Name: rapid golgi stain for dendritic spine visualization in hippocampus and prefrontal cortex
Uploaded: 2021-12-03T14:00:00
Description: Watch this Scientific Journal Video about Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex at JoVE.com

这项工作得到了圣心大学本科生研究计划Grants的支持。

所有实验程序均由圣心大学机构动物护理和使用委员会批准，并符合NIH动物护理和使用指南。
1. 脑组织的分离和浸润
<ol><li>使用前24小时高尔基体染色试剂盒的预混溶液A和B，并保存在深色瓶中和/或黑暗中。制作约80 mL溶液A和B混合物，足以在24小时后更换溶液。存放在密封瓶中。 注意：不需要用盐水或多聚甲醛灌注。</li>
	<li>在二氧化碳安乐死后通过断头台?...

<ol>
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E., Luine, V., Khandaker, H., Villafane, J. J., Frankfurt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adolescent+bisphenol-A+exposure+decreases+dendritic+spine+density:+role+of+sex+and+age.">Adolescent bisphenol-A exposure decreases dendritic spine density: role of sex and age.</a> Synapse. 68 (11), 498-507 (2014).</li><li>Bowman, R. E., 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=Bisphenol-A+exposure+during+adolescence+leads+to+enduring+alterations+in+cognition+and+dendritic+spine+density+in+adult+male+and+female+rats.">Bisphenol-A exposure during adolescence leads to enduring alterations in cognition and dendritic spine density in adult male and female rats.</a> Hormones Behavior. 69, 89-97 (2015).</li><li>Eilam-Stock, T., Serrano, P., Frankfurt, M., Luine, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol-A+impairs+memory+and+reduces+dendritic+spine+density+in+adult+male+rats.">Bisphenol-A impairs memory and reduces dendritic spine density in adult male rats.</a> Behavioral Neuroscience. 126 (1), 175-185 (2012).</li><li>Inagaki, T., Frankfurt, M., Luine, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-induced+memory+enhancements+are+blocked+by+acute+bisphenol+A+in+adult+female+rats:+role+of+dendritic+spines.">Estrogen-induced memory enhancements are blocked by acute bisphenol A in adult female rats: role of dendritic spines.</a> Endocrinology. 153 (7), 3357-3367 (2012).</li><li>Jacome, L. F., 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=Gonadal+Hormones+Rapidly+Enhance+Spatial+Memory+and+Increase+Hippocampal+Spine+Density+in+Male+Rats.">Gonadal Hormones Rapidly Enhance Spatial Memory and Increase Hippocampal Spine Density in Male Rats.</a> Endocrinology. 157 (4), 1357-1362 (2016).</li><li>Frankfurt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol-A:+a+plastic+manufacturing+compound+disrupts+critical+brain+structures+and+impairs+memory.">Bisphenol-A: a plastic manufacturing compound disrupts critical brain structures and impairs memory.</a> Research Features. , (2021).</li><li>Wallace, M., Luine, V., Arellanos, A., Frankfurt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovariectomized+rats+show+decreased+recognition+memory+and+spine+density+in+the+hippocampus+and+prefrontal+cortex.">Ovariectomized rats show decreased recognition memory and spine density in the hippocampus and prefrontal cortex.</a> Brain Research. 1126 (1), 176-182 (2006).</li><li>Wallace, M., Frankfurt, M., Arellanos, A., Inagaki, T., Luine, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+recognition+memory+and+decreased+prefrontal+cortex+spine+density+in+aged+female+rats.">Impaired recognition memory and decreased prefrontal cortex spine density in aged female rats.</a> Annals of the New York Academy of Science. 1097, 54-57 (2007).</li><li>Bowman, R. E., Hagedorn, J., Madden, E., Frankfurt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+adolescent+Bisphenol-A+exposure+on+memory+and+spine+density+in+ovariectomized+female+rats:+Adolescence+vs+adulthood.">Effects of adolescent Bisphenol-A exposure on memory and spine density in ovariectomized female rats: Adolescence vs adulthood.</a> Hormones Behavior. 107, 26-34 (2019).</li><li>Novaes, L. S., Dos Santos, N. B., Perfetto, J. G., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+prevents+acute+restraint+stress-induced+anxiety-related+behavior+but+not+changes+in+basolateral+amygdala+spine+density.">Environmental enrichment prevents acute restraint stress-induced anxiety-related behavior but not changes in basolateral amygdala spine density.</a> Psychoneuroendocrinology. 98, 6-10 (2018).</li><li>Trzesniewski, J., Altmann, S., J&#228;ger, L., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+Purkinje+cell+size+is+compatible+with+near+normal+morphology+and+function+of+the+cerebellar+cortex+in+a+mouse+model+of+spinocerebellar+ataxia.">Reduced Purkinje cell size is compatible with near normal morphology and function of the cerebellar cortex in a mouse model of spinocerebellar ataxia.</a> Experimental Neurology. 311, 205-212 (2019).</li><li>Zemmar, 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=Oligodendrocyte-+and+Neuron-Specific+Nogo-A+Restrict+Dendritic+Branching+and+Spine+Density+in+the+Adult+Mouse+Motor+Cortex.">Oligodendrocyte- and Neuron-Specific Nogo-A Restrict Dendritic Branching and Spine Density in the Adult Mouse Motor Cortex.</a> Cerebral Cortex. 28 (6), 2109-2117 (2018).</li></ol>

本方案描述了一种高尔基体浸渍方法，该方法允许快速同时处理许多切片。这是对前面描述的5 种劳动密集型方法的改进，并且始终产生浸渍神经元进行分析。此外，接触高尔基体浸渍中使用的有毒化学物质较少。该过程中最具挑战性的部分是使切片在幻灯片上保持平坦，这需要大量的练习。使用冷冻喷雾保持所有东西尽可能冷是至关重要的。
一旦载玻片干...

使用重铬酸钾和硝酸银标记神经元的方法首先由Camillo Golgi1，2 描述，随后由Santiago Ramon y Cajal使用，以产生大量区分神经元和神经胶质亚型的工作。最近出版的一本附有他的插图的书现已出版3.在100多年前发表的Ramon y Cajal的研究之后，很少使用高尔基体浸渍。高尔基体浸渍是一个费力的过程，允许用光学显微镜对神经元进行三维可视化。多年来，高尔基体方法进行了多次修改，使方法更容易，染色更一致4。1984年，Gabbott和Somogyi5 描述了单段高尔基体浸渍程序，该程序允许更快速的加工。这种高尔基体浸渍方法需要用4%多聚甲醛和1.5%苦味酸灌注，固定后，然后振动切片到3%重铬酸钾浴中。切片安装在载玻片上，盖玻片的四个角粘合在一起，以便当浸入硝酸银中时，扩散是渐进的。然后将盖玻片弹出，部分脱水，并最终用安装介质永久滑落盖板。该技术被成功地用于标记海马体中的神经元和神经胶质细胞6，7，8 。这里描述的快速高尔基体方法是一种改进，因为重铬酸钾和硝酸银的暴露要少得多，并且不使用多聚甲醛和苦味酸。此外，尽管可以使用Gabbott和Somogyi5 方法的修饰来浸渍的细胞进行分析，但在脱水步骤中，切片通常过度或未充分暴露或从载玻片上掉落，并且通常必须合并几个实验以具有足够的细胞进行分析。
本方案描述了使用高尔基染色试剂盒（见 材料表）来标记大鼠海马体和内侧前额叶皮层（mPFC）中的树突和树突状棘。与以前的方法相比，这种方法的优点是它速度快，研究人员接触有毒化学物质较少，并且神经元染色一致。在许多研究中，下面描述的方案已被用于进行微小的修改，以评估大鼠海马体和mPFC中的树突状脊柱密度9，10，11，12，13，14，15。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cardboard slides trays</td>
			<td>Fisher Scientific</td>
			<td>12-587-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 24 x 60mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-M</td>
			<td></td>
		</tr>
		<tr>
			<td>FD Rapid GolgiStain kit</td>
			<td>FD Neurotechnologies</td>
			<td>PK 401</td>
			<td>Stable at RT in the dark for months; Golgi staining kit</td>
		</tr>
		<tr>
			<td>Freezing Spray</td>
			<td>Fisher Scientific</td>
			<td>23-022524</td>
			<td></td>
		</tr>
		<tr>
			<td>HISTO-CLEAR</td>
			<td>Fisher Scientific</td>
			<td>50-899-90147</td>
			<td>clearing agent</td>
		</tr>
		<tr>
			<td>NCSS Software</td>
			<td>Kaysville, UT, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific</td>
			<td>SP-15-100</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek CTYO OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td>Used to mount brains on cryostat chuck</td>
		</tr>
	</tbody>
</table>

rapid golgi stain for dendritic spine visualization in hippocampus and prefrontal cortex

高尔基体浸渍，使用具有轻微适应的高尔基染色试剂盒，用于浸渍大鼠海马体和内侧前额叶皮层中的树突状棘。这种技术比以前的高尔基体浸渍方法有了显着的改进，因为预混合的化学物质使用起来更安全，神经元始终浸渍良好，背景碎片少得多，并且对于给定的区域，实验之间的脊柱密度存在极小的偏差。此外，大脑可以在某一点后积累并保持冷冻，直到进一步处理。使用这种方法可以研究任何感兴趣的大脑区域。一旦染色并覆盖滑动，树突状脊柱密度通过计算树突长度的棘数量来确定，并表示为每10μm树突的脊柱密度。

使用快速高尔基体方法，细胞始终被很好地浸渍，以便有足够的细胞进行分析。与以前的方法相比，这是一个显着的改进，在以前的方法中，必须将实验合并以具有足够的数据进行分析。因此，可以一次处理更多的样品，并且可以冷冻储存大脑直到处理。 图3显示了海马体CA1区域中高尔基体浸渍细胞在低功率和高功率下的例子。对给定区域中的棘进行计数可以产生具有小标?...

Watch this Scientific Journal Video about Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex at JoVE.com

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

该协议描述了快速高尔基体方法的修改，该方法可以适应神经系统的任何部分，用于染色大鼠海马体和内侧前额叶皮层中的神经元。

快速高尔基染色用于海马体和前额叶皮层树突状脊柱可视化

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial ...

虽然相对简单，但高尔基体浸渍技术确实有一些棘手的步骤，如果不能正确完成这些步骤，会导致细胞不适合分析。该技术为高尔基体浸渍神经元提供了快速，可重复的标记。此外，均值的小标准误允许在实验之间进行比较。关于这种技术最具挑战性的部分是切割低温恒温器部分，因为存在不寻常的一致性和固定，因此将它们放在幻灯片平面上是一个需要练习的挑战。首先，将少量组织培养基放在预冷的低温恒温器卡盘上。将脑阻滞物安装在低温恒温盘上，方法是用戴手套的手稍微解冻积木块的一侧，并将其放在组织培养基上。在刀和块上使用冷冻喷雾。使用防滚板或刷子在切割时保持截面平坦。在零下22摄氏度的低温恒温器上切割100微米的部分。如果可能的话，通过使用室温载玻片来解冻安装，并迅速将其与截面相对。或者，将载玻片保存在低温恒温器中，使它们冻结。用冷镊子或冷漆刷转移该部分，然后解冻安装座。将三到四个日冕部分安装到下沉上。用纸巾擦拭刀片之间的刀。如果刀具需要进一步清洁，请在切割下一片之前使用100%乙醇并干燥。将载玻片放在机架中，间隔足够远，以便解决方案能够进入各个部分。将切片放入蒸馏水中两次，持续四分钟，然后将其放入高尔基体浸渍溶液中10分钟。分离盖玻片，以确保各部分上只放置一个盖玻片。在载玻片上放置玻璃盖玻片之前，用大量的安装介质覆盖载玻片。一旦盖子滑落，干燥的载玻片在任何无孔纸上标记三到五天，稍微移动它们，特别是在第一天之后，以避免粘附。然后将载玻片转移到载玻片架上，理想情况下，在检查载玻片之前至少干燥三周。为了分析内侧前额叶皮层和海马体CA1区域的锥体神经元中的树突状脊柱密度，使用图像分析程序测量树突状长度。使用手动计数器计算树突上的棘，并记录棘的长度和数量。对于分析，选择浸渍时具有细胞体和树突的神经元，以及连续且可与相邻细胞区分开来的树突。该图显示了海马体CA1区域高尔基体浸渍细胞在低功率和高功率下的示例。该图说明了一项实验，其中在CA1和内侧前额叶皮层中环境富集后，青春期雄性和雌性大鼠锥体细胞上的基底树突状脊柱密度增加。切割时保持足够的温度是具有挑战性的，为此我们使用了大量的冷冻喷雾。然后，很难将这些部分放到载玻片上并将它们干燥平放。该技术用于检查神经解剖学特征。它可以单独使用或与其他神经解剖学示踪方法结合使用。

Research

JoVE Journal

Neuroscience

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

科研

神经科学

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

关联从人类前额叶皮层的fMRI信号的行为反应：检查的认知过程，使用任务分析

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

侧前额叶皮质联合经颅磁刺激与脑电图的研究

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

Abbiategrasso 脑库协议，用于收集、处理和描述老化的大脑

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

莱纳尔皮质-希波坎普斯有机切片文化中的癫痫发生模型

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...