这项工作由 Inserm, 大学蒙彼利埃, 视网膜法国, 联合国家 des Aveugles et Déficients Visuels (UNADEV), 协会综合征钨, 基金会倾吐 la 研究 Médicale, 法兰西基金会和卓越实验室支持EpiGenMed 计划。

《实验议定书》获得了国家 #233 和 #233;d icale (Inserm; 研究);蒙彼利埃, 法国), 符合欧洲的指示, 并符合帕特声明的使用动物在眼科研究。它是根据《动物实验 (CEEALR; nuCEEA-LR-12123) 》中的《伦理道德的一致性的协议进行的. 
 1. 设备设置和预成像准备 注意: 在这里, OCT 是利用光谱域 (SD) 眼科成像系统 ( 图 1A ) 在小鼠视网膜上进行的。SD OCT 设备包括一个基地和动物成像坐骑 (AIM) 与啮齿动物对齐阶段 (RAS) ( 图 1B )。该底座包括计算机、oct 引擎、SD oct 探头和鼠标专用镜头。探头安装在目标上, 其中包括 Z 转换器。该 RAS 用于鼠标定位感谢表与 X 和 Y 翻译, 磁带可以旋转和旋转, 以及可移动的叮咬酒吧与鼻带。制造商提供的软件允许对 OCT 文件进行获取和分析, 尽管后者也可以通过开源图像处理程序完成. 
<ol> <li> 将探测器牢牢地放置在目标中. </li>
	<li> 将鼠标特定的镜头连接到探测器. </li>
	<li> 调整特定镜头的参考臂设置 (此处为 086, 位置为 964). </li>
	<li> 确保鼠标特定的镜头离卡带足够远, 将咬杆与鼻带连接到卡带上. 
	注: 镜头与卡带之间的距离可与 Z 型转换器螺钉一起调整, 位于目标背面。鼻带是一个商业上可用的弹性带, 其张力应根据鼠标大小调整. </li>
	<li> 打开 "电源 (购物车右下角)" 和 "计算机". </li>
	<li> 若要启动图像处理程序, 请双击屏幕上相应的快捷方式. 
	注: 这取决于透镜的类型;这里, 老鼠视网膜. </li>
	<li> 创建新的临床研究并添加所需的协议。否则, 使用现有的临床研究和/或协议. 
	<ol> <li> 通过选择和 #34 添加新的研究; 研究名称和 #34;; #34; ID 和 #34;; #34; 处理臂规格和 #34; (这里, #34; 未和 #34;); 预定义和 #34; 扫描协议和 #34; 如果它们存在. </li>
	</ol> </li> <li> 选择 #34; 考官和 #34; 在和 #34; 临床研究和 #34; 部分. 
	注意: 对于一个新的 #34; 考官和 #34;, 去和 #34; 设置考官和 #38; 医生和 #34; 定义它. </li>
	<li> 通过单击添加患者, 在患者/考试部分添加新病人; 进入 #34; 身份证和 #34;, #34; 姓名和 #34;、#34; 姓氏和 #34;; #34; 性与 #34; #34;d 吃了生与 #34; (可选). 
	注意: 在测试每只鼠标之前, 如果更方便的话。请确保该 ID 不长于10字符。对于鼠标, 两个眼睛的折射误差为 0, 轴向长度为 23.0. </li>
	<li> 单击和 #34; 添加考试和 #34; 选择所需的和 #34;P rotocol 和 #34; 通过添加和 #34;P 重置扫描和 #34; 从列表中, 从将首先测量的眼睛开始 (这里, 右眼), 或通过自定义扫描. </li>
	<li> 若要自定义扫描, 请使用现有的扫描作为模板并对其进行编辑, 或从头开始通过和 #34 创建它; 添加自定义扫描和 #34; 选项。将所有扫描添加到列表后, 使用 "#34" 定义新协议; 输入和 #34; 新协议名称和 #34; 选项 (OS:26 险恶, 左眼;26: 德克斯特, 右眼). 
	注意: 在这里, 协议涉及三扫描: (i) 径向扫描, 具有1.4 毫米直径, 0 毫米水平和垂直偏移, 1000 行 a 扫描/b 扫描, 100 b 扫描/卷, 1 帧/b 扫描, 80 行非活动 a 扫描/b 扫描, 和1卷;(二) 矩形扫描, 长度和宽度为1.4 毫米, 0 和 #176; 角度, 0 毫米水平和垂直偏移, 1000 行 a 扫描/b 扫描, 100 B 扫描, 1 帧/b 扫描, 80 行非活动 a 扫描/b 扫描, 和1卷;和 (iii) 环形扫描, 最大直径为0.4 毫米和0.6 毫米, 0 毫米水平和垂直偏移, 1000 行扫描/b 扫描, 3 b 扫描/卷, 48 帧/b 扫描, 80 行非活动 a 扫描/b 扫描, 和1卷. </li>
</ol> 2。鼠标准备 <ol> <li> 眼球扩张和固定 <ol> 在数据采集前至少15分钟, 将10% 肾上腺素滴眼液注入老鼠眼中, 除去多余的, 并注入0.5% 卡滴眼液. <li>
		注: 第一次扩张可以做一次对所有的老鼠, 将在会议上进行测试。确保液体在室温下. </li>
		<li> 在诱导全麻 (步骤 2.2) 后, 立即管理0.4% 盐酸因滴眼液, 使其保持在3秒的位置以麻醉和固定眼睛。然后, 擦掉掉滴, 再重复10% 肾上腺素和0.5% 卡的灌注, 以确定眼睛是否有很好的扩张. 
		注意: 确保液体在室温下, 并且老鼠不会吞下因. </li>
	</ol> </li> <li> 全麻 <ol> <li> 在盐水中制备氯胺酮 (20 毫克/毫升) 和嗪 (1.17 毫克/毫升) 的麻醉液。存储在4和 #176; C 最多2周. </li>
		<li> 大约5分钟前测试, 腹腔注射 5-10 和 #181; L 的麻醉液每1克的身体质量, 取决于年龄和大小的鼠标. 
		注意: 幼鼠和/或瘦老鼠需要较少的麻醉剂, 少花点时间麻醉, 但也能更快地苏醒。这里, 8 和 #181; 升/克 (160 毫克/千克氯胺酮, 9.33 毫克/千克的嗪). </li>
		<li> (可选) 用粘稠滴眼药水或眼部凝胶润滑眼睛, 以避免角膜干燥. </li>
	</ol> </li> </ol> 3。鼠标定位 <ol> <li> 用粘性 glycol-based 眼药水润滑眼睛以提供角膜水化. 
	注意: 如果在考试中眼睛看起来很干燥, 请重新申请眼药水. </li>
	<li> 将鼠标包裹在一张外科纱布上以保持体温. </li>
	<li> 使用海绵或棉芯, 在每只眼睛上应用一层薄薄的眼部凝胶0.3% 甲, 以减少光的折射, 避免混浊, 并确保角膜水化。这样做的时候, 把睫毛和胡须移到一边. </li>
	<li> 将鼠标置于盒式磁带中, 并将其头部伸直并向前指向. </li>
	<li> 轻轻打开咬杆钳, 将杆放在嘴里; 用鼻带固定位置. 
	注意: 确保咬杆位于卡带的中间. </li>
	<li> 如果右 (左) 眼正在测试, 请在右侧 (左) 侧放置一个棉卷. </li>
	<li> 同时在鼠标特定的镜头上保持夹紧的瞄准头, 通过逆时针旋转 Z 转换器螺钉将其对准眼睛. </li>
	<li> 通过转动和旋转卡带, 并通过转动咬杆和 X 转换器螺钉, 预先设定鼠标的位置; 目标是让右眼直视镜头, 从而使眼睛和镜片的光轴对准. 
	注意: 如果鼠标太高或过低, 应首先使用 Y 型转换器螺钉. </li>
	<li> 选择 #34 中的第一个扫描; 考试和 #34; 单击 #34; 开始瞄准和 #34; 并对视网膜成像进行进一步的人工调整. 
	<ol> <li> 使用 Z 转换器螺钉, 在左侧面板上垂直移动视网膜 ( 图 2 , 水平 B 扫描对齐), 并水平在 r 上可选面板 ( 图 2 , 垂直 B 扫描对齐). </li>
		<li> 旋转盒式磁带, 通过将 ONH 向上或向下移动, 使 ONH 位于右侧面板的中间。使用咬杆螺钉在右侧面板上伸直视网膜。旋转卡带, 将 ONH 放置在左侧面板的中间. </li>
		<li> 使用 X 转换器螺钉在左侧面板上水平视网膜。记住每个调制器的主要功能, 进一步调整视网膜的位置以集中 ONH. 
		注: 如有必要, 使用 Y 型转换器螺钉在右侧面板上上下移动 ONH。该位置可以在扫描之间的任何时间被细化. </li>
	</ol> </li> </ol> 4。ONH 和视网膜的 SD oct 成像 <ol> <li> 一旦内容具有调整, 请单击 #34; 启动快照和 #34; 开始 sd oct 扫描. 
	<ol> <li> 如果不需要3D 成像, 请取消选中 OCU 选项. </li>
	</ol> </li> <li> 保存扫描和报表. </li>
	<li> 继续进行下一次扫描. </li>
	<li> 图像的第二眼, 在缩回镜头后, 相应地打开卡带, 重复步骤 3.6-4.3. </li>
</ol> 5。购置完成 <ol> <li> 当采集完成时, 将鼠标从卡带上取出, 用0.3% 甲的眼部凝胶涂抹在每只眼睛上, 并将鼠标放在加热板上唤醒. </li>
	<li> 上次购置后, 关闭软件并关闭计算机和 OCT 计算机 (电源按钮). </li>
	<li> 用消毒剂清洗卡带. </li>
</ol> 6。分析 <ol> <li> 用于视网膜层厚度测量, 请使用制造商提供的自动分割软件. 
	<ol> <li> 单击 和 #34;P atient 和 #34; , 选择所需的 和 #34; 考试和 #34; 然后单击 和 #34; 查看考试和 #34; 选项. 
		<li> 通过右键单击所需扫描的文件夹图标, 将所需的 oct 数据加载到 oct 软件中. </li>
		<li> 右键单击 B 扫描; 通过启用10测量卡尺来配置卡尺; 并输入它们的名称、角度和颜色. </li>
		<li> 通过右键单击 B 扫描来选择所需的卡尺; 将其相应放置到视网膜上进行测量. 
		注意: 对于以 ONH 为中心的扫描, 在它的每一侧设置5卡尺, 彼此等距。对于 peripapillary 分析, 请确保卡尺没有放置太远的 ONH。对于径向扫描, 分析 OCT 软件选择的每扫描10图片。在 和 #34; 报告和 #34; 文件夹中可以找到它们的编号. </li>
		<li> 通过右键单击 B 扫描并单击 并 #34 将结果保存在电子表格软件程序中进行分析; 保存结果和 #34; 
		注意: 结果可以在与扫描数据相同的文件夹中找到. </li>
	</ol> </li> <li> 也可以使用自制的卡尺宏, #34; MRI 视网膜工具和 #34, 为开源图像处理程序开发 (参见材料表). 
	<ol> <li> 确保 #34; MRI 视网膜工具和 #34; 和 #34; 修改多边形剖面工具和 #34; 宏处于活动状态。加载图像。单击 m 按钮开始测量. 
		注意: 宏自动在 ONH 的两侧创建一个0.2 毫米长的测量盒。每个卡带包含5测量点, 功能作为卡尺。它们的横向位置是不可更改的, 并为径向扫描中的 peripapilla 定制。盒式磁带的水平位置是预先定义的, 用来测量纤维/协变厚度, 但可以很容易地修改以测量 GC 复杂层。如果扫描质量较差, 则需要调整水平位置, 或者需要从分析中排除图片. </li>
		<li> 对于水平调整, 单击 "e" 按钮。在新开的 #34; ROI 管理器和 #34; 窗口中, 选择第一个卡带. </li>
		<li> 单击蓝色多边形按钮, 然后通过单击图片中测量图层的边框来调整第一个卡带的位置. </li>
		<li> 对第二个卡带进行重复, 首先在 #34 中选择它; ROI 管理器和 #34; 窗口, 然后单击相应的图像。点击 r 按钮测量。查看 #34 中的结果; 测量和 #34; 窗口. 
		注意: 结果可以随时复制到电子表格软件程序中。它们包含了正确的卡带 (r), 左卡式 (l) 和总计: Intden-综合密度在卡带内的以下值;面积: 测量面积, 以 mm 2 ;长度的卡尺内的卡带, 在毫米;平均值: 在卡带内的平均信号强度;和 Std: 信号平均强度的标准误差. </li>
		<li> 继续执行下一图像. </li>
		<li> 进一步分析电子表格软件程序中的数据. 
		注意: 要查找层的平均厚度, 请取十 Len 值. </li>
	</ol>
	</li>
</ol>

<ol>
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作者没有什么可透露的。

OCT 系统是一种非侵入性的活体成像方法, 它提供了高分辨率的视网膜横断面扫描。因此, 它的主要优势是它的详细分析的潜力, 与极好的机会, 转置协议常规适用于人类的鼠标模型。
在 Opa1delTTAG突变小鼠的例子中, SD OCT 结果显示了纤维和 GC 复合层厚度的增加, 这使得对 DOA 病理生理学的进一步探索9。这是不可能完全通过组织学分析。相比...

OCT 是一种诊断工具, 有助于检查视网膜结构1, 包括视神经头 (ONH)。多年来, 它已经成为人类疾病进展的可靠指标2,3, 以及啮齿动物4,5。它使用干涉法来创建2µm 轴向分辨率的视网膜层的横断面图像。最内层是视网膜神经纤维层 (纤维), 含有研资局轴突, 其次是神经节细胞层 (协鑫), 其中大部分为研资机构。其次是内部状层 (彩光), 在那里, 王国的树突满足两极, 水平, 和突细胞轴突。这些, 连同水平的细胞, 形成内部核层 (INL), 并且他们的突起连接与感光轴突在外部状层 (组织)。其次是外核层 (只), 与感光细胞体, 并通过外部限制膜 (OLM) 与感光层分离, 也称为内段/外段 (是/OS) 层。最后, 在小鼠视网膜的最后可见层是视网膜色素上皮 (RPE) 和脉络膜 (C)。单独的纤维通常太薄, 不能用老鼠来测量;因此, 分析纤维/协鑫是最好的4,5。另一个可能性是 GC 复合层, 它包含了后者, 除了光子, 使它更厚, 从而更容易测量 OCT 扫描4。因此, OCT 可以提供洞察的病理状态的视网膜, 如在 OPNs。
另一种方法是, 用剖检组织学来分析小鼠视网膜的厚度。然而, 这项技术面临的限制与组织收集, 固定, 切割, 染色, 安装,等因此, 一些缺陷, 如微妙的厚度变化, 无法检测到。最后, 由于同一鼠标不能在多个时间点测试, 所以每项研究的动物数量大大增加, 不像 OCT。所有的侵入、高分辨率、重复的可能性、时间的监控以及 OCT 技术的易用性使其成为视网膜疾病研究的首选方法。
小鼠模型用于识别基因缺陷并阐明 retinopathies6的分子机制。OPN 是一种视网膜病变, 对视神经 (ON) 有很大的损害, 由大约120万王国的神经轴突构成。OPN 可以集中在上, 也可以是次要的其他疾病, 先天或不7, 导致视觉领域的损失, 后来, 失明。OPN 的特征性状为研资损失和损害, 可在人类 OCT 中观察到为纤维和协变细化2,3。同时, OPN 的病理生理学尚不清楚, 因此需要对小鼠视网膜进行检测。
这份手稿描述了视网膜层厚度的成像和量化, 使用 Opa1delTTAG鼠标线8,9的例子, 一个显性光学萎缩 (DOA)10的模型。评估研资局的病理生理学、放射、矩形和环形扫描的数量。这是通过 OCT 软件提供的标准卡尺或为开源图像处理程序开发的自制宏完成的。标准卡尺是难以操作, 往往比纤维/协进会厚, 而自制卡钳是易于使用, 重现性, 更精确。该宏在 peripapillary 区域的 ONH 的两侧对自动检测到的层进行了测量, 在5点和固定位置上。所提出的协议的目的是描述 OCT 扫描获得指定视网膜定位, 重点是视网膜。

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optical coherence tomography: imaging mouse retinal ganglion cells in vivo

视网膜结构改变是眼科疾病的常见表现。光学相干断层扫描 (OCT) 使他们的识别在体内-迅速, 反复, 并在高分辨率。该协议描述 OCT 成像在鼠标视网膜作为一个强大的工具, 研究光学神经病 (OPN)。OCT 系统是一个 interferometry-based, 非侵入性的替代一般的验尸组织学化验。它提供了一个快速和准确的评估视网膜厚度, 使可能跟踪变化, 如视网膜变薄或增厚。我们以 Opa1delTTAG鼠标线为例介绍了成像过程和分析。提出了三种扫描方式, 采用两种定量方法: 标准和自制卡尺。后者是最好的使用在 peripapillary 视网膜的径向扫描;更精确, 更适合分析更薄的结构。这里描述的所有方法都是为视网膜神经节细胞 (王国) 设计的, 但很容易适应其他细胞的数量。总之, OCT 是有效的小鼠模型分, 并有可能用于可靠的评价治疗干预。

SD OCT 技术使视网膜的想象和厚度分析可以与组织学相媲美, 但速度更快且更详细 (图 3)。正如野生 C57Bl/6 小鼠所提出的, 即使 SD OCT 扫描 (图 3A, 右) 的质量不如视网膜横断面 (图 3A, 左) 的图像好, 它可视化更多的层 (例如, OLM)。此外, 它只需要大约40分钟, 包括小鼠准备, 与几天或几周的组织学分析。最?...

Watch this Scientific Journal Video about Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo at JoVE.com

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

这篇手稿描述了一个协议为在体内成像的小鼠视网膜与高分辨率光谱领域光学相干断层扫描 (SD OCT)。它的重点是视网膜神经节细胞 (王国) 在 peripapillary 地区, 有几个扫描和量化的方法描述。

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 ...

optical-coherence-tomography-imaging-mouse-retinal-ganglion-cells

Research

JoVE Journal

Neuroscience

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

19.1K 次观看.  INSERM U1051, Institut of Neurosciences of Montpellier. 这篇手稿描述了一个协议为在体内成像的小鼠视网膜与高分辨率光谱领域光学相干断层扫描 (SD OCT)。它的重点是视网膜神经节细胞 (王国) 在 peripapillary 地区, 有几个扫描和量化的方法描述。

视频: Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

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

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

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

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

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

科研

神经科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

蟹电压敏感染料Stomatogastric神经节神经元的光学成像

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

稀疏的头骨皮层的窗口技术<em在体内</em光学相干断层成像

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

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 ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

光相一致性断层扫描血管造影评估的视网膜血管反应

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

在活体钙成像小鼠致病性甘里昂神经元对味觉刺激的反应

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...