Name: visualization of cortical modules in flattened mammalian cortices
Uploaded: 2018-01-22T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Cortical Modules in Flattened Mammalian Cortices at JoVE.com

这项工作得到了洪堡大学祖柏林, 伯恩斯坦计算神经科学中心柏林, 德国神经退行性疾病中心 (DZNE), 德国联邦教育和研究部 (BMBF, Förderkennzeichen01GQ1001A), NeuroCure, 和戈特弗里德的 DFG 奖。我们感谢藤新平西山优秀的平面设计和 Juliane Diederichs 优秀的技术援助。

所有的实验程序都是按照德国在地方伦理委员会 (LaGeSo) 监督下的动物福利准则进行的。人和蝙蝠脑数据来自瑙曼et al.5对雄性成年大鼠 (菌株: RJHan: WI) 执行以下步骤。
1. 灌注和脑部提取
注: 为了获得一个 homogenously 固定和无血脑, transcardial 灌注的动物是高度鼓励, 因为残血增加不背景信号在染色。然而, 也可以从非灌注标本中获得扁?...

<ol>
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在大脑皮层的模块化已被确定使用各种技术。最早的研究通常通过可视化细胞稠密区或缺乏纤维1来识别皮质模块。随后的方法利用了树突丛的存在24, 传入从特定区域25, 或富集神经递质26。在这里我们展示了两种技术, (i) 细胞色素氧化酶组织化学和 (ii) 免疫组织化学染色。
细胞色素氧化酶染色是最常用的...

在脑皮质中, 可以观察到脑组织在系统发展史上最显著的变化。尽管有很大的差异, 动物的皮层遵循一个共同的模式, 可以大致分为两种不同的方式, 由层和区域1。皮质层与大脑的表面平行, 在爬行动物皮层的3层中有不同的数量,2到哺乳动物皮层的6层, 即1。另一方面, 皮质区是皮层的不同区域, 主要对应于不同的功能,例如, 体感皮层参与触觉的感觉或视觉输入过程中的视皮层。这些皮质区通常可以细分为补丁或模块3, 这是定期重复解剖结构, 基本上发现平行于大脑的脑膜表面。皮质模块可以被限制在一个特定的层4, 或者延伸到多个层5。
大脑的标准切片方法涉及大脑表面正常的部分, 如冠状或矢状。虽然这些方法可以用来可视化皮层模块, 许多有趣的功能可以揭示, 当皮质模块的可视化, 在一个平面平行的大脑表面。例如, 在啮齿动物大脑中代表胡须的体感模组, 在大脑表面正常的情况下显示为桶, 因此区域会产生名称桶皮层。然而, 在以切线方向对桶进行可视化时, 它们揭示了一个晶须图, 其中的桶被布置在一个地形方向上, 反映了外部体表的晶须的精确布局。在某些情况下, 当以非切线的方式进行可视化时, 模块式排列甚至有相当长的时间逃脱检测。内侧嗅皮层, 是已知的存在的网格细胞, 神经元在正常的六边形模式时, 动物正在穿越环境。尽管这是一个研究范围很大的区域, 直到最近, 在内侧嗅皮层中存在的补丁或单元模块, 它们在六角形图案6中被物理地布置, 却逃过了检测。这些模块的存在和安排, 在大鼠大脑中, 通过制作内侧嗅皮层的切线部分, 并以一层聪明的方式调查细胞。
在切片之后, 皮层模块可视化的特定方面也可以通过多种方式实现。典型地, 研究根据细胞密度或纤维布局1划定了模块。另一个流行的方法是使用细胞色素氧化酶组织化学, 这揭示了更高的活动领域8。新的方法包括寻找基因上确定的细胞类型, 根据他们的蛋白质表达谱6,8进行区分。
在这项研究中, 我们强调的方法, 以分离的皮层从哺乳动物的大脑, 获得扁平切切面, 并可视化皮层模块的细胞色素氧化酶组织化学和免疫组化的特定蛋白。

<table>
	<tbody>
		<tr>
			<td>Cytochrome oxidase staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c from equine heart</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;Diaminobenzidine tetrahydrochloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>D5637</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Saccharose</td>
			<td>Carl Roth&#160;</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium nickel(II) sulfate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A1827</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antigen retrieval</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phosphate buffer/phosphate-buffered saline/prefix/PFA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-Sodium hydrogen phosphate dihydrate</td>
			<td>Carl Roth</td>
			<td>4984.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>0335.3</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON-X 100</td>
			<td>Carl Roth</td>
			<td>3051.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin D-28k puriefied from chicken gut, Mouse monoclonal</td>
			<td>Swant</td>
			<td>RRID: AB_10000347</td>
			<td></td>
		</tr>
		<tr>
			<td>Calbindin D-28k from recombinant rat calbindin D-28k, Rabbit polyclonal</td>
			<td>Swant</td>
			<td>RRID: AB_10000340</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraction V, biotin free</td>
			<td>Carl Roth</td>
			<td>0163.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mounting or freezing media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount (immunofluorescence)</td>
			<td>Sigma-Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt (histochemistry)</td>
			<td>Sigma-Aldrich</td>
			<td>03989</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue freezing medium</td>
			<td>Leica Biosystems</td>
			<td>NC0696746</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Alcohol dehydration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Carl Roth</td>
			<td>9065.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Carl Roth</td>
			<td>P075.3</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Carl Roth</td>
			<td>6752.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>Fluka</td>
			<td>78475</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Devices/tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microm HM 650V</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jung RM2035</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps - Inox</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps - Inox Biology Tip</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps - Inox Super Fine Tip</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Shears - 24 cm</td>
			<td>Fine Science Tools</td>
			<td>16150-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16000-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt Scissors</td>
			<td>Fine Science Tools</td>
			<td>14000-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Large Loops</td>
			<td>Fine Science Tools</td>
			<td>14101-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>Fine Science Tools</td>
			<td>14001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris Scissors</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of cortical modules in flattened mammalian cortices

哺乳动物大脑的皮质被 parcellated 成不同的子结构或模块。皮质模块通常与皮质板平行, 可以用某些组织化学和免疫组织化学方法来描述。在这项研究中, 我们突出了一种方法, 分离的皮层从哺乳动物的大脑, 并铺平他们获得的部分平行的皮质板。我们进一步强调选定的组织化学和免疫组织化学方法, 以处理这些扁平切切面, 以可视化皮层模块。在各种哺乳动物的体感皮层, 我们执行细胞色素氧化酶组织化学, 以揭示身体的地图或皮层模块代表动物身体的不同部位。在嗅皮层中, 我们利用免疫组织化学方法来突出细胞, 在皮层片中排列成一个网格模式, 在多个物种中形成一个晶格单元。总的来说, 我们提供了一个框架, 以隔离和准备层明智的扁平皮层切片, 并可视化皮质模块使用组织化学和免疫组化方法在哺乳动物的大脑广泛。

我们获得了各种大脑体感皮层的扁平皮层切片, 并对其进行细胞色素氧化酶组织化学处理, 以可视化 somatotopic 模块代表不同的身体部位。这种比较方法允许研究形状皮层的进化力,例如, 显示 mystacial 触须在啮齿动物和兔中的高度保守的表示, 如桶21 (图 2)。相比之下, 其他身体部位, 如爪子和生殖器显示其相对大小的变化, 并反映...

Watch this Scientific Journal Video about Visualization of Cortical Modules in Flattened Mammalian Cortices at JoVE.com

Visualization of Cortical Modules in Flattened Mammalian Cortices

本文介绍了一个详细的方法, 以获得从哺乳动物皮层扁平切切面和可视化皮层模块使用组织化学和免疫组织化学方法。

扁平哺乳动物皮层皮质模块的可视化研究

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Research

JoVE Journal

Neuroscience

Automated Interactive Video Playback for Studies of Animal Communication

自动化研究动物交流的交互式视频播放

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

科研

神经科学

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

活大鼠皮层神经元线粒体膜电位和活性氧的测定

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Functional Calcium Imaging in Developing Cortical Networks

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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

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

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 ...

Isolation and Culture of Mouse Cortical Astrocytes

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Subtype-selective Electroporation of Cortical Interneurons

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

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

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

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 ...