Name: imaging ca2+ dynamics in cone photoreceptor axon terminals of the mouse retina
Uploaded: 2015-05-06T13:00:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina at JoVE.com

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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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><tbody><tr><td>High vacuum grease</td><td>Dow Corning</td><td>1018817</td><td>&lt;a href=&quot;http://www.dowcorning.com/&quot;&gt;http://www.dowcorning.com&lt;/a&gt;</td></tr><tr><td>Cover slips</td><td>R. Langenbrinck</td><td></td><td>24 x 60 mm; 

&lt;a href=&quot;http://www.langenbrinck.com&quot;&gt;http://www.langenbrinck.com&lt;/a&gt;</td></tr><tr><td>Glass slides</td><td>R. Langenbrinck</td><td></td><td>76 x 26 mm; 
&lt;a href=&quot;http://www.langenbrinck.com&quot;&gt;http://www.langenbrinck.com&lt;/a&gt;</td></tr><tr><td>Blades for tissue chopper</td><td>MARTOR KG</td><td>ARGENTAX No. 1044</td><td>0.25 mm; 

&lt;a href=&quot;http://www.martor.de&quot;&gt;http://www.martor.de&lt;/a&gt;</td></tr><tr><td>Tissue chopper</td><td>Custom-build</td><td></td><td>Based on a design by F. Werblin&lt;sup&gt;23&lt;/sup&gt;, 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 &amp;micro;m;

&lt;a href=&quot;http://www.emdmillipore.com&quot;&gt;http://www.emdmillipore.com&lt;/a&gt;</td></tr><tr><td>Isoflurane CP</td><td>CP pharma</td><td>AP/DRUGS/220/96</td><td>&lt;a href=&quot;http://www.cp-pharma.de/&quot;&gt;http://www.cp-pharma.de&lt;/a&gt;</td></tr><tr><td>UV/green LED-based full-field stimulator</td><td>Custom-build</td><td></td><td>for details, see&lt;sup&gt;10&lt;/sup&gt;</td></tr><tr><td>Open source microprocessor board</td><td>Arduino</td><td></td><td>&lt;a href=&quot;http://www.arduino.cc/&quot;&gt;http://www.arduino.cc&lt;/a&gt;</td></tr><tr><td>Imaging software CfNT</td><td>Custom-written</td><td></td><td>developed by Michael M&amp;uuml;ller, MPI for Medical Research, Heidelberg, Germany</td></tr><tr><td>IgorPro</td><td>Wavemetrics</td><td></td><td>&lt;a href=&quot;http://www.wavemetrics.com/&quot;&gt;http://www.wavemetrics.com&lt;/a&gt;</td></tr><tr><td>VC3-4 System focal perfusion system</td><td>ALA Scientific Instrument</td><td></td><td>with 100 &amp;mu;m-diameter tip manifold; 

&lt;a href=&quot;http://www.alascience.com/&quot;&gt;http://www.alascience.com/&lt;/a&gt;</td></tr><tr><td>Mai Tai HP DeepSee (Ti:Sapphire laser)</td><td>Newport Spectra-Physics</td><td></td><td>&lt;a href=&quot;http://www.spectra-physics.com/&quot;&gt;http://www.spectra-physics.com&lt;/a&gt;</td></tr><tr><td>Movable objective microscope (MOM),&lt;br/&gt; Designed by W. Denk, MPImF, Heidelberg, Germany</td><td>Sutter Instruments</td><td></td><td>&lt;a href=&quot;http://www.sutter.com/MICROSCOPES/index.html&quot;&gt;http://www.sutter.com/MICROSCOPES/index.html&lt;/a&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>XLUMPlanFL 20X/0.95w objective</td><td>Olympus</td><td></td><td>&lt;a href=&quot;http://www.olympusamerica.com/&quot;&gt;http://www.olympusamerica.com&lt;/a&gt;</td></tr><tr><td>Vaporizer 100 series</td><td>Surgivet</td><td></td><td>&lt;a href=&quot;http://www.surgivet.com/&quot;&gt;http://www.surgivet.com&lt;/a&gt;</td></tr><tr><td>Ionomycin, Calcium salt</td><td>Life technologies</td><td>I24222</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/&quot;&gt;http://www.lifetechnologies.com&lt;/a&gt;</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td>&lt;a href=&quot;https://www.sigmaaldrich.com/&quot;&gt;https://www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>101191250</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>Leica MZ95</td><td>leica microsystems</td><td></td><td>&lt;a href=&quot;http://www.leica-microsystems.com/&quot;&gt;http://www.leica-microsystems.com/&lt;/a&gt;</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

10.7K 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

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 ...

科研

神经科学

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

上丘的双光子和宽场钙成像

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

作者聚焦:揭示上丘的神经编码和视觉处理机制 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

小鼠上丘的钙成像

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

成像钙

摘要

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小鼠上丘的钙成像

小鼠上丘的钙成像

小鼠上丘的钙成像

成像钙

摘要

探索更多视频

此视频中的章节

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

在Wholemount大鼠视网膜免疫组化和钙成像方法

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

小鼠视网膜的经瞳双光子体内成像

在体内 小鼠劣质橄榄中的钙成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

小鼠上丘的钙成像

小鼠上丘的钙成像

小鼠上丘的钙成像

在体内小鼠劣质橄榄中的钙成像