是支持的威卢克斯基金会的AK是支持的奥本海姆基金会和相对湿度。我们感谢一Krumbein球形的技术和K.普德进行组织学支持，和J.施密特（莱布尼茨研究所神经生物学，马格德堡，德国）就TIMM染色技术咨询。

所有动物的干预按照欧洲公约动物保护实验动物和使用以及ARVO声明在眼科和视觉研究用动物进行的。所有的实验都是由当地伦理委员会批准。对损伤的小鼠中的程序是在别处9所述。 1，玻璃体内注射锰<ol><li>执行中Mn 2 +注射24小时的MR扫描与助手的帮助之前。腹腔注射5％水合氯醛溶液（420-450毫克/千克体重在无菌PBS中）麻醉动物。有关其他局部麻醉，应用一滴液体conjuncain（0.4％奥布卡因盐酸盐）来之前，眼睛穿刺角膜。注入15纳摩尔Mn 2 +的每只眼睛准备一个7.5毫米的MnCl 2溶液， 例如 ，通过稀释1升1米的MnCl 2原液在132 LH 2 &lt;/ SUB&gt; O。负载5微升的最终解决方案到5微升汉密尔顿注射器连接到34 g小枢纽可拆卸针（RN针）的。 </li><li>当与右眼开始，请将鼠标左键双面双目显微镜下，轻轻打开并修复你的左手的拇指和食指之间的右眼。拿起注射器用你的右手，并采取针靠近尖端的保持。为眼部灯泡无创伤穿刺，小心将针刺入玻璃体在infero颞周长大约1mm远端的角膜缘，从而不遗余力巩膜血管。 </li><li>接着，助手慢慢适用的2微升的总体积，同时控制Hamilton注射器的刻度。在此过程中，监测在显微镜下最优的针的位置，并避免液体的透镜或溅出的穿刺。保持静态地插入另外的30秒的针，然后撤回它慢慢地减少液体从注射部位的泄漏。 </li><li>在整个过程中，特别应注意避免压到眼球。同样，避免恶劣或多次试图刺破眼球灯泡。因为Mn 2 +摄取到视网膜神经节细胞，并沿对运输已经饱和在15纳摩尔的MnCl 2，这稍微不精确的进样量最小化的信号变化。对于视觉投影的双边信号增强，重复注射过程用于左眼。 </li><li>申请氧氟沙星的含（3毫克/毫升）滴眼液和含有泛醇-软膏后，一旦程序，以防止眼部感染和眼干燥。返回小鼠正常居住条件下它们的笼子，直到MR扫描的开始。 </li></ol> 2，动物准备的MRI <ol><li>由2％/ 98％异氟烷/氧气气体混合物的施用麻醉小鼠。安装鼠标鼠标持有人在一个几乎水平，无捻位置。插件ERT成的MR线圈，然后将其在MR扫描仪内进行调整。监测呼吸和心脏率合适的系统。有关技术详情，请参阅赫尔曼等人 20。 </li><li>在MRI，供给麻醉经由由集成管连接到鼠标头部保持器的蒸发器的最初的1.5％/ 98.5％异氟烷/氧气气体混合物的连续吹入。在扫描过程中，根据记录的重要参数（ 即目标为约每分钟40次呼吸平稳呼吸速率）调整麻醉深度。使用加热装置保持在35和37℃下的体表温度稳定的，如通过定位于小鼠的腹腔内有热传感器测量。有关技术详情，请参阅Herrmann 等[17]。 </li><li>扫描之后，从支架释放鼠标和纯氧供应，以加速从麻醉中恢复。另外，通过使用保持体温稳定红灯加热源。 </li></ol> 3，MRI协议<ol><li>该协议被验证为3特斯拉扫描器配备专用的，信噪比高效，小动物线圈（线偏振绞线线圈）与视35毫米×38毫米直径的一个有效字段。操作在发射 - 接收模式线圈。 </li><li>在其最终位置的动物，调节线圈的同一个频率分析仪的辅助下调谐和匹配。手动调整发射机的基准电压和电流的垫片，以优化图像的均匀性和质量。 </li><li>收购Ŧ1加权谢2D图像为0.5毫米，分辨率×0.5㎜×2毫米的规划矢状和横向视图。使用规划MR扫描，获取在冠状测量方向上的MEMR图像，旋转成平行于动物的头沿左右方向的相位编码。为了尽量减少采集时间，使用的视图的矩形区域调整到实际的头部尺寸。基础矩阵256，视场54㎜×50.65毫米×14.08毫米，利用相位编码方向的视图93.8％的矩形区域，和128片0.11毫米：使用以下参数采用一个被宠坏的3D FLASH序列（VIBE 3D）切片厚度与片的分辨率设置为61％。 </li><li>激活平面内插值到512×480×128创建最终图像，提供0.21㎜×0.21毫米* 0.18毫米的有效分辨率（0.1毫米×011㎜×0.09毫米插值），回波时间T E = 6.51毫秒，重复时间T R = 16毫秒，带宽= 160赫兹/像素，翻转角= 22°。应用两个平均值和三个重复，实现约30分钟，总采集时间（T A）。 </li></ol> 4，MRI数据分析<ol><li>使用该软件的syngo fastView分析数据。对于定量信号增强，选择定义的区域Ø在2D平面的MRI录音f兴趣，并决定加强结构（SI MEMRI），组织背景（SI backgr），噪音（SD N）的标准偏差的信号强度（SI）。如有需要，使用鼠标脑图谱，以促进神经解剖定位为外膝体和SC的结构。计算出的对比度噪声比（CNR），使用下式： </li><li> CNR =（SI MEMRI - SI backgr）/ SDÑ </li><li>量化连续三个图像的平均CNR计算每个样品。在双边注射的动物，独立分析每个半球。 </li><li>对于水平，冠状面和矢状面图像的描述，计算从原来的3D MRI数据集多平面重建。这些处理过的图像，不建议进行定量分析。要创建眼膜的动画三维重建（最大强度投影，得到的MIP）- 顶盖投影，使用血管造影后处理软件模块。 </li></ol> 5，Mn 2 +的自显影技术（TIMM染色） <ol><li>对于Mn 2 +的跟踪大脑结构的下列MEMRI TIMM染色，注入的15-150纳摩尔Mn 2 +的ivit 24小时前成像剂量。 </li><li>后的MR扫描，灌注动物用30ml冰冷的0.325％的Na 2 S的PBS（pH 7.4）中。视网膜解剖，冷冻样品中冰冻切片。 </li><li>剪下的cryotome 15微米厚的顺序，赤道部分。 </li><li>根据Angenstein 等[22]在没有固定剂和冷冻保护的执行TIMM染色21。 </li></ol> 6，统计分析使用Student-t检验进行单比较，接着事后方差分析进行统计分析。数据以平均值±标准误差。各个N个数字分别为每个实验给出。结果到达压力 ≤0.05顷认为有统计学显著性（P≤0.05，*，P≤0.01，**，P≤0.001，***）。

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作者宣称，他们有没有竞争的财务权益。

视觉系统的MEMRI延伸常规神经生物学技术的幼稚和病理条件下评估功能。除了 ​​提供独特的洞察到一个孤立的中枢神经纤维束的完整性，这种成像方法可以很容易地辅以行为测试， 例如 ，验光和视觉水性任务，调查一个给定的范例视觉感知的直接后果。它还连接电生理和病理调查与体内功能的视觉表征。该技术是高度可靠和可重复使用相同的内群体未成年人的个体差异（参见<stro...

基于其良好的神经解剖结构的啮齿动物视觉系统提供了独特的机会，以评估药物的化合物和它们的能力，调解神经保护1或促再生作用2,3。此外，它允许对小鼠突变体的功能及神经解剖特点的研究，如最近列举的缺乏突触前支架蛋白巴松管4只小鼠。此外，辅助工具的广谱提供了额外的视网膜神经节细胞（RGC）和RGC轴突数量，以及研究资助局的活动， 如为特色，通过电图和行为测试，和皮质重排由内在信号光学成像的决心。在激光显微镜的最新技术发展在视神经（ON）和脑整装标本深部组织荧光成像使在研资局再生原位可视化。在这种histologiCal的方法，基于四氢呋喃组织清算与光片荧光显微镜组合允许单纤维的分辨率，重新进入deafferented ON和视束5。虽然这种技术可能是决心和意志的增长模式优越，他们不使个人成长活动，这是特别理想，以评估长期再生的过程中重复和纵向分析。 对比增强MRI已用于小鼠和大鼠6,7的眼膜，顶盖投影的微创可视化。这可以通过直接的眼内递送顺磁离子（ 如 Mn 2 +的）的视网膜细胞来实现。作为钙类似物，Mn 2 +的结合到RGC胞体通过电压门控钙通道，并沿着完整ON和视束的轴突细胞骨架的主动转运。虽然它积聚在脑细胞视觉投影， 即在外侧膝状体（LGN）和上丘（SC），跨突触传播到初级视觉皮层出现8,9可以忽略不计，虽然它可能会出现10,11。在MR测序，顺Mn 2 +的增强磁共振造影主要是通过缩短Ŧ1自旋-晶格弛豫时间12。该Mn 2 +增强磁共振成像（MEMRI）已在大鼠的各种神经解剖和功能的研究，包括轴突再生和变性受伤13,14后的评估中得到成功应用，将眼膜-顶盖突起15的精确解剖标测，以及轴突转运特性的药物治疗16后的决心。在剂量，毒性和神经元Mn 2 +的吸收和运输，以及改进的MRI方案动力学近期的改进已经扩大其应用到转基因研究9小鼠用3特斯拉的扫描仪在临床实践17常用。 在这里，我们提出了适用于纵向一MEMRI协议在鼠标眼膜-顶盖投影的体内成像和评估天真和各种神经退行性变的条件下Mn 2 +的依赖性信号增强体现了它的适用性。我们的协议的地方特别强调磁共振数据采集在一个温和的3个T磁场通常比动物专用扫描仪更加方便。在天真的小鼠中，我们说明了如何系特有的信号强度可以大大而重复地成为后玻璃体（ivit）Mn 2 +的应用增加。定量，Mn 2 +的传播沿着视觉投影独立地发生正常的老化过程（3和26个月大的小鼠之间测得）和增强是难治的视觉刺激和适应黑暗。与此相反，锰<s高达&gt; 2 +富集丘脑和中脑中心是减少急性上挤压伤18以及在NFKB1敲除小鼠（P50 KO）从自发凋亡RGC的死亡和ON变性19痛苦。因此，在扩大传统的组织学分析，动物个体的纵向MEMRI分析，可以为神经变性过程的独特的动力学分析。这应该证明与药物或基因干预相关的神经保护和轴突再生的研究很有用。

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			<td height="21" style="height:21px;width:363px;">Manganese (II) chloride solution 1M</td>
			<td style="width:269px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">M1787</td>
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			<td>Dr. Mann Pharma, Berlin, Germany</td>
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			<td>0.4% oxybuprocaine hydrochloride</td>
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			<td>Dr. Mann Pharma, Berlin, Germany</td>
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			<td>420-450 mg/kg body weight</td>
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			<td>Actavis, Munich, Germany</td>
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			<td>SYR 5 &#181;l, 75 RN, no NDL</td>
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			<td style="width:269px;">Hamilton Company, Reno, NV, USA</td>
			<td style="width:119px;">207434/00</td>
			<td>removable needle RN, 34 gauge, lenght 38.1 mm, point style 4</td>
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			<td style="width:269px;">Zeiss, Oberkochen, Germany</td>
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			<td>Doty Scientific Inc., Columbia, SC, USA</td>
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			<td height="42" style="height:42px;width:363px;">Frozen section medium NEG-50</td>
			<td style="width:269px;">Thermo Fisher Scientific, Schwerte, Germany</td>
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			<td>tissue embedding for cryo-sections</td>
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			<td height="21" style="height:21px;width:363px;">Sodium sulfide nonahydrate (Na2S &#215; 9 H2O)</td>
			<td>Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">208043</td>
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			<td height="21" style="height:21px;width:363px;">gum arabic</td>
			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
			<td style="width:119px;">4159</td>
			<td rowspan="5">for TIMM staining</td>
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			<td style="width:269px;">Roth, Arlesheim, Switzerland</td>
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in vivo imaging of optic nerve fiber integrity by contrast-enhanced mri in mice

啮齿动物视觉系统包含形成视神经进入丘脑和中脑中心和突触后投射到视觉皮层的视网膜神经节细胞和它们的轴突。基于其独特的解剖结构和便利的交通，它已成为青睐的结构对神经元的存活，轴突再生，突触可塑性研究。在MR成像的最新发展，使体内使用的可视化介导锰对比度增强（MEMRI）这款投影的眼膜，顶盖部分。在这里，我们提出了一个MEMRI协议说明视觉投影在小鼠中，其中（200微米）3分辨率可以使用常见的3特斯拉的扫描仪来实现的。我们将演示如何玻璃体腔注射15纳摩尔的MnCl 2单剂量导致24小时内完整投影的饱和增强。与异常的视网膜，改变信号强度都INDEPEN凹痕恰逢视觉刺激或生理老化。我们还应用此技术来监测纵向轴索变性响应急性视神经损伤，范式其中Mn 2 +的运输完全逮捕的病变部位。相反，有源Mn 2 +的传输是定量相称的生存能力，数量和轴突纤维的电活动。对于这样的分析，我们举例说明的Mn 2 +转运动力学以及在转基因小鼠模型（NF-κBp50的KO）显示的感官自发萎缩，包括视觉，突出视觉路径。在这些小鼠中，MEMRI表示减少，但不会延迟Mn 2 +的传输相比，野生型小鼠，从而通过NF-κB突变揭示的结构和/或功能障碍的迹象。 综上所述，方便MEMRI桥梁体内试验和验尸组织学的characterizati对神经纤维的完整性和活性。它是在轴突变性和再生，突变小鼠真正的或可诱导的表型调查纵向研究是非常有用的。

这种成像技术的准确评估视觉投影的活力和功能的能力依赖于一个无毒的Mn 2 +的用量精准应用的玻璃体，其吸收的视网膜神经节细胞。这主要假设在图1中，其中层具体Mn 2 +的摄取证明了自显影技术（TIMM染色）21进行测试。视网膜节，在24小时后，无论是15纳摩尔或150纳摩尔的Mn 2 +，或PBS作为对照的ivit的应用进行了分析。在该时间点时，Mn 2 +<...

Watch this Scientific Journal Video about In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice at JoVE.com

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

该视频演示了一种方法，利用临床3个T的扫描仪，对天真的鼠标视觉投影对比增强磁共振成像和重复性和纵向体内视神经病变的研究，急性视神经挤压伤和慢性视神经变性相关敲除小鼠（P50 KO）。

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

in-vivo-imaging-optic-nerve-fiber-integrity-contrast-enhanced-mri

Research

JoVE Journal

Neuroscience

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

13.2K 次观看.  Jena University Hospital. 该视频演示了一种方法，利用临床3个T的扫描仪，对天真的鼠标视觉投影对比增强磁共振成像和重复性和纵向体内视神经病变的研究，急性视神经挤压伤和慢性视神经变性相关敲除小鼠（P50 KO）。 

视频: In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

在哺乳动物的中枢神经系统视神经横断后的实验操作方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

科研

神经科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

小鼠视网膜神经节细胞的转染在体内电穿孔

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

高分辨率结构磁共振人类皮质下的成像<I&gt;在体内</I&gt;与死亡

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

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.