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，因为对疾病的易感性可以与年龄和性别的神经发育障碍的不同的鼠标型号而异。 </li><li>选择动物的适当数量达到统计学显着性（至少10只小鼠基因型的行为研究和4只小鼠基因型的免疫组织化学实验的）。 </li></ol> 2. Morris水迷宫（MWM） <ol><li>使用一个圆形罐（直径为100厘米，高度为50厘米）。 <ol><li>放置一些视觉提示对周围的坦克的房间隔断。 </li><li>放置一个直径10厘米的平台到罐在四个假想象限之一的中心的固定位置。保持在同一象限的所有培训课程的所有审判隐藏的平台。 </li><li>填用自来水池，直到平台为1cm的水面上方。平衡水的温度应接近22℃;这个步骤可能需要几个小时。加入热水，以缩短平衡水温度所需要的时间。 </li><li>添加大约1.5-2升白，无毒涂料，使水不透明。 </li></ol></li><li>轨道征收程序<ol><li>设置MWM的跟踪系统与标准的CCD摄像头和视频板。选择一个视频跟踪系统，以监视和分析几个变量，如路径长度，ESCAPE延迟和时间花费在每个象限中。 </li><li>划分舞台四个象限，调整检测设置，以确保动物和背景之间最佳的对比度进行现场跟踪。 </li><li>指定平台区域。 </li><li>设置最大的试验时间为60秒。 </li></ol></li><li> MWM测试<ol><li>前行为测试，允许小鼠在20-25分钟的适应期在一个地区，他们不能看到游泳池或空间线索。 </li><li> （一天连续两次审判，它们之间20-30分钟间隔）训练每只小鼠9天。 </li><li>切换计算机和摄像机并开始播放视频跟踪系统软件。 </li><li>就拿老鼠的尾巴，将其放入水中，用它的头指向了游泳池边，从四个起始位置（北，南，东，西）中的一个开始。对于每个小鼠，开始位置应该是不同的，并且在各试验pseudorandomized。 </li><li>让鼠标游泳，搜索platfORM最多60秒。一旦动物达到平台，允许它停留在平台上，持续20秒。 </li><li>如果鼠标没能找到时间内平台上，轻轻引导鼠标的平台，通过它的尾巴抱着它，让它呆在那里，持续20秒。一旦动物已完成全部两项试验，用毛巾擦干其关闭，并把它放回笼子。 </li><li>训练期间中的每一天，重复相同的过程（步骤2.3.4-2.3.6）。 </li><li>最后一次训练后的步道上进行9天的探索实验。 </li><li>从池中取出平台。 </li><li>让鼠标游泳和搜索平台，最多60秒。 </li><li>定期检查水温度，以便它应该是相同的（22℃）和清理出池。 </li></ol></li></ol> 3.准备从灌流动物的节<ol><li>牺牲小鼠在MWM的末端。每四个基因型的小鼠之前必须要麻醉euthaniz根据当地和国际准则通货膨胀。 </li><li> transcardially灌注动物（切右auriclewith剪刀，以允许出血和引入一个蝴蝶套管进入左心室灌注）用磷酸缓冲盐水（PBS）为4-5分钟，随后在PBS中的4％多聚甲醛为10-15分22。 </li><li>解剖和后固定在4％多聚甲醛的大脑在4℃下至少12小时。 </li><li>直到所需的切片放置在含有在4℃在0.1M PBS中的15ml 0.02％的叠氮化钠和存储50毫升聚苯乙烯锥形管的大脑。 </li><li>剪切和vibratome切割之前删除的小脑。 </li><li>胶大脑直立与切割部位，用强力胶，到vibratome的固定板。 </li><li>切脑入20-40微米厚的冠状切片，在串行顺序整个背海马，适当的vibratome。 </li><li>收集vibratome片，用画笔和转移到24多孔板包含用于每个井1毫升的0.1M PBS中。 </li><li> （组织可能被用于最多一年）店铺部分为短期的PBS或长期在0.02％叠氮化钠的PBS中，在4ºC。 </li></ol> 4.免疫组化<ol><li>转印3-5背海马切片每只小鼠，于24多孔板含有500微升的0.1M PBS中每个孔中。 </li><li>用500微升的0.1M PBS（5分钟，3次，用温和振荡）洗涤包含在每个部分好。 </li><li>淬灭内源过氧化物酶活性，通过将切片在1％的H 2 O 2的PBS 20分钟，轻轻摇动。 </li><li>在0.1M PBS中，轻轻摇动（3×5分钟）洗涤切片。 </li><li>组织的通透性：在PBS中孵育切片5分钟在0.2％的Triton X-100。 </li><li>在上述步骤过程（4.4; 4.5），使足够封闭缓冲液（10％山羊血清和0.1％的Triton-X100的PBS）在整个实验（numbe段 - [R×100微升×3步）。 </li><li>注意：从在其中二次抗体（缀合到生物素）中的由相同物种使用血清。 </li><li>防止非特异性抗体结合，通过将切片在封闭缓冲液（100μl/孔部）1小时，在室温（RT）下温和搅拌。 </li><li>孵育稀释在封闭缓冲液中过夜，在4℃，温和搅拌在初级抗体（的pERK）部分。 </li><li>在0.1M PBS中，轻轻摇动（3×5分钟）洗涤切片。 </li><li> （如在步骤4.6中使用相同）处理1小时，在室温下缓慢搅拌稀释在封闭缓冲液：孵育缀合有生物素（250 1）的次级抗体。 </li><li>制备ABC试剂同时，因为抗生物素蛋白和生物素至少需要30分钟，在室温下，以复合物。 </li><li>准备根据制造商的协议ABC试剂的所需体积。 </li><li>在0.1M PBS中，轻轻摇动（3×5分钟）洗涤切片。 </li><li> IncubatË部分在室温与ABC试剂45分钟。 </li><li>在0.1M PBS中，轻轻摇动（3×5分钟）洗涤切片。 </li><li>准备工作DAB解决方案，涉及的每一步DAB必须做通风柜作为DAB为致癌和致畸。 </li><li> DAB传输解决方案，以一个新的板块由塑料移液管。 </li><li>在含DAB手工工作溶液慢慢旋流板的板放置节，以允许承受的最大部分。 </li><li>监测显微镜DAB反应，直到有足够的信号开发（2-5分钟）。 </li><li>停止在所希望的颜色强度的反应洗涤在0.1M的PBS（3×5分钟）该组织。 </li><li>使用漂白剂停用通风柜任何剩余的DAB底物溶液中过夜，并根据实验室规定进行处置。 </li><li>明胶山段浮在PBS涂幻灯片。然后干燥它们在室温下至少3-4小时。 </li><li>在50％的脱水段顺序地，70％，95％和100％的乙醇，每次2分钟，并明确在100％二甲苯5分钟。 </li><li>盖玻片用封固剂和在通风橱离开干燥过夜。 </li></ol> 5.细胞计数<ol><li>算的pERK阳性细胞，三到五节，在背侧海马的水平应每只动物进行分析。 </li><li>从各部分中，在放大200倍的使用适当的显微镜获取多个亮场图像，然后使用图像处理软件将它们组装。 </li><li>关于使用ImageJ的免费软件TIFF转换拼接图像进行细胞计数。 </li><li>计数结束二点五十八计数为100×100微米的每个箱子的pERK染色的细胞在门区及颗粒细胞层。 </li><li>细胞密度将表示为每计数窗口（100×100微米）标记的细胞的数目。 </li><li>执行统计分析，以评估基因型之间显著差异。这可以用学生t测试或ANOVA，然后通过使用商业软件适当事后检验。在P &lt;0.05设置统计显着性水平。 </li></ol>

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作者什么都没有透露

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 ...

immunohistochemical-visualization-hippocampal-neuron-activity-after

Research

JoVE Journal

Neuroscience

11.2K 次观看.  University of Trento. 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。 

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

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

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 in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

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

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of 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

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

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

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

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

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

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

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups 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

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

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

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 in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

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

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use 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

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 undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

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 ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.