器细胞死亡的一个触发器，以Ca的活性2+依赖性蛋白酶型蛋白酶的变性过程2,3中上调支持的假说。然而，到目前为止，这种假设不被钙离子的生理测量支持。对钙通道阻滞剂在重效应在一些研究中的不一致tinal疾病进一步挑战钙在细胞死亡4-6的参与，呼吁方法在哺乳动物锥感光细胞直接评估的Ca 2+。 以前，大多数电子记录和钙成像研究已经因为在对视锥细胞的7-9更容易获得两栖爬行动物模型的表现。然而，哺乳动物感光生理学可能与非哺乳动物的10和尤其是不同，在人的遗传性视网膜变性的范围内，更好地了解哺乳动物感光生理学的是一个关键的新颖治疗方法的发展。许多小鼠模型模仿人类视网膜疾病也有，但很少有人知道钙动力学在小鼠锥11。电技术不适合用于从锥体高通量的录音，尤其不小鼠，其中杆（〜97％）显 ​​著多于锥（〜3％）12 </s向上&gt;。此外，如全细胞膜片钳记录敏感电生理技术扰乱细胞内环境，并提供关于绝对细胞内Ca 2+浓度对Ca 2+电流的数据，但并非如此。因此，尽管在较低的时间分辨率，成像是选择的方法解决对锥体的Ca 2+动力学问题时。与成像的关键问题是如何有选择地与荧光钙指示剂染料标记的视锥细胞。适当的条块和细胞特异性是很难实现的“批量装入”合成的Ca 2+指示剂染料进入组织。作为结果，标记的锥体，杆13,14，和米勒胶质细胞不能可靠地区分。另外，合成染料趋于渗漏出细胞，从而防止一致的条件下延长的录音。此外，在他们的AM-酯形式装载合成的Ca 2+的指标是有问题的，因为它需要去tergents（ 如 DMSO）中，并生成甲醛15。对于绝对的Ca 2+测量，比率指标是强制性的。然而，最好的目前可用的合成比率指示剂Fura-2需要在700〜760纳米范围内的激发光（对于双光子激发），这取决于它的强度，可以在本身刺激视锥细胞，并且因此阻碍研究锥钙的生理光照条件下的动态2+。 不像合成染料，遗传编码的Ca 2+指标可以表示在一个细胞类型选择性方式。它们不漏细胞出来，因此，如果避免漂白，长时间和可靠的比例测量是可能的。细胞类型选择性的钙离子的生物传感器的表达，当与双光子显微镜组合，表示一个强大的工具下基本上生理条件13,16,17，以评估和研究亚细胞的Ca 2+ </SUP&gt;。在这里，我们描述了一个协议，以研究光刺激诱 ​​发锥钙在转基因钙的生物传感器鼠标线2+动力学（HR2.1：TN-XL），它体现了基于FRET的钙传感器TN-XL 18选择性地锥，在人红视蛋白启动子HR2.1 19。要访问锥端，一个离体切片准备20被雇用。该协议已经成功地运用在锥功能三个研究健康小鼠10,21,22。此外，该协议允许研究锥钙离子在特定的遗传条件， 如信号，通过杂交小鼠模型的遗传性视网膜变性HR2.1：TN-XL小鼠。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>High vacuum grease</td>
			<td>Dow Corning</td>
			<td>1018817</td>
			<td><a href="http://www.dowcorning.com/">http://www.dowcorning.com</a></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>R. Langenbrinck</td>
			<td></td>
			<td>24 x 60 mm; http://www.langenbrinck.com</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>R. Langenbrinck</td>
			<td></td>
			<td>76 x 26 mm; http://www.langenbrinck.com</td>
		</tr>
		<tr>
			<td>Blades for tissue chopper</td>
			<td>MARTOR KG</td>
			<td>ARGENTAX No. 1044</td>
			<td>0.25 mm; http://www.martor.de</td>
		</tr>
		<tr>
			<td>Tissue chopper</td>
			<td>Custom-build</td>
			<td></td>
			<td>Based on a design by F. Werblin23, adapted by T. Schubert</td>
		</tr>
		<tr>
			<td>Nitrocellulose filter membrane gridded</td>
			<td>Merck Millipore</td>
			<td>AABG01300</td>
			<td>Filter type: 0.8 &#181;m; http://www.emdmillipore.com</td>
		</tr>
		<tr>
			<td>Isoflurane CP</td>
			<td>CP pharma</td>
			<td>AP/DRUGS/220/96</td>
			<td><a href="http://www.cp-pharma.de/">http://www.cp-pharma.de</a></td>
		</tr>
		<tr>
			<td>UV/green LED-based full-field stimulator</td>
			<td>Custom-build</td>
			<td></td>
			<td>for details, see10</td>
		</tr>
		<tr>
			<td>Open source microprocessor board</td>
			<td>Arduino</td>
			<td></td>
			<td><a href="http://www.arduino.cc/">http://www.arduino.cc</a></td>
		</tr>
		<tr>
			<td>Imaging software CfNT</td>
			<td>Custom-written</td>
			<td></td>
			<td>developed by Michael M&#252;ller, MPI for Medical Research, Heidelberg, Germany</td>
		</tr>
		<tr>
			<td>IgorPro</td>
			<td>Wavemetrics</td>
			<td></td>
			<td><a href="http://www.wavemetrics.com/">http://www.wavemetrics.com</a></td>
		</tr>
		<tr>
			<td>VC3-4 System focal perfusion system</td>
			<td>ALA Scientific Instrument</td>
			<td></td>
			<td>with 100 &#956;m-diameter tip manifold; http://www.alascience.com/</td>
		</tr>
		<tr>
			<td>Mai Tai HP DeepSee (Ti:Sapphire laser)</td>
			<td>Newport Spectra-Physics</td>
			<td></td>
			<td><a href="http://www.spectra-physics.com/">http://www.spectra-physics.com</a></td>
		</tr>
		<tr>
			<td>Movable objective microscope (MOM), 
			Designed by W. Denk, MPImF, Heidelberg, Germany</td>
			<td>Sutter Instruments</td>
			<td></td>
			<td><a href="http://www.sutter.com/MICROSCOPES/index.html">http://www.sutter.com/MICROSCOPES/index.html</a></td>
		</tr>
		<tr>
			<td>XLUMPlanFL 20x/0.95w objective</td>
			<td>Olympus</td>
			<td></td>
			<td><a href="http://www.olympusamerica.com/">http://www.olympusamerica.com</a></td>
		</tr>
		<tr>
			<td>Vaporizer 100 series</td>
			<td>Surgivet</td>
			<td></td>
			<td><a href="http://www.surgivet.com/">http://www.surgivet.com</a></td>
		</tr>
		<tr>
			<td>Ionomycin, Calcium salt</td>
			<td>Life technologies</td>
			<td>I24222</td>
			<td><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com</a></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td><a href="https://www.sigmaaldrich.com/">https://www.sigmaaldrich.com</a></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>101191250</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com</a></td>
		</tr>
		<tr>
			<td>Leica MZ95</td>
			<td>leica microsystems</td>
			<td></td>
			<td><a href="http://www.leica-microsystems.com/">http://www.leica-microsystems.com/</a></td>
		</tr>
	</tbody>
</table>

imaging ca2+ dynamics in cone photoreceptor axon terminals of the mouse retina

视网膜光感受器锥（锥）担任日光视野，并歧视色彩的基础。他们受变性，往往导致失明，在许多视网膜疾病。钙（Ca 2+），在感光体信令和代谢的关键的第二信使，已经提出了被间接地与光感受器变性在各种动物模型相联系。系统的学习生理学锥的这些方面和病理生理学已经阻碍了从这些小细胞在视网膜由视杆细胞为主的鼠标电记录，特别的困难。为了避免这个问题，我们使用表达上述基因编码的钙生物传感器的TN-XL只在锥和可与小鼠模型为光感受器变性可杂交的转基因小鼠系建立了双光子钙成像协议。这里描述的方案涉及制备垂直部分（R20;切片小鼠和锥钙水平光刺激诱 ​​发的变化光学成像视网膜“）。该协议还允许“在片测量”绝对的钙离子浓度;作为录音可被随后的校准。这个协议使研究成功能性锥体性能和预期有助于锥体的Ca的理解2+信令以及钙离子的光感受器的死亡和视网膜变性的潜在参与。

通过组合垂直切片（ 图1）从HR2.1制备：TN-XL小鼠视网膜与双光子成像（ 图2），我们能够进行在锥光感受器轴突终末的光刺激诱 ​​发的Ca 2+信号比例测量。这种方法是在访问和以上合成的Ca 2+指标预先存在的光学成像方法可视化小鼠锥体一个显著改善。例如，该基因编码的比例的Ca的圆锥特异性表达2+生物传感器的TN-XL大大便利锥轴突终端的识别（参见感兴趣区中图3A）。因此，我们的协议消除了来源不明的记录信号的风险，例如“混合”的捐款来自锥和视杆轴突末端的信号。 我们的方法允许在INDIVI的水平解决审单的响应双锥轴突终末。的Ca 2+从锥形终端光诱发挤压带标记的增加及在供体和受体荧光，分别为（ 图3B）的降低。降低荧光比（R）对应于钙 ​​离子从锥形终端（ 图3C），引起的锥体和电压门控的Ca 2+通道的随后关闭的光致超极化的挤压。因为几个锥形轴突终端可以同时监视（ 图3A），数据采集是非常有效和数百锥体的Ca 2+响应可以被收集在一个单一实验。通过评估这些反应定量（ 图4），锥的Ca 2+信号进行比较和在各种条件下的（见讨论）来评价。 它往往是足够用FRET的受体（柠檬色）和端供体（ECFP）荧光之间的比（F A / F D;有关详细信息，请参见3.4）作为细胞内钙离子水平的相对度量。然而，在必要时，其比例是可以校准，以获得绝对细胞内Ca 2+浓度（[Ca2 +浓度）（ 图5）。在这个协议中使用的背景照明（详见10和图3的传说），校准产生一个估计的绝对休息的[Ca 2+]在“野生型”HR2.1：TN-XL鼠标锥端243±159纳米（平均值±标准差），这是报告的小鼠在文献11的范围内。 <img alt="图1" src="/files/ftp_upload/52588/52588fig1.jpg" /> 图1.准备的垂直切片视网膜。 （A）孤立和被压扁的视网膜准备切片。（B）一块长方形的视网膜（See值在一个红色框）切片后安装在滤纸膜（深灰色）。（C）的膜-安装的视网膜切片固定在使用润滑脂的玻璃盖玻片的顶视图。（D）一种视网膜切片的一部分的示意图（见C红色框）。锥光感受器显示为绿色。五，腹; D，背; N，鼻; T，时间; OS，外段; ONL，外核层; OPL，外网状层; INL，内核层; IPL，内网层;保利协鑫，神经节细胞层。 <a href="https://www.jove.com/files/ftp_upload/52588/52588fig1large.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/52588/52588fig2.jpg" /> 图2.双光子视网膜切片成像：插图记录配置 上：水浸泡物镜聚焦的是点扫描激光（红色向下箭头）到视网膜，并收集发射的荧光（向上箭头）。该透镜还收集透射红外光从照明LED使用CCD照相机成像的切片时安装在冷凝器下方中心 ：视网膜切片安装在记录室中，并灌流细胞外溶液底部 ：聚光用透镜聚焦的光由刺激发光二极管（与用于CCD相机成像的红外LED）通过记录室的透明底部进入视网膜组织。红外灯，红外发光二极管;的CCD，电荷耦合器件; BP，带通。 <a href="https://www.jove.com/files/ftp_upload/52588/52588fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52588/52588fig3.jpg" /> 图3.光呼魂ð 钙在小鼠锥感光细胞轴突终末反应。 （A） 左图：录音集中在光感受器锥体（绿色）的视网膜切片突触终端（红色框） 右：例一记录区域的7个人终端。使用两个信道的荧光被记录：（535 BP 50上）和一个用于FRET的供体ECFP（底部; 480 BP 32）一个用于FRET的受体黄水晶（B）的光诱发的变化柠檬色（F A）和ECFP（F D）荧光记录在一个单一的投资回报率。（C） 钙离子反应（如比F A / F D）圆锥终端的一系列1秒耀眼的光芒在闪烁5秒间隔。投资回报率，感兴趣区域; BP，带通滤波器;男，荧光强度; AU，任意单位; FRET，福斯特共振能量转移; ECFP，增强青色荧光蛋白。刺激强度（如对HOTO异构化率在10 3·P·-1每锥）：13.0和12.8 M和S-视蛋白，分别加入到背景水平（=指示灯熄灭，激发激光〜10扫描） <a href="https://www.jove.com/files/ftp_upload/52588/52588fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52588/52588fig4.jpg" /> 光诱发锥钙离子的响应图4.典型的定量分析（一）光诱发的Ca 2+从单一的终端锥反应（如ΔR）（单独审判：灰色痕迹，N = 16;平均： 黑色痕迹）（B）的参数确定的平均值的Ca 2+响应：静息钙水平（R 基本 ）表示前光刺激的基线，响应大小作为芳EA-下的曲线（R A）和峰值幅度（R 放大器 ），响应发病动力学（叔上升 ，从20％至80％R 放大器的时间间隔）和偏移（叔衰减 ，时间间隔从80％至R 安培的20％）。 <a href="https://www.jove.com/files/ftp_upload/52588/52588fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52588/52588fig5.jpg" /> 图5.估计绝对Ca 2+浓度。静息钙离子浓度（ 钙离子浓度 ，因为比F A / F D）记录在8锥轴突终末在TN-XL鼠标（圆圈，平均±SD）对于不同的胞外溶液：在标准外培养基两次（Ctrl键1和2），然后以最小的（“零”）的[Ca 2+]（10毫米EGTA）的ð具有较高的钙离子浓度（2.5毫米），在5微米的存在霉素平衡细胞内外钙离子后两个条件。 <a href="https://www.jove.com/files/ftp_upload/52588/52588fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina at JoVE.com

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

我们描述了一个协议，以监控使用前体切片准备小鼠视网膜锥感光细胞的轴突终末钙离子动力。该协议允许锥钙离子信号的一个重要哺乳动物模型系统全面的研究，鼠标。

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

imaging-ca2-dynamics-cone-photoreceptor-axon-terminals-mouse

Research

JoVE Journal

Neuroscience

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

10.6K 次观看.  University of Tübingen. 我们描述了一个协议，以监控使用前体切片准备小鼠视网膜锥感光细胞的轴突终末钙离子动力。该协议允许锥钙离子信号的一个重要哺乳动物模型系统全面的研究，鼠标。 

视频: Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

科研

神经科学

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

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

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

血管再生在中枢神经系统中使用的小鼠视网膜评估

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

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

的vibratome切片小鼠视网膜准备感光文化

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

在肠神经系统的原位 钙成像

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...