Name: imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy
Uploaded: 2014-05-04T15:00:00
Description: Watch this Scientific Journal Video about Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy at JoVE.com

这项工作的经费由荷兰科学研究组织（NWO）以中央公积金的VIDI授权号H64.09.016。 CPF是感激医生Silvina A. Fratantoni她的批评意见，并在最后的手稿更正。 GMRDL / EMMM公司受荷兰技术基金会STW（项目12151和11350），这是苏国的一部分，这部分是由经济事务部资助支持。我们感谢尼康仪器Europe BV公司的凯瑟琳·基茨和彼得·德​​兰特的援助和支持。被授予荷兰科学研究组织（补助金820.02.006）支持HX是支持艺术和科学的荷兰皇家科学院（补助11CDP10）和WT。

所有涉及动物实验的程序进行了优化，以减少动物的痛苦，并获得证监会核准的动物实验，阿姆斯特丹大学，赤纬协议＃DED204和DED250。 1，准备盖玻片<ol><li>切下来的盖玻片为15毫米×15毫米用碳化物或金刚石划片的尺寸，以使它们放入一个12孔板的孔中。 </li><li>而在通风橱内工作时，通过充分浸没的盖玻片在浓硝酸溶液（70％重量/重量）在玻璃容器中开始盖玻片涂层。为了?...

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput,+detailed,+cell-specific+neuroanatomy+of+dendritic+spines+using+microinjection+and+confocal+microscopy.">High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy.</a> Nat Protoc. 6, 1391-1411 (2011).</li><li>Fitzsimons, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+the+glucocorticoid+receptor+alters+functional+integration+of+newborn+neurons+in+the+adult+hippocampus+and+impairs+fear-motivated+behavior.">Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.</a> Mol Psychiatry. , (2012).</li><li>van Hooijdonk, L. 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在这篇文章中的工作协议从大鼠原代海马神经元使用SIM卡在体外培养的图像树突棘描述。在原代海马神经元培养方法是通过Kaech和庄家18中描述的原始方法的改编。的主要差别是使用Neurobasal/B27培养基中，从而消除了星形胶质细胞培养物进料器的要求，并且除了在第3天的有丝分裂抑制剂FUDR能促进神经元的存活，同时抑制神经胶质增生的，如所描述由Brewer 等人 19。 <...

一个树突棘是神经元膜的小突起。这种结构的特点是专业，如从单一的突触接收输入并代表两个神经元之间的物理接触面积。功能最成熟的树突棘包括一个球状尖，称为头和颈细，头部连接到树突状轴。然而，刺不是静态的，积极移动，不断改变自己的形态，甚至在成人大脑2。在规定时间内有2周的时间，从胚胎后期或出生后早期的时间得出大鼠原代海马神经元培养开发复杂的树突状乔木与众多的膜突起，从早期的filipodia演变为棘状结构3。在此基础上动态行为等特点，树突棘被认为是提供了一个解剖基板的内存存储和突触传递4,5。 鉴于日Ë关键作用的树突棘的大小和形状都在突触功能，它准确地测量它们的尺寸是很重要的。刺从约200到2,000纳米，长度各不相同，可以通过共焦激光扫描荧光显微镜很容易识别。然而，长度比其他脊椎尺寸通常低于传统的光学系统的分辨率，理论上固定衍射约200纳米的6。这些解决权力是不够的成像更精细的细节，如脊柱的脖子和头的宽度。大量的工作一直致力于解决这个问题，许多相对较新的超高分辨率显微技术提供了实质性的进展。特别是，能够实现超越经典极限分辨率，不通过在一个宽视场，非共聚焦显微镜7-10采用横向结构照明显微镜（SIM）丢弃任何发射光。与非线理组合使用这种技术Ř显微技术，它在理论上是可能通过一个无限因子11，以提高光学显微镜的横向分辨率。然而，在大多数实验的情况下，SIM允许将超过由两个1的因子的分辨率极限。如受激发射损耗（STED）显微镜12和光激活定位显微镜（PALM）12其他超分辨率光学显微技术已被应用到树突棘的影像。定位为基础的方法，如PALM需要非常大的数字原始图像，实现超分辨率的，因此在有限的速度。另一方面，受激发射损耗可以达到高的成像速度，但是在相对 ​​低的光子数和视场小，这可能不是SIM卡13的情况。 在本文中，目的是从大鼠原代海马神经元在体外培养提供了一个工作协议到图像树突棘</EM&gt;使用SIM卡。该协议由两个可区分的阶段：一个初始组成的建立，发展，转染大鼠原代海马神经元培养的免疫组化和专用采样成像的后期阶段。

<table>
	<tbody>
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			<td>Fine forceps</td>
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			<td>Big forceps</td>
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			<td>Fine scissor</td>
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			<td>Big scissor</td>
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			<td>Blunt spatula</td>
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			<td>Dissecting microscope with illumination</td>
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			<td>Light microscope</td>
			<td></td>
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		</tr>
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			<td>37 &#176;C water bath</td>
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		</tr>
		<tr>
			<td>Laminar flow cell culture hood</td>
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			<td>High-temperature dry-oven</td>
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		</tr>
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			<td>Bunsen burner</td>
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		<tr>
			<td>Cell culture incubator (5% CO2, 37 &#176;C)</td>
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			<td>Microcentrifuge</td>
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			<td>Orbital shaker</td>
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			<td>Nikon structured illumination microscope setup consisting of:</td>
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		</tr>
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			<td>Nikon Eclipse Ti research inverted microscope with Perfect Focus System</td>
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			<td>Nikon CFI Apo TIRF 100x oil objective lens (N.A. 1.49)</td>
			<td></td>
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			<td>4 Coherent Sapphire Lasers (458, 488, 514 and 561 nm exitation wavelength)</td>
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			<td>SIM Illuminator</td>
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			<td>Nikon Stage Controller</td>
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			<td>MCL Nano-Drive piezo controller</td>
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			<td>Nikon Intensilight C-HGFIE mercury lamp</td>
			<td></td>
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		</tr>
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			<td>SIM Microscope Enclosure temperature control</td>
			<td></td>
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		</tr>
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			<td>Andor EM-CCD Camera iXon DU897</td>
			<td></td>
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			<td></td>
		</tr>
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			<td>PC with Microsoft Windows 7 Home Edition</td>
			<td></td>
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		</tr>
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			<td>Nikon&#8217;s NiS Elements 6.14 SIM software package</td>
			<td></td>
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			<td></td>
		</tr>
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			<td>Nikon type A immersion oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy

树突棘是新兴突起从一个神经元的树突和代表在脑兴奋性输入端的初级突触后指标。技术进步已经确定这些结构在神经元连通性和突触可塑性的关键要素。利用光学显微镜棘形态的定量分析仍然是由于光的折射内在限制相关的技术限制，一个重要的问题。树突棘可以很容易地确定共聚焦激光扫描荧光显微镜。然而，测量在刺的形状和大小的细微变化是困难的，因为比长度其他脊柱的尺寸通常比固定为200nm的光镜的理论分辨率极限传统的光学分辨率小。 几个最近开发的超分辨率技术已被用来对图像蜂窝结构比200纳米，包括树突棘小。 Ŧ淡褐色技术是基于经典的远场操作，因此允许使用现有的样品制备方法及图像以外的试样的表面上。这里描述的是工作协议，超分辨率结构照明显微镜（SIM）适用于原代海马神经元培养树突棘的影像。 SIM卡可能的应用重叠与激光共聚焦显微镜。然而，这两种技术提出不同的适用性。 SIM卡可提供更高的有效横向分辨率，而共焦显微镜中，由于一个物理针孔的使用量，实现了分辨率的改善以除去离焦光的费用。在这个协议中，原代神经元是使用标准协议，转染的DNA的质粒编码的荧光蛋白质和可使用SIM成像的玻璃盖玻片上培养的。这里所描述的整个协议需要大约2周，因为树突棘是体外后16-17天，当成像树突发育是最优的。协议完成后，树突棘可以在3D系列从使用专门的软件，SIM卡图像堆栈的重建。

这里描述的是一种标准化的工作协议，用于从成像使用的SIM 体外大鼠原代海马神经元树突棘。该协议的工作流程，其关键步骤示于图1。总体而言，协议需要大约2周的实验工作中的样品制备的第一阶段，包括文化，发展和大鼠原代海马神经元和免疫组化染分离，并且第二相使用SIM卡的样品成像。大鼠原代海马神经元的文化，当神经元已经开发出复杂的树突状乔木承载无数树突?...

Watch this Scientific Journal Video about Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy at JoVE.com

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

本文介绍了使用结构照明显微镜（SIM卡） 在体外海马神经元的工作协议，以形象的树突棘。使用SIM卡超高分辨率显微镜提供的图像分辨率显著超越在所有三个空间维度的光的衍射极限，允许单个树突棘与改进的细节的影像。

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

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Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

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Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

具有突触可塑性树突脊柱的三维建模

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...