Name: monocular visual deprivation and ocular dominance plasticity measurement in the mouse primary visual cortex
Uploaded: 2020-02-08T15:00:00
Description: Watch this Scientific Journal Video about Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex at JoVE.com

这项研究得到了国家自然科学基金（81571770、81771925、81861128001）的支持。

本方案从四川省医学科学院实验动物研究所和四川省人民医院获得雄性C57Bl/6小鼠。所有动物护理和实验程序均获得中国电子科技大学动物护理与使用委员会的批准。
1. 小鼠产后第28天的单眼剥夺（MD）
<ol><li>将手术工具、缝合针（直径0.25毫米，弦直径0.07毫米）和棉签放入铝盒中，在120°C下高压灭菌，用75%乙醇消毒发动机罩。在干燥炉中干燥手术工具。</li>
	<li>制备2%的甘蔗?...

<ol>
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E., Gordon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+period+for+the+monocular+deprivation+effect+in+rats:+assessment+with+sweep+visually+evoked+potentials.">Critical period for the monocular deprivation effect in rats: assessment with sweep visually evoked potentials.</a> Journal of Neurophysiology. 81 (1), 121-128 (1999).</li><li>Wang, L., Sarnaik, R., Rangarajan, K. V., Liu, X., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+receptive+field+properties+of+neurons+in+the+superficial+superior+colliculus+of+the+mouse.">Visual receptive field properties of neurons in the superficial superior colliculus of the mouse.</a> Journal of Neuroscience. 30 (49), 16573-16584 (2010).</li><li>Niell, C. 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我们提出了一个详细的协议，MD和测量OD可塑性通过单单元记录。该协议在视觉神经科学中得到了广泛的应用。虽然MD协议并不复杂，但有一些关键的外科手术必须小心遵循。首先，有两个重要的细节，以确保缝合的质量。如果缝合集中在眼睑的中段，缝合线就足够稳定。此外，3 μL的胶水被涂在结的头部，以增加结的稳定性，以防止眼睛重新开放。其次，应采取一些关键步骤改善伤口愈合，减少?...

单目视觉剥夺是检验V1可塑性的绝佳实验范例。为了研究视觉体验在神经发育中的重要性，大卫·胡贝尔和托斯滕·威塞尔1，2在不同时间点和不同时间段的一只眼睛中剥夺正常视力的小猫。然后，他们观察了贫困和非贫困眼睛V1反应强度的变化。他们的结果显示，在头三个月里，对眼睛有异常低反应的神经元数量是被缝合的。然而，小猫的神经元在各方面的反应仍然与正常成年猫的眼睛的反应相同，而正常成年猫的眼睛被缝合了一年，小猫没有恢复。MD在成年猫不能诱导OD可塑性。因此，视觉体验对 V1 布线的影响在短暂、明确定义的开发阶段非常强烈，前后相同的刺激影响较小。这种对视觉输入的易感性增加的阶段被称为视觉皮层的关键时期。
虽然小鼠是夜间动物，但小鼠V1中的单个神经元具有与猫3、4、5的神经元相似的特性。近年来，随着转基因技术的飞速发展，越来越多的视觉神经科学研究将小鼠作为实验模型6、7、8。在小鼠视觉研究中，神经科学家使用突变体和敲除小鼠线，从而控制小鼠的基因组成。尽管小鼠 V1 缺少 OD 柱，但 V1 双目区域中的单个神经元显示出显著的 OD 属性。例如，大多数细胞对反向刺激的反应比对边侧刺激的反应更强烈。在关键时期暂时关闭一只眼睛，导致OD指数分布9、10、11发生显著变化。因此，MD可用于建立OD可塑性模型，以研究神经发育障碍中涉及的基因如何影响体内皮质可塑性。
本文介绍了一种医学实验方法，并推荐了一种常用的方法（电生理记录），用于分析单眼视觉剥夺期间OD可塑性的变化。该方法已广泛应用于许多实验室超过20年12，13，14，15，16。在测量OD可塑性时也使用其他方法，如慢性视觉唤起电位（VEP）记录17，和内在光学成像（IOI）18 。这种急性方法的显著优点是易于遵循，结果非常可靠。

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			<td>66 Vision Tech Co., Ltd.</td>
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			<td>66 Vision Tech Co., Ltd.</td>
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			<td>RWD Life Science Co.,Ltd</td>
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			<td>Microscope</td>
			<td>SOPTOP</td>
			<td>SZMT1</td>
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			<td>Noninvasive Vital Signs Monitor</td>
			<td>Mouseox</td>
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			<td>Oil hydraulic micromanipulator</td>
			<td>NARISHIGE International Ltd.</td>
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			<td>Cambridge Electronic Design, Cambridge, UK</td>
			<td>Spike2 Version 9</td>
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			<td>Surgical scissors</td>
			<td>66 Vision Tech Co., Ltd.</td>
			<td>54010</td>
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			<td>Surgical scissors</td>
			<td>66 Vision Tech Co., Ltd.</td>
			<td>54002</td>
			<td></td>
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			<td>Suture Needle</td>
			<td>Ningbo Medical Co.,Ltd</td>
			<td>3/8 arc 2.5*8</td>
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			<td>Tungsten Electrode</td>
			<td>FHC, Inc</td>
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monocular visual deprivation and ocular dominance plasticity measurement in the mouse primary visual cortex

单目视觉剥夺是诱导原发性视觉皮质反应可塑性的一个很好的实验范式。一般来说，皮层对反向眼睛对刺激的反应比小鼠主要视觉皮层（V1）的双目部分的侧边眼的反应要强得多。在哺乳动物的关键时期，缝合反向眼睛将导致V1细胞对反向眼部刺激的反应迅速丧失。随着转基因技术的不断发展，越来越多的研究利用转基因小鼠作为实验模型，研究特定基因对眼部支配力（OD）可塑性的影响。在这项研究中，我们介绍了单目视觉剥夺的详细方案，并计算了小鼠V1中OD可塑性的变化。在关键时期单眼剥夺（MD）4天后，测量每个神经元的方向调谐曲线，并比较V1中第四层神经元的调谐曲线。可使用每个细胞的眼部 OD 评分来计算反向偏置指数 （CBI），以指示 OD 可塑性的程度。该实验技术对于研究关键时期OD可塑性的神经机制和特定基因在神经发育中的作用具有重要意义。主要局限性是，急性研究无法研究同一小鼠在不同时间的神经可塑性变化。

此处描述的实验结果使被剥夺和非剥夺小鼠在关键时期（P19_P32）成功进行了MD和OD可塑性测量。图 1显示了如何在 V1 的双目区域的第 4 层中执行单单元录像，以比较 MD 后 4 天在益边眼和反向眼中的响应。图 2显示了用于刺激侧边和反向眼睛的尖峰排序和方向调谐测量值。对于尖峰排序，通过聚类尖峰的主要组件权重建立了尖峰模板。例如（<strong class="xf...

Watch this Scientific Journal Video about Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex at JoVE.com

Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex

本文提出了单眼视觉剥夺和眼主导性可塑性分析的详细方案，是研究关键时期视觉可塑性的神经机制和特定基因对视觉发展。

小鼠主视觉皮质中的单眼视觉剥夺和眼部优势可塑性测量

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the ...

单目视觉剥夺是诱导初级视觉皮质反应可塑性的一个很好的实验方案。通常，小鼠主要视觉皮层双目部分的神经元对反侧眼刺激的反应是同侧眼的两倍。然而，在眼部占重可塑性的关键时期，停止闭合的反向眼会导致V1神经元对反向眼刺激的迅速反应丧失，随后对无边眼的反应也随之而来。在这里，我们介绍了单眼剥夺的实验方案，并建议一种常用的方法来分析视觉剥夺期间眼部支配的趋势。将手术工具放入铝制箱中，并在120摄氏度以下进行半小时解塞。在水浴水壶中准备 2%的加糖溶液，并保持在 75 摄氏度。在两只眼睛上涂抹一层薄薄的眼药膏。在具有照明的解剖显微镜下，缝合眼睑。缝大约四针。创建线程的两到三个节点。在节点头涂抹三个微升的速溶干燥胶502，以提高节点的稳定性。然后将螺纹切成足够短。当鼠标完全清醒时，将它们放入分离的保持架中。在进行电生理记录之前，使用异氟兰麻醉小鼠。取出缝线，露出眼球。小心修剪眼睑。使用隐形眼镜溶液冲洗眼睛，并检查眼睛的清晰度。只有眼球状况良好的鼠标才能使用。麻醉鼠标后，将鼠标放在立体轴框架上。在整个过程中，使用加热垫保持体温。在眼睛表面涂抹眼药膏，以保持其湿润。取下鼠标的头发以露出其皮肤。将8毫米乘以两只耳朵之间的皮肤面积乘以8毫米，以暴露头骨并去除头皮组织。然后用过氧化氢去除上皮结缔组织。在小脑上方的头骨上钻一个洞。将孔中的小骨螺钉固定为参考。在 V1 双目区域进行直径为 1 毫米的小颅骨切除术。小心地取出头骨碎片，而不会伤害大脑。用2%的加糖覆盖外露的皮质表面，以防止干燥。将钨电极固定到立体轴框架上。将钨电极垂直放置到双目 V1 区域，以确保记录的细胞对两只眼睛有反应。刺激在随机块中相隔 30 度。每次测量共提出了三到五个区块。用不透明的塑料板遮盖一只眼睛。提供 LCD1 以在距离鼠标眼睛 23 厘米的位置。通过油液压微操纵器缓慢推进微电子。当电极提前到第四层并开始测量时，停止推进高信号噪声比信号信号。我们放大尖峰活动在1000倍，并过滤从300到10千赫，然后采样在40千赫。然后遮盖另一只眼睛并测量反面眼睛的反应。单电极测量的信号是种群活动。使用软件分隔不同单元格的峰值。首先，将信号从500赫兹过滤到5000赫兹。设置两个光标，一个用于正偏转，另一个用于负偏转尖峰。设置峰值模板。设置模板区域，其中显示不同峰值类别之间的最大差异。使用主体组件分析将它们分离为群集。使用 K 手段算法对边界的峰值进行分类。将方向与尖峰发射速率关联，并绘制用于双面眼和反向眼睛的方向调整曲线。然后计算单个细胞的 OD 指数，该指标表示反向和异方响应，根据 OD 索引为每个细胞分配 OD 分数。根据公式计算反向偏置指数。此图显示了单目缺位和非被侵占小鼠中同形眼和反向眼的方向调谐曲线。该协议使在关键时期从贫困和非剥夺小鼠成功的单眼视觉剥夺和眼部支配性测量。我们刚刚向您展示了如何完成这个实验，并分析了视觉剥夺期间眼部支配性的变化。在实验过程中，在电生理记录时避免在苏化过程中感染和相互作用是十分重要的。感谢您观看我们的视频。

Research

JoVE Journal

Developmental Biology

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

科研

发育生物学

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

从鼠耳和尾组织生成主成纤维细胞培养

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Live Imaging of Mouse Secondary Palate Fusion

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

多光子时间推移成像实时可视化发展： 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

Induction and Validation of Cellular Senescence in Primary Human Cells

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

达尼奥胚胎发生过程中的上眼硫可视化

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...