Name: ballistic labeling of pyramidal neurons in brain slices and in primary cell culture
Uploaded: 2020-04-02T14:00:00
Description: Watch this Scientific Journal Video about Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture at JoVE.com

这项工作由NIH拨款HD043680、MH106392、DA013137和NS100624资助。

所有动物协议均由南卡罗来纳大学动物护理和使用委员会审查和批准（联邦保证号：D16-00028）。
1. 准备 DiI/钨珠管
<ol><li>用 10 mL ddH2O. Vortex 轻轻溶解 100 毫克聚乙烯丙烯酮 （PVP）。</li>
	<li>用 PVP 溶液填充油管（参见材料表），并保留 20 分钟。然后，使用 10 mL 注射器通过管道的另一端排出 PVP 溶液。</li>
	<li>将 170 毫克钨微载珠与 250 μL 的二甲苯氯化物混...

<ol>
	<li>Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+"DiOlistic"+labeling+of+the+nervous+system+using+lipophilic+dye+combinations.">Multicolor "DiOlistic" labeling of the nervous system using lipophilic dye combinations.</a> Neuron. 27, 219-225 (2000).</li><li>Gan, W. B., Grutzendler, J., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ballistic+delivery+of+dyes+for+structural+and+functional+studies+of+the+nervous+system.">Ballistic delivery of dyes for structural and functional studies of the nervous system.</a> Cold Spring Harbor Protocol. 2009 (4), 5202 (2009).</li><li>Seabold, G. K., Daunais, J. B., Rau, A., Grant, K. A., Alvarez, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiOLISTIC+labeling+of+neurons+from+rodent+and+non-human+primate+brain+slices.">DiOLISTIC labeling of neurons from rodent and non-human primate brain slices.</a> Journal of Visualized Experiments. (41), (2010).</li><li>Spacek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+Golgi+method:+a+time-lapse+study+of+the+early+stages+of+impregnation+in+single+sections.">Dynamics of the Golgi method: a time-lapse study of the early stages of impregnation in single sections.</a> Journal of Neurocytology. 18 (1), 27-38 (1989).</li><li>McLaurin, K. A., Li, H., Booze, R. M., Mactutus, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Timing:+NeuroHIV+Progression+in+the+Post-cART+Era.">Disruption of Timing: NeuroHIV Progression in the Post-cART Era.</a> Science Reports. 9 (1), 827 (2019).</li><li>Spruston, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+neurons:+dendritic+structure+and+synaptic+integration.">Pyramidal neurons: dendritic structure and synaptic integration.</a> Nature Reviews Neurosciences. 9 (3), 206-221 (2008).</li><li>Megias, M., Emri, Z., Freund, T. F., Gulyas, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+number+and+distribution+of+inhibitory+and+excitatory+synapses+on+hippocampal+CA1+pyramidal+cells.">Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells.</a> Neuroscience. 102, 527-540 (2001).</li><li>Peters, A., Kaiserman-Abramof, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+small+pyramidal+neuron+of+the+rat+cerebral+cortex.+The+perikaryon,+dendrites+and+spines.">The small pyramidal neuron of the rat cerebral cortex. The perikaryon, dendrites and spines.</a> American Journal of Anatomy. 127, 321-355 (1970).</li><li>Harris, K. M., Sultan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+the+number,+location,+and+size+of+synaptic+vesicles+provides+an+anatomical+basis+for+the+nonuniform+probability+of+release+at+hippocampal+CA1+synapses.">Variation in the number, location, and size of synaptic vesicles provides an anatomical basis for the nonuniform probability of release at hippocampal CA1 synapses.</a> Neuropharmacology. 34, 1387-1395 (1995).</li><li>Sorra, K. E., Fiala, J. C., Harris, K. 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A., 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=Synaptic+connectivity+in+medium+spiny+neurons+of+the+nucleus+accumbens:+A+sex-dependent+mechanism+underlying+apathy+in+the+HIV-1+transgenic+rat.">Synaptic connectivity in medium spiny neurons of the nucleus accumbens: A sex-dependent mechanism underlying apathy in the HIV-1 transgenic rat.</a> Frontiers in Behavior Neurosciences. 12, 285 (2018).</li><li>Roscoe, R. F., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+transgenic+female+rat:+synaptodendritic+alterations+of+medium+spiny+neurons+in+the+nucleus+accumbens.">HIV-1 transgenic female rat: synaptodendritic alterations of medium spiny neurons in the nucleus accumbens.</a> Journal of Neuroimmune Pharmacology. 9 (5), 642-653 (2014).</li><li>Li, H., Aksenova, M., Bertrand, S. J., Mactutus, C. F., Booze, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Filamentous+Actin+(F-actin)+Puncta+in+Rat+Cortical+Neurons.">Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.</a> Journal of Visualized Experiments. (108), e53697 (2016).</li><li>Rodriguez, A., Ehlenberger, D. B., Dickstein, D. L., Hof, P. R., Wearne, S. 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在此协议中，我们描述了一种针对大鼠大脑和体外生长的神经元的通用标记技术。此外，我们报告利用神经元重建软件和神经元重建定量分析软件来评估神经元形态和树突脊柱的方法。对神经元形态学和树突脊柱的评估提供了一个机会，以确定树突分枝的复杂性、神经元树干复杂性、树突脊柱形态和突触连通性的变化。
在进行协议时，研究人员应特别注意几个步骤。首先，?...

2000年，Gan等人描述了神经系统中单个神经元和胶质的快速标记技术，将各种嗜脂染料结合在一起，允许同时标记许多不同颜色的脑细胞11，2。2最近，Seabold等人3描述了一种弹道标记技术，该技术将荧光染料（Dil）引入脑切片的神经元。弹道标记是一种多功能染色技术，因其在多种动物种类和不同年龄范围内使用的能力而广受赞赏。此外，它可以与免疫染色相结合，以识别脑细胞的亚群3。与传统技术（如Golgi-Cox银浸渍、微注射）4相比，弹道标记提供了一个更清晰地区分形态特征的机会，包括树突脊柱，这一特征对于绘制关于神经元复杂性和突触连通性5的推论至关重要。
外泄金字塔神经元的特点是单个，大的树突，多个较短的基底树突，和数以千计的树突脊柱6。金字塔神经元存在于与高阶认知处理相关的多个大脑区域，包括前额叶皮质（PFC）和海马。在PFC中，金字塔神经元在II/III层和第五层被观察到，每个神经元都表现出独特的形态。具体来说，PFC第二/III层中的金字塔神经元具有比第五层6中的金字塔神经元较短的尖树突和更少的分支。在海马区内，金字塔神经元位于CA1和CA3区域，每个区域都显示不同的形态。具体来说，CA1 区域中的金字塔神经元表现出更独特的尖突，与 CA3 区域6相比，分支远离躯体。
在PFC和海马的金字塔神经元上的腺突脊柱是兴奋突触7的主要部位。树突脊柱的形态特征，被经典地分为三大类（即薄、小根或蘑菇8），与兴奋突触9的大小有关。细脊椎，其特点是长，薄的脖子，小球状头，和较小的脑后密度，是更不稳定的，并发展较弱的连接。然而，蘑菇脊柱，有一个更大的树突脊柱头，被认为是形成更强的突触连接，其更大的尺寸所产生的效果。与此形成鲜明对比的是，短根脊柱没有脊柱颈部，头部和颈部体积比约为8。在海马区内，也可以观察到分支脊柱，即脊柱有多个头，从同一树突脊柱颈部10中浮现出来。因此，树突脊柱的形态变化可以反映功能和结构能力。此外，研究表明，树突椎的大小和形状与结构可塑性有关，导致小脊柱参与学习和注意力，而更大，更稳定的脊柱，参与长期过程，包括记忆11。此外，树突状脊柱沿树突的分布可能与突触连接55，1212相关。
因此，本方法论文件有三个目标：1） 提出我们的弹道标记方案，其成功率（即符合选择标准的神经元和适合分析的神经元）为 83.3%5,5、12、13,13和跨多个大脑区域（即 PFC、核积液、海马区）;2） 证明该技术的通用性及其在体外生长的神经元中的应用;3） 详细说明神经元重建软件中使用的方法以及可以从此类数据中提取的推论。

<table border="0" cellpadding="0" cellspacing="0" style="width:868px;" width="869">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">20Gx25mm PrecisionGlide needle</td>
			<td style="width:263px;">BD</td>
			<td style="width:143px;">305175</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well cell culture plate</td>
			<td>Costar</td>
			<td>3562</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic-Antimycotic solution</td>
			<td>Cellgro</td>
			<td>30004CI</td>
			<td>100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 supplement</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>50X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barrel liner</td>
			<td>BIO-RAD</td>
			<td>165-2417</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borax</td>
			<td>Sigma</td>
			<td>B9876</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Boric acid</td>
			<td>Sigma</td>
			<td>B0252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cartridge holder</td>
			<td>BIO-RAD</td>
			<td>165-2426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Confocal imaging software</td>
			<td style="width:263px;">Nikon</td>
			<td style="width:143px;">EZ-C1</td>
			<td>version 3.81b</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Confocal microscope</td>
			<td style="width:263px;">Nikon</td>
			<td style="width:143px;">TE-2000E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass</td>
			<td>VWR</td>
			<td>637-137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DilC18(3)</td>
			<td>Fisher Scientific</td>
			<td>D282</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 medium</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14095</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #7 Forceps</td>
			<td>World Precision Instruments</td>
			<td>14097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">F344 rat</td>
			<td style="width:263px;">(Harlan Laboratories, Indianapolis, IN)</td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>VWR</td>
			<td>101174Y</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Sigma</td>
			<td>H4641</td>
			<td>10X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios diffusion screens</td>
			<td>BIO-RAD</td>
			<td>165-2475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios gene gun kit</td>
			<td>BIO-RAD</td>
			<td>165-2411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios gene gun system</td>
			<td>BIO-RAD</td>
			<td>165-2431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Helium hose assembly</td>
			<td>BIO-RAD</td>
			<td>165-2412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris Scissors</td>
			<td>World Precision Instruments</td>
			<td>500216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene chloride</td>
			<td>Fisher Scientific</td>
			<td>D150-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Neurolucida 360 software</td>
			<td style="width:263px;">mbf bioscience</td>
			<td style="width:143px;"></td>
			<td>dendritic spine analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P9155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvinylpyrrolidone</td>
			<td>Fisher Scientific</td>
			<td>5295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
			<td>Fisher Scientific</td>
			<td>P36930</td>
			<td>mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat brain matrix, 300 - 600g, Coronal, 0.5mm</td>
			<td>Ted Pella</td>
			<td>15047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sevoflurane</td>
			<td>Merritt Veterinary Supply</td>
			<td>347075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Life Technologies</td>
			<td>25080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperFrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-154%</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe kit</td>
			<td>BIO-RAD</td>
			<td>165-2421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tefzel tubing</td>
			<td>BIO-RAD</td>
			<td>165-2441</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing cutter</td>
			<td>BIO-RAD</td>
			<td>165-2422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing Prep station</td>
			<td>BIO-RAD</td>
			<td>165-2418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tungsten M-25 Microcarrier 1.7 &#181;m</td>
			<td>BIO-RAD</td>
			<td>165-2269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas Scissors</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
	</tbody>
</table>

ballistic labeling of pyramidal neurons in brain slices and in primary cell culture

据报道，树突椎的大小和形状与其结构可塑性有关。为了识别金字塔神经元和树突脊柱的形态结构，可以使用弹道标记技术。在本协议中，金字塔神经元标有DilC18（3）染料，并使用神经元重建软件进行分析，以评估神经元形态和树突脊柱。为了研究神经元结构，进行了树突分支分析和Sholl分析，使研究人员能够分别对树突分支的复杂性和神经元树体的复杂性进行推论。树突脊柱的评估使用重建软件不可或缺的自动辅助分类算法进行，该算法将脊柱分为四类（即薄、蘑菇、短根、纤维素）。此外，还选择另外三个参数（即长度、头部直径和体积）来评估树突脊柱形态的变化。为了验证弹道标记技术的广泛应用潜力，成功地标记了体外细胞培养的金字塔神经元。总体而言，弹道标记方法是独特的，可用于可视化大鼠不同大脑区域的神经元，与复杂的重建软件相结合，使研究人员能够阐明潜在的机制神经认知功能障碍。

在图2A中，大鼠大脑部分海马区域的典型金字塔神经元通过弹道标记技术进行识别，其特点是在躯体周围有一个大的树突和几个较小的基底树突。图2B显示了检测到躯体后神经元重建定量分析软件中的神经元，追踪树突状分支，并检测到脊柱。随后，利用神经元重建定量分析软件对数据进行了分析，为评估树突分支复杂性（图2C）?...

Watch this Scientific Journal Video about Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture at JoVE.com

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

我们提出了一种用于标记和分析金字塔神经元的协议，这对于评估神经元和树突脊柱的潜在形态变化至关重要，这些变化可能是神经化学和行为异常的基础。

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

该协议使得评估神经元和树突脊柱的潜在形态变化成为可能，这些改变可能突出神经化学和行为异常。它对于可视化大鼠不同大脑区域的神经元是独特和有用的，它结合复杂的重建软件，使研究人员能够阐明神经认知功能障碍背后的可能机制。首先根据手稿说明准备 PVP 解决方案。用 PVP 填充油管，并离开 20 分钟。用10毫升注射器将它通过另一端排出。将170毫克钨微载体珠与250微升的氯化甲基汞混合。然后彻底涡流悬浮。接下来，加入6毫克的脂亲D二钙18（3）染料到300微升的二氯甲烷和涡流。将钨珠悬浮在玻璃幻灯片上的 250 微升的管道。等待悬浮液风干，并加入300微升的染料溶液。干燥后，使用剃须刀将混合物分成两个1.5毫升的离心管，并加注水管。在水浴中对混合物进行声波化，直到均匀。确保声波尖端直接位于管子上。将两种混合物混合在15毫升锥形管中。再声化三分钟，确保没有大面积的染料涂层珠子。声化后，用10毫升注射器将混合物抽入PVP涂层管中。将油管喂入制备站。旋转油管一分钟。使用注射器小心地取出所有水。打开氮气，将氮气流量调整到每分钟约 0.5 升。在制备站中旋转油管，用氮气干燥 30 分钟。从站中拆下油管，用管切割机将其切割成 13 毫米段。确保大鼠对有毒刺激没有反应，并且没有反射。然后把它固定在上位。沿着胸腔中线切开。将隔膜分开，用剪刀打开胸部。然后将 20 量表 25 毫米针插入左心室。立即用剪刀切割右中庭，并用每分钟 5 毫米的流量对 15 毫升 100 毫升 PBS 进行测。然后在 PBS 中缓冲 100 毫升 4%PFA。灌注后，取出整个大鼠大脑。并后修复它与4%PFA10分钟。使用大鼠大脑矩阵切割500微米厚的日冕部分。做一个拳头切割，并保持刀片到位。然后用第二个刀片进行第二次切割，并垂直取出第一个刀片，将组织放在刀片表面上。将大脑切片放在24井板中，每个井中有1毫升PBS。并重复此过程，直到所有切片都已切割。从每个目标井中删除 PBS。用迪尔/钨管将软骨装入施用器中。在两个网屏之间放一张滤纸。将施用器连接到氦软管。然后将氦气的输出压力调整到每平方英寸90磅。将施用器垂直放置在样品和网格屏幕之间 1.5 厘米的目标井的中心。然后发射迪尔/钨管。用下一个管子加载软骨，并在剩余切片上连续从管子上发射珠子。用 100 毫摩尔 PBS 填充 24 井板。用500微升新鲜PBS洗三次切片。确保切片在洗涤过程中不会翻转。完成后，向切片中添加 500 微升新鲜 PBS。在黑暗中四摄氏度下孵育它们三个小时。孵育后，使用细刷将大脑切片转移到玻璃幻灯片上。并立即添加一毫升的防淡安装介质到每个部分。将 22 到 15 毫米的盖玻片放在各部分上，在黑暗中擦干滑梯。打开共体显微镜系统并切换到 60 倍目标。根据手稿方向调整成像设置。根据神经元的脑区边界和形态特征，获取靶向神经元类型的Z-堆栈图像。在产后第一天从F344/N大鼠身上分离出原发性皮质神经元，并在35毫米玻璃底盘培养它们一周。隔离后的第三天刷新一半的介质。用1毫升100毫摩尔PBS洗两次。在室温下用4%PFA固定细胞15分钟。如前面所述，以弹道标记细胞。用1毫升PBS洗三次。将500微升PBS加入细胞，在黑暗中4摄氏度下孵育3小时。然后添加 200 微升防淡安装介质。并获取每个目标神经元的 Z 堆栈图像。大鼠大脑部分海马区域的典型金字塔神经元被识别为弹道标记技术，其特点是一个大的皮毛树突和周围几个较小的基底树突。神经元重建定量分析软件用于追踪树突状分支和检测脊柱。随后，该软件被用来评估树突分支的复杂性和神经元树干的复杂性。用长度、体积和头部直径评估树突脊柱的形态变化。然后脊柱被分为薄，存根，蘑菇脊柱。并检查了每个半径之间的脊柱数的相对频率。此外，在细胞培养的初级金字塔神经元上验证了弹道标记技术。根据索玛和大的脂肪树突的三角形来识别金字塔神经元。然后使用神经元重建软件来分析细树突脊柱和树突状脊柱长度的分布。在尝试此协议时，重要的是避免大团团或弹道染料涂层钨珠开始制备。团块不允许区分单个神经元。结合神经元重建软件，这种方法使我们能够检查海马金字塔神经元的神经元和树突状脊柱形态。神经元重建软件利用一种算法提供树突状脊柱的自动辅助分类。多个神经元参数的量化提供了一个机会，以更好地了解神经元认知功能障碍背后的机制。

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Primary Culture of Mouse Dopaminergic Neurons

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Whole-cell Patch-clamp Recordings in Brain Slices

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...