Name: use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain
Uploaded: 2017-10-29T13:00:00
Description: Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

这项工作得到了国家研究资源中心 (2G12RR03060-26A1) 的赠款的支持;国立卫生研究院的少数群体健康和健康差异 (8G12MD007603-27);纽约市立大学专业工作人员代表大会和教职员研究津贴 (悬浮角速度陀螺仪 II) 美国沙迦大学。我们感谢维德雅瑟格 Sriramoju 向我们介绍这些方法。

以下协议遵循由纽约市城市学院机构动物保育和使用委员会 (IACUC) 建立的动物保育指导方针, 该准则符合所有适当的州和联邦准则。麻醉适合于雪貂, 并应根据所研究的物种进行修改. 
 <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56547/56547fig1.jpg"/> 图 1: 流程图概述了本协议3阶段涉及的主要步骤以及完成每个步骤所需的时间. 要求部分?...

<ol>
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目前的研究采用了基于改进版本的 Danscher (1982) 方法10的组织化学技术, 据此可以在大脑中检测到突触锌的定位并进行可视化。这种方法基本上是通过注射合剂酸钠 (Na2SeO3) (15 毫克/千克) 的动物。在注射后, 亚硒酸向脑部移动, 并与游离锌结合, 前囊泡中含有锌的神经元。锌离子与突触囊泡内的分子结合沉淀, 并可通过物理发育2,<sup class="xre...

组织学上的污点传统上被用来帮助鉴别不同物种的皮质区, 揭示不同的建筑特征。联合使用的组织化学技术, 如尼物质, 细胞色素氧化酶 (CO) 的反应性, 或髓鞘可以证明是卓有成效的, 因为他们揭示了类似的区域边界在成人的大脑。然而, 这些组织化学染色并不总能充分揭示未成熟大脑皮层区和层间的明显界限。
在中枢神经系统, 锌有几个关键的功能, 包括稳定 DNA 结构, 作为一个酶因子, 参与许多调节功能, 并作为一个调通过其存在的突触囊泡1. 突触锌是独一无二的, 因为它可以使用组织学方法进行可视化, 而蛋白质结合的锌则不能被形象化2。该功能已被用来揭示不同皮层区的突触锌模式, 并在许多研究中使用突触锌组织化学。大脑皮层中谷氨酸神经元的一个子集在其轴突末端的前泡内含有锌,3,4。组织化学研究揭示了脑皮层突触锌的异质分布5,6,7。在不同皮层区 (如, 视觉与体感皮层) 或层 (如如, 锌含量在 supragranular 和infragranular 层的初级视觉皮层大大高于丘脑输入层 IV 与相对较低的突触锌水平)5,8,9。在皮层中观察到的突触锌染色的异质性特别有利, 因为它能促进区域和层流的识别。
在这里, 我们提出了一个突触锌组织化学过程的详细描述, 这是一个修改版本的 Danscher 的1982方法10。该方法利用亚硒酸腹腔 (IP) 作为螯合剂。亚硒酸的移动到大脑, 以反应池的自由锌发现在囊泡的一个子集的谷氨酸突触在大脑中。这种反应产生的沉淀, 可以提高随后由银开发2,10,11。
这一过程揭示了突触锌染色的层流和区域模式;密度分析可用于评估这些模式的质量和数量在成人和未成熟的大脑, 以研究影响的其他干预, 如感官, 环境, 药理, 或基因的操纵。此外, 人们还可能希望评估其他模型系统中其他皮层或皮层下结构中的突触锌分布的潜在发展变化。密度分析提供的定量信息对后续的脑发育具有一定的优势。该协议提供了一个同伴的其他免疫和组织化学标记, 以揭示层流和区域边界。

<table>
	<tbody>
		<tr>
			<td>Euthasol (Euthanasia solution)</td>
			<td>Henry Schein</td>
			<td>710101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma-Aldrich</td>
			<td>214485</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Ketaved)</td>
			<td>Henry Schein</td>
			<td>48858</td>
			<td>100 mg/ml injectables</td>
		</tr>
		<tr>
			<td>Xylazine (Anased)</td>
			<td>Henry Schein</td>
			<td>33198</td>
			<td>100 mg/ml injectables</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Dilute to 4%</td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Sigma-Aldrich</td>
			<td>G9752-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Sigma-Aldrich</td>
			<td>W302600</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroquinone</td>
			<td>Sigma-Aldrich</td>
			<td>H9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver lactate</td>
			<td>Sigma-Aldrich</td>
			<td>85210</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish gelatine</td>
			<td>Sigma-Aldrich</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td>(Type III, from equine heart)</td>
		</tr>
		<tr>
			<td>Catalse</td>
			<td>Sigma-Aldrich</td>
			<td>C10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Domino</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>X5P-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>A406-20</td>
			<td>Used for dehydration prior to slide mounting</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Brain Research Laboratories</td>
			<td>#3660-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted unsubbed slides</td>
			<td>Brain Research Laboratories</td>
			<td>#3875-FR</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>American Optical Company</td>
			<td>860</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Adope Photoshop</td>
			<td>Adobe Systems, San Jose, CA</td>
			<td></td>
			<td>To assemble images</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td>
			<td></td>
			<td>For densometric measurements</td>
		</tr>
		<tr>
			<td>Plastic tray</td>
			<td>Any standard plastic tray may be used</td>
			<td></td>
			<td>to immerse slides in developer solution</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Any standard hotplate may be used</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain

解剖和功能性脑组织和发展的特征, 需要准确识别不同的神经回路和未成熟和成人大脑的区域。在这里, 我们描述了一个锌组织化学染色程序, 揭示不同的染色模式之间的各层和脑区。其他人利用这一程序不仅揭示了含锌神经元的分布和大脑中的电路, 而且成功地描绘了几个物种的发育中和成年大脑的区域和层流边界。在这里, 我们说明这个染色程序与图像从开发和成人鼬脑。我们揭示了一个锌染色模式, 作为一个解剖标记的地区和层, 并可以可靠地用于区分视觉皮层区域的发展和成人视觉皮层。本议定书的主要目的是提出一种组织化学方法, 允许准确地识别发育中和成人大脑中其他方法无法做到的层和区域。其次, 结合密度图像分析, 这种方法可以评估突触锌的分布, 以揭示整个开发过程中的潜在变化。本协议详细描述了连续染色冰冻脑切片所需的试剂、工具和步骤。虽然这个协议被描述使用鼬脑组织, 它可以很容易地被适应用于啮齿目动物, 猫, 或猴子并且在其他脑子区域。

在图 1的流程图中, 本协议中涉及的主要步骤是为突触锌的大脑切片着色。该协议可分为三阶段: 1) 灌注和组织收集, 2) 组织制备和染色, 3) 锌组化学。简单地, 在协议的第一阶段, 动物被麻醉并且注射了 IP 以适当的剂量亚硒酸钠。在一个充足的时间段 (理想情况下 60-90 min) 之后, 该动物随后被安乐死, 用 0.9% NaCl 溶液灌流, 然后是4% 甲醛溶液。头部被斩首...

Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

我们描述了一个组织化学过程, 揭示特征的层流和区域锌染色模式在不同的脑区。锌染色模式可与其他解剖标记一起使用, 以可靠地区分发育中和成人大脑的层和区域。

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有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

探索深空-揭开人脑侧脑室的脑室结构解剖

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

发育中大脑运动皮质的非侵入性调制和机器人映射

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...