Name: immunohistochemical visualization of hippocampal neuron activity after spatial learning in a mouse model of neurodevelopmental disorders
Uploaded: 2015-05-12T15:00:00
Description: Watch this Scientific Journal Video about Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders at JoVE.com

我们要感谢CIBIO（特伦托大学）和中国北车研究院神经科学的行政人员寻求帮助。乔瓦尼·普罗文扎诺是由韦罗内西基金会（米兰，意大利）博士后奖学金支持。这一工作是由意大利教育部大学和研究（2008年PRIN补助＃200894SYW2_002和打2010至2011年批＃2010N8PBAA_002到YB），特伦托大学（CIBIO创业补助金，以YB）和马拉松式节目基金会（拨款＃GGP13034到YB）。

所有实验均符合欧盟指令六十三分之二千〇一十/ EU进行，并批准了卫生意大利外交部。 1，动物保健，住房和治疗<ol><li>执行使用小鼠按照各自的机构动物护理准则的所有实验方案。 </li><li>维持动物在12小时光照/黑暗周期的食物和水可随意获得。 </li><li>众议院在小鼠2-4组，在标准尺寸的聚碳酸酯鼠标笼锯末垫料每周更换一次。 </li><li>使用年龄和性别匹配的小鼠，因?...

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J., 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=A+mouse+model+for+the+learning+and+memory+deficits+associated+with+neurofibromatosis+type.">A mouse model for the learning and memory deficits associated with neurofibromatosis type.</a> I. Nature. 15, 281-284 (1997).</li><li>Morris, R. G., Garrud, P., Rawlins, J. N., O'Keefe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Place+navigation+impaired+in+rats+with+hippocampal+lesions.">Place navigation impaired in rats with hippocampal lesions.</a> Nature. 297, 681-683 (1982).</li><li>Praag, H., Kempermann, G., Gage, F. 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在这里，我们提供了一个的pERK免疫协议为揭示神经元激活在EN2以下MWM - / -小鼠，神经发育障碍的小鼠模型。被检测EN2的CA3区振作水平降低- / -突变体相比，WT。不同于在CA3区，pERK的阳性神经元的普遍增加什么观察物代替在两个门区检测和EN2的GCL - / -小鼠与WT相比。在ERK磷酸化水平这一趋势相反的一种可能的解释可能依赖的pERK感应以下MWM子的具体机制。 <p class="jove_content"...

神经发育障碍包括多种异构疾病如唐氏综合征，脆性X染色体综合征（FXS），Rett综合征，神经纤维瘤病，结节性硬化症和ASD，其中中枢神经系统（CNS）的发育和成熟期间早期干扰的基胎儿期1。这些发育的大脑功能障碍可引起运动功能，语言，学习和记忆过程中深刻的，终身的影响。遗传和环境因素的过多，在过去的几年里2,3-已牵涉在神经发育障碍的发病机制。即使临床表型的分子机制仍然是未知的，上面提到的研究结果已允许的这些疾病的几个小鼠模型的发展。学习和记忆的赤字已经确定了一些，这些小鼠模型，如+/- TSC1，TSC2的+/-，NF1 +/-和EN2 - / -小鼠2,4-7。在神经发育障碍的领域的一个重要的挑战是底层记忆和学习功能障碍细胞和分子过程的识别。学习或记忆时激活所选的信号通路可诱导特异性基因的转录，并最终导致从头蛋白质的合成。立即早期基因（即早基因）活化和蛋白依赖性突触修饰迅速诱导的脑的神经元响应于神经元活动和行为训练8,9。 在涉及的信号通路神经纤维瘤蛋白赤字已与神经发育障碍学习障碍有关。神经纤维瘤是NF1基因的产物，其突变导致神经纤维瘤病1型，一个复杂的遗传综合征的特点是神经系统的肿瘤，行为和电机的延迟，和认知迪萨bilities 10。小鼠杂合NF1缺失局限于抑制性神经元表现出的长时程增强的早期阶段（LTP），以及在MWM 5,11,12损害空间学习障碍。有趣的是，NF1缺陷在该小鼠模型导致过度活化在抑制性Ras信号在学习期间，导致增加ERK磷酸化，最后在GABA的释放，从这些神经元5的异常强化。 基于这些发现，神经元活动的行为任务之后的可视化表示的方式来重建涉及神经发育疾病的具体电路。这里所描述的免疫组化协议旨在评估和量化的ASD小鼠模型的认知缺陷以下MWM海马ERK的磷酸化水平。 MWM被广泛用来研究在啮齿类动物13,14海马依赖性的空间学习和记忆</s向上&gt;。我们决定使用ERK磷酸化作为任务依赖性海马学习的分子读出，由于ERK显示出具有在学习和记忆形成15了至关重要的作用。此外，ERK途径是必要的经验依赖性可塑性在显影视觉皮层16。最后，缺乏两个ERK亚型（ERK2）在CNS节目之一的小鼠标记在认知，情绪和社会行为17的异常，这表明ERK信号可能在神经发育障碍，如房间隔缺损的发病中起关键作用。 我们使用ENGRAILED 2淘汰赛（EN2 - / - ）小鼠神经发育障碍的典范。 EN2 - / -小鼠显示出解剖和行为“ASD样”特征，包括前脑interneurons 18的损失，ASD相关基因19表达降低，降低的社交性，和受损的认知灵活性6,7,20。空间学外贸纳克和存储器的缺陷，如那些在MWM检测，尤其稳健在EN2 - / -小鼠6,7-和可能相关于在ASD患者21中观察到的认知障碍。此外，我们发现，空间学习MWM中受损与神经纤维瘤缩小表达有关，增加的pERK水平EN2的门区- / -成年小鼠7。在这里，我们提出详细的协议振作这个ASD小鼠模型以下MWM的免疫组化特征。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EthoVision XT 8</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td>This software platform is not a requirement &#8211; there are many other behavioral softwares on the market.</td>
		</tr>
		<tr>
			<td>Tempera Paint</td>
			<td>Giotto - Fila Group Company</td>
			<td></td>
			<td>White and liquid, non toxic. Used to prepare opaque water in the Morris water maze.</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>Equivalent models from other companies can be used.</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sigma</td>
			<td>CLS3524</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Fisher Scientific</td>
			<td>A406-20</td>
			<td>Used to make ethanol gradient for dehydration prior to slide mounting.</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>66004-950</td>
			<td>Toxic - to be used under hood. Change xylene every month depending on use.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>&#160;S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Hydrogen Peroxide</td>
			<td>Sigma</td>
			<td>H1009-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Abcam</td>
			<td>G9023-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ABC kit Vectastain&#160;</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>Add in a volume of 5 ml of PBS 2 drops of reagent A, mix and then add 2 drops of reagent B and mix.</td>
		</tr>
		<tr>
			<td>DAB peroxidase substrate</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>Add in a volume of 5 ml ddH2O: 2 drops of buffer stock solution and mix; 4 drops of DAB and mix; 2 drops of H2O2 and mix.</td>
		</tr>
		<tr>
			<td>pERK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>4370</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Biotinylated Goat Anti-Rabbit IgG Antibody</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>Dilution 1:250</td>
		</tr>
		<tr>
			<td>SuperFrost Slides&#160;</td>
			<td>Carl Roth</td>
			<td>1879</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-548-B</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX</td>
			<td>Sigma</td>
			<td>317616</td>
			<td>Mounting medium for slides. Equivalent mounting medium can be used.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>&#160;Zeiss&#160;</td>
			<td>Axio Imager.M2</td>
			<td>Equivalent microscope can be used.</td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe Systems, San Jose, CA</td>
			<td></td>
			<td>To assemble images.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institute of Health</td>
			<td></td>
			<td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>SigmaPlot 11.0</td>
			<td>Systat Software Inc. (USA)</td>
			<td></td>
			<td>Equivalent softwares for statistical analysis can be used.</td>
		</tr>
		<tr>
			<td>Prism 6</td>
			<td>GraphPad Software, Inc. (La Jolla, CA, USA)</td>
			<td></td>
			<td>Equivalent softwares for statistical analysis can be used.</td>
		</tr>
	</tbody>
</table>

immunohistochemical visualization of hippocampal neuron activity after spatial learning in a mouse model of neurodevelopmental disorders

感应磷酸化细胞外调节激酶（的pERK）是学习相关神​​经元激活的可靠的分子读出。这里，我们描述一个的pERK免疫协议来研究海马神经元激活的轮廓在暴露于一个空间学习任务的特征在于神经起源的认知缺陷小鼠模型。具体来说，我们使用的pERK免疫研究神经元激活以下Morris水迷宫（MWM，经典的海马依赖学习任务）中ENGRAILED-2基因敲除（EN2 - / - ）小鼠，自闭症谱系障碍模型（ASD）。相比于野生型（WT）对照，EN2 - / -小鼠显示出在MWM显著空间学习障碍。 / - - MWM后，在EN2的特定海马子场中检测到在的pERK阳性神经元的数目显著差异小鼠相比，WT动物。因此，我们的协议可以稳健地检测在不同在ASD的小鼠模型相关联的海马依赖性学习障碍的pERK阳性神经元。更一般地，我们的协议可以应用于调查其特征在于认知缺陷都遗传或药理学小鼠模型的海马神经元激活的轮廓。

这里所描述的协议被设计来可视化，通过免疫组织化学，海马神经元活动的MWM之后的特定标志物在神经发育障碍的小鼠模型中的表达。所有本文中所示的实验数据取自我们最近的工作7。男性和女性WT和EN2 - / -年龄匹配的同窝成人（3-5个月;重量= 25-35克）用杂交配获得的使用。二十二个小鼠（11％基因型）被用于MWM。 8只（4％基因型）处死在MWM探针试验结束并用于免疫组织化学实验?...

Watch this Scientific Journal Video about Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders at JoVE.com

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

我们描述了一个协议，免疫组织化学研究海马神经元激活的轮廓暴露于空间学习任务为特征的神经发育起源的认知缺陷小鼠模型。此协议可应用到特征的认知缺陷都遗传或药理学小鼠模型。

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Research

JoVE Journal

Neuroscience

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 ...

科研

神经科学

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

使用近红外光谱MazeSuite和功能研究学习空间导航

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

海马胰岛素显微注射和<em在体内</em&gt;微透析过程中空间记忆测试

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

性别差异在小鼠海马星形胶质细胞<em&gt;在体外</em&gt;缺血

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

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 ...

Low-Density Primary Hippocampal Neuron Culture

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

评估空间学习记忆小鳞类爬行动物

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...