We wish to thank Yvonne Ducho and Kathrin Pohlmann for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 779) and by the State Saxony-Anhalt / European Regional Development Fund (ERDF) via the Center for Behavioral Brain Sciences (CBBS).

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The authors have nothing to disclose.

这项研究提出了学习和小鼠不同脑区记忆的巩固过程中的突触蛋白表达变化的准确的定量分析进行优化的方法流程。该装置提供了学习的机会，在一个动物的水平蛋白的表达，尽管每个样品至少有三个技术重复进行质谱分析所需的应用程序的。 该方法考虑到前和postsynapse选自高分子量支架蛋白的特定的蛋白质组合物也是重要的介体蛋白承载介质或更低的分子量。突触制剂的在溶液消化导致有效地产生，因此，支架衍生肽的过表达。这，反过来，可能抑制较小或低丰度蛋白的分析。从SDS-PAGE馏分的建议制备每个样品与平行的在凝胶内消化过程合并的等分试样促进中，低丰度蛋白质的分析和表示高度推荐的互补方法。后从样品衍生的所有级分的分离质谱应用（ 例如 ，在溶液消化，凝胶内消化，结合磷富集馏分）可以结合在相应的MS / MS数据集，并进一步用于蛋白质鉴定和量化由PEAKS计算软件或替代流行的软件包。 可替代地，在溶液中消化的样品的产生的样品的凝胶内消化衍生馏分（样品泳道分开处理的凝胶区域）和级分的个人应用（通过离子交换色谱，例如 ），以质谱可以增加分析深度。然而，这种延长的工作流显着地增加对于LS-MS / MS数据采集所需的时间。对于generatio学习和记忆形成的蛋白质组分析的指定时间期间的突触蛋白的重排的详细分子序列的n个是必需的。这个时候当然会，直到动物的性能达到学习曲线的渐近水平约后后甚至第一堂训练课中立即启动，涵盖了近网状的时间框架。 8 - 10天的培训（ 见图2细节）。 突触蛋白的磷酸化的变化分析需要FMTD学习过程中特别注重所选择的时间框架。在启动已知由蛋白质磷酸化dephosphorylations触发突触蛋白重组，一方面信号通路有望在动物训练的早期阶段。另一方面，也有其调节S内的连接和装配已知多种磷酸突触蛋白的长期持久的修改ynaptic结构19,20。这些翻译后修饰甚至在记忆巩固稍后的时间点的预期。 该蛋白质组的工作流程产生的复杂的数据集需要处理生物信息学，以确定参与的分子途径和关键分子。荟萃分析显示显著任职人数过多的途径，从而起到在学习和记忆过程中发挥作用。

学习是基于记忆痕迹及其维护的形成。它已被广泛接受，一个潜在的机制可以表示神经元之间的现有突触联系的新的和/或重排的活性依赖性形成。在分子水平上，各种蛋白质修饰，亚细胞relocalizations和突触蛋白的周转变化已经描述1-4（Lamprecht的，2004＃8）。然而，大多数的研究迄今针对选定的蛋白质，而不是对全球但复杂的突触蛋白质组合物。本方法允许一个学习实验后小鼠脑区突触蛋白质组变化公正的审查。合适的是使突触架构的学习诱导重组时间点相关的分子的快照。所描述的工作流程，需要不同的专家在动物行为，蛋白质生物化学，质谱和bioi一个特定的团队合作nformatics。 所选择的学习范例， 即频率调制的音调鉴别（FMTD），是在啮齿类动物5充分表征听觉辨别任务。学习和长期记忆的形成这个梭箱GO / NO-GO-任务涉及取决于增加皮质多巴胺信号和蛋白质合成机制。因此，最近对沙土鼠和小鼠的蛋白质组学研究揭示多巴胺和皮质突触部件的学习诱导塑料重排，而且在更基础的大脑区域FMTD学习和记忆6-8期间，理应相互作用。这表明，记忆的形成涉及不同脑区之间复杂的相互作用，因此，可能在蛋白质水平这些区域内差异调节。因此，选择的皮层和皮层下小鼠的大脑区域的解剖被包括在工作流程。 此外，可靠characterizati在偶数的突触蛋白组合物弱变化需要前和突触后隔室的富集而非匀浆或粗膜部分9的分析。蛋白质组学分析之前因此，突触体的制备利用建立的协议，以增加检测电平与动态范围为特定突触蛋白10,11进行说明。 使用无标签的高分辨率质谱定量目的的基本前提是高度的蛋白质样品的相似性。由于突触蛋白质组成，而细微的变化，预计学习，无标签的方法将是适当的比较，从相应的培训和天真的小鼠获得的蛋白样品后出现。可替代地，使用稳定同位素（ 例如 TMT，iTRAQ的，ICPL和SILAC）以及基于MS2-标签自由QUA蛋白/肽的特定条件下的标签的策略ntification（SWATH）是有用的，但它们比选择无标记方法更昂贵或需要特别的质谱硬件。 因为蛋白质组掩护往往产生复杂的数据集，建议适当的数据解释生物信息的处理。其他荟萃分析可以支持更好地了解潜在的范式相关的变更，涉及的关键细胞过程和信号通路的识别潜在的分子机制。适当的方法也被描述如下。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3M Empore Solid Phase Extraction- Filter</td>
			<td>3M Bioanalytical Technologies</td>
			<td>4245SD</td>
			<td>7 mm/3 ml</td>
		</tr>
		<tr>
			<td>Acclaim PepMap 100</td>
			<td>Dionex/Thermo Scientific</td>
			<td>164564</td>
			<td>100 &#181;m x 2 cm, C18</td>
		</tr>
		<tr>
			<td>Acclaim PepMap 100</td>
			<td>Dionex/Thermo Scientific</td>
			<td>164569</td>
			<td>75 &#181;m x 25 cm, C18</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Carl Roth GmbH</td>
			<td>3738.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile (ACN)</td>
			<td>Carl Roth GmbH</td>
			<td>AE70.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (30%)</td>
			<td>AppliChem</td>
			<td>A0951</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydrogen carbonate&#160;&#160;</td>
			<td>Fluka</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide&#160;</td>
			<td>Fluka&#160;</td>
			<td>44273</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate&#160; (APS)</td>
			<td>AppliChem</td>
			<td>A2941</td>
			<td></td>
		</tr>
		<tr>
			<td>Biofuge pico</td>
			<td>Heraeus GmbH</td>
			<td>75003280</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue R-250&#160;</td>
			<td>SERVA Electrophoresis GmbH</td>
			<td>17525</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Pharmacia Biotech</td>
			<td>17132901</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Charles River&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cantharidin</td>
			<td>Carl Roth GmbH</td>
			<td>3322.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes for MLS-50</td>
			<td>Beckman Coulter</td>
			<td>344057</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes for TLA 100.1 rotor</td>
			<td>Beckman Coulter</td>
			<td>343776</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>AppliChem</td>
			<td>A1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5417R centrifuge</td>
			<td>VWR</td>
			<td>22636138</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf A-8-11 rotor</td>
			<td>VWR</td>
			<td>5407000317</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Fluka</td>
			<td>14265</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneCodis</td>
			<td>http://genecodis.cnb.csic.es/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gephi</td>
			<td>https://gephi.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>AppliChem</td>
			<td>A1123</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>AppliChem</td>
			<td>A1067</td>
			<td></td>
		</tr>
		<tr>
			<td>HALT Phosphatase Inhibitor Cocktail&#160;</td>
			<td>Pierce /Thermo Scientific</td>
			<td>78420</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer solution&#160;</td>
			<td>PAA Laboratories GmbH</td>
			<td>S11-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenization vessel 2 mL</td>
			<td>Sartorius AG</td>
			<td>854 2252</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>Ingenuity Pathway Analysis</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory drilling drive &#160;K-ControlTLC 4957&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Kaltenbach &#38; Vogt GmbH</td>
			<td>182997</td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ Tune Plus 2.7.0.1112 SP2</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos Pro</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Macs-mix tube rotator</td>
			<td>Miltenyi Biotech</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr>
			<td>Magic Scan 4.71</td>
			<td>UMAX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth GmbH</td>
			<td>AE71.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MLS-50 rotor</td>
			<td>Beckman Coulter</td>
			<td>367280</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima MAX Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>364300</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Scientific</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>PEAKS 7.5</td>
			<td>Bioinformatic Solutions&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail 3</td>
			<td>Sigma-Aldrich</td>
			<td>P0044</td>
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			<td>PhosphoRS 3.1</td>
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			<td>Roche</td>
			<td>4906845001</td>
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			<td>Plunger/pestle made of PTFE</td>
			<td>Sartorius AG</td>
			<td>854 2651</td>
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			<td>Carl Roth GmbH</td>
			<td>274.2</td>
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			<td>SONOREX RK 156 Ultrasonic Bath</td>
			<td>BANDELIN electronic GmbH &#38; Co. KG</td>
			<td>305</td>
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			<td>Soundproof chamber</td>
			<td>Industrial Acoustics Company</td>
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			<td>Carl Roth GmbH</td>
			<td>4621.2</td>
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			<td>Tetramethyl ethylene -1,2-diamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
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			<td><a name="RANGE!A60">Titansphere TiO 5&#181;m</a></td>
			<td>GL Sciences Inc. Japan</td>
			<td>502075000</td>
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			<td>TLA 100.1 rotor</td>
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			<td>Tris ( hydroxymethyl) aminomethane (TRIS)</td>
			<td>AppliChem</td>
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			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
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			<td>Trypsin Gold</td>
			<td>Promega</td>
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			<td>Ultimate 3000 Ultra HPLC&#160;</td>
			<td>Dionex/Thermo Scientific</td>
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			<td>Ultracentrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>343776</td>
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			<td>Unijet II Refrigerated Aspirator</td>
			<td>Uniequip Laborger&#228;te- und Vertriebs GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UNIVAPO 100 H Concentrator Centrifuge&#160;</td>
			<td>Uniequip Laborger&#228;te- und Vertriebs GmbH</td>
			<td></td>
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			<td>Urea</td>
			<td>AppliChem</td>
			<td>A1049</td>
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			<td>Water (high quality purifed)</td>
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			<td>Resistivity: &#62; 18.2 M&#937;*cm at 25 &#176;C&#160; Pyrogens: &#60; 0.02 EU/ml&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; TOC: &#60; 10 ppb</td>
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			<td>Xcalibur 3.0.63</td>
			<td>Thermo Scientific</td>
			<td></td>
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			<td>ZipTipC18 Pipette Tips</td>
			<td>MILLIPORE</td>
			<td>ZTC18S960</td>
			<td></td>
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	</tbody>
</table>

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.

图1总结了听觉辨别学习后，小鼠的大脑区域的定量蛋白质组突触分析的完整工作流程。它开始在梭箱动物训练。在图2所示的例子中，小鼠开始表现出在第4 次训练显著FM音源歧视，指示有效的学习。动物在脑区域解剖选定的时间点处死。突触的需要富集可以通过突触的制备来实现或者通过一个PSD富集级分的制备，无论在细节在图3中描述的PSD-富集方法已为低组织量的发展， 例如，1 -从大鼠脑12，18 2海马。它需要的小管，聚四氟乙烯杵拟合这些管，并为杵供电实验室钻车程。由于突触的特定蛋白质组合物，强烈建议在两个不同的但互补的方式执行的样品制备。的PSD的支架往往是高的化学计量存在的非常高的分子量的蛋白质。在溶液摘要是有效地提取他们，但可能会导致所产生的肽混合物的过采样的最佳方法。中凝胶消化并联同一样品进行可以排除这些高分子量蛋白质和有利于具有中等和低分子量的蛋白质的分析。对于一个全面的分析建议这两种类型的蛋白酶消化的。 的不同量的调查了脑区的组织中的所需要的施加材料的更好的比较的调整。内的四个研究脑区听觉皮层通常是限制事实要么。所有其他脑区的材料应仔细制备突触体或PSD富集级分后进行调整，以听觉皮层的量（见3.1.1）。从小鼠新鲜制备的脑区的典型权重如下:听觉皮质（AC）:〜50毫克;海马（HIP）:〜90毫克;纹状体（STR）:〜120毫克和额叶皮质（FC）:〜100毫克。 在2.3节中所描述的PSD-富集法允许的约1500种不同的蛋白质和每个脑区的约250个不同的磷酸化的肽上的单个动物（ 表1）的水平的鉴定。蛋白质组分析的第一次训练后24小时，发现该鉴定的蛋白质的7.3％和磷酸化肽的5.8％，表现出显著（P &lt;0.05）相比天真对照（ 表1）中其突触表达量的变化。对于下一个边条显眼的趋势突触支架的化可以在FMTD学习的早期阶段指向突触结构的明显重排。绝大多数调节蛋白在大脑特定区域的方式被改变，而只有22％的被发现在两个或更多个脑区，以进行调节。六选择的实施例示于图4。 通过IPA复杂结果的Meta分析提供了证据以下规范途径的具体参与/操控:"网格蛋白介导的内吞信号"，"轴突指导信号"，"钙信号"，"RhoA的信号"，"Notch信号"，"上皮粘着结的重塑"，"谷氨酸受体信号"，"GABA受体信号"，"多巴胺受体信号"和"突触长时程增强"。 图5）显著任职人数过多的生物过程。在听觉皮层的生物过程，包括离子传输，翻译，表达运输，蛋白转运和学习是引人注目。海马的蛋白质级分的分析显著检测与离子迁移，细胞周期，翻译，磷酸化和神经系统发育富集过程。在纹状体，人数过多生物学过程，包括mRNA的转运，囊泡介导的运输，axonogenesis，蛋白水解，蛋白质运输和内吞作用被发现了。 <img alt="图1" src="/files/ftp_upload/54992/54992fig1.jpg" /> 图1: 系统WorkfloW上的方法论途径。这个数字示意图总结脑区特异性的突触蛋白质组成的高分辨率定量分析的工作流程。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54992/54992fig2.jpg" /> 图2: 在FM音辨别任务小鼠性能的例子。动物显示命中的速度越来越快（蓝色曲线），并在培训课程的过程中误报的下降率（黑线）。显著歧视，第四届会议发生。误差棒被提供作为扫描电镜。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig2large.jpg" target="_blank">请点击这里查看LAR</a>GER版本这个数字。 <img alt="图3" src="/files/ftp_upload/54992/54992fig3.jpg" /> 图 3: 突触和PSD的富集级分的制备。 答:突触准备。 A:PSD富集级分的准备。这两个数字说明准备从脑组织突触体或替代PSD富集分数的详细工作流程。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54992/54992fig4.jpg" /> 图4: 选择 的 定量蛋白质组学结果。选择蛋白的相对突触丰度的小鼠TRA之间比较独立非执行董事对FMTD任务（AV，N = 6）和天真的对照组（NV中，n = 6），24小时后的第一堂训练课。的丰度值计算成的蛋白质的三个最激烈的肽的峰面积的值。与显著丰度的变化（AV / NV，t检验）的蛋白质标记的曲线内:* P &lt;0.05，** P &lt;0.01，*** P &lt;0.005。误差棒被提供作为SD。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54992/54992fig5.jpg" /> 图 5: 由GeneCodis / Gephi额叶生物学途径的可视化。不仅关系到"生物过程"三个最低蛋白质数量的基因本体论（GO）数据库（http://geneontology.org）的显著条款<html>此处显示。节点表示的GO术语，节点的大小，有一定的节点的连接的线宽度和数量描绘的蛋白质，其中共享此与其他节点GO术语的数目。由于Gephi的"强制地图集"的方法，相关节点正在紧密集群。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54992/54992fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 大脑区域 </td><td> AC </td><td> FC </td><td> HIP </td><td><html> 000"FACE ="宋体"大小="3"&gt; STR </td><td> Σ </td></tr><tr><td> 鉴定的蛋白质 </td><td> 1435 </td><td> 1758 </td><td> 1572 </td><td> 1507 </td><td> 6272 </td></tr><tr><td> 调节的蛋白质（P &lt;0.05） </td><td> 59 </td><td> 130 </td><td> 162 </td><td> 108 </td><td>FACE ="宋体"大小="3"&gt; 459 </td></tr><tr><td> ↑AV / NV </td><td> 8 </td><td> 4 </td><td> 76 </td><td> 35 </td><td> 123 </td></tr><tr><td> ↓AV / NV </td><td> 51 </td><td> 126 </td><td> 86 </td><td> 73 </td><td> 336 </td></tr><tr><td> 确定phosphomoti<html> FS </td><td> 197 </td><td> 361 </td><td> 273 </td><td> 278 </td><td> 1109 </td></tr><tr><td> 监管phosphomotifs（P &lt;0.05） </td><td> 8 </td><td> 22 </td><td> 21 </td><td> 14 </td><td> 65 </td></tr><tr><td> ↑AV / NV </td><td> 4 </td><td>00000"FACE ="宋体"大小="3"&gt; 17 </td><td> 五 </td><td> 9 </td><td> 35 </td></tr><tr><td> ↓AV / NV </td><td> 4 </td><td> 五 </td><td> 16 </td><td> 五 </td><td> 三十 </td></tr></tbody></table> 表1:蛋白质组学结果的总结。下表总结中训练的小鼠的代表蛋白质组学实验（AV，N = 6）相比，他们的天真控制第一堂训练课后24小时（NV中，n = 6）。该459调节蛋白的总和，包括重叠的规定。 283不同的规定，确定脑具体。详细地说，57蛋白在两个脑区调节，在三个脑区域中检测到18蛋白法规和仅2蛋白在所有四个研究脑区调控。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 误差容限 </td></tr><tr><td>前体的质量（傅立叶变换质谱法） </td><td> 10ppm的</td></tr><tr><td>碎片离子质量（线性离子阱） </td><td> 0.6大</td></tr><tr><td> 每个肽最大遗漏分裂 </td><td> 3 </td></tr><tr><td> 固定修改 </td></tr><tr><td>在凝胶消化的样品</td><td>半胱氨酸Carbamidomethylation </td></tr><tr><td>对于在溶液中消化的样品</td><td> Methylthiolatio半胱氨酸的n个</td></tr><tr><td> 变量修改 </td><td>蛋氨酸氧化</td></tr><tr><td></td><td>天冬酰胺和/或谷氨酰胺Deamidations </td></tr><tr><td> 数据库 </td><td> UNIPROT / Sprot </td></tr><tr><td>分类</td><td>老鼠</td></tr><tr><td> 统计鉴定验收设置 </td></tr><tr><td> 从头当地平均信心（ALC） </td><td> &gt; 50％ </td></tr><tr><td>肽假发现率（FDR，基于EST，诱骗融合） </td><td> &lt;1％ </td></tr><tr><td>蛋白意义（-10logP，基于改性T-检验） </td><td> &gt; 20 </td></tr><tr><td>独特的多肽/蛋白质</td><td> ≥1 </td></tr><tr><td> 量化设置: </td></tr><tr><td>用于定量如果肽: </td></tr><tr><td>肽意义（-10logP） </td><td> &gt; 30 </td></tr><tr><td>肽鉴定</td><td>样品≥50％ </td></tr><tr><td>肽信号质量</td><td> &gt; 1 </td></tr><tr><td>肽平均面积</td><td> &gt; 1E5 </td></tr><tr><td>多肽保留时间公差</td><td> &lt;5分钟</td></tr><tr><td>正常化</td><td>由总离子电流（TIC） </td></tr></tbody></table> 表2:蛋白质鉴定的设置（步骤4.2.2）。

Watch this Scientific Journal Video about High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning at JoVE.com

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

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 ...

high-resolution-quantitative-synaptic-proteome-profiling-mouse-brain

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.4K 次观看.  Leibniz Institute for Neurobiology (LIN). 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.

视频: High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

科研

神经科学

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the cortical networks involved in dynamical perceptual and cognitive tasks, such as attending to different sounds in a cocktail party. Many past studies have employed data recorded at the sensor level only, i.e., the magnetic fields or the electric potentials recorded outside and on the scalp, and have usually focused on activity that is time-locked to the stimulus presentation. This type of event-related field / potential analysis is particularly useful when there are only a small number of distinct dipolar patterns that can be isolated and identified in space and time. Alternatively, by utilizing anatomical information, these distinct field patterns can be localized as current sources on the cortex. However, for a more sustained response that may not be time-locked to a specific stimulus (e.g., in preparation for listening to one of the two simultaneously presented spoken digits based on the cued auditory feature) or may be distributed across multiple spatial locations unknown a priori, the recruitment of a distributed cortical network may not be adequately captured by using a limited number of focal sources.
Here, we describe a procedure that employs individual anatomical MRI data to establish a relationship between the sensor information and the dipole activation on the cortex through the use of minimum-norm estimates (MNE). This inverse imaging approach provides us a tool for distributed source analysis. For illustrative purposes, we will describe all procedures using FreeSurfer and MNE software, both freely available. We will summarize the MRI sequences and analysis steps required to produce a forward model that enables us to relate the expected field pattern caused by the dipoles distributed on the cortex onto the M/EEG sensors. Next, we will step through the necessary processes that facilitate us in denoising the sensor data from environmental and physiological contaminants. We will then outline the procedure for combining and mapping MEG/EEG sensor data onto the cortical space, thereby producing a family of time-series of cortical dipole activation on the brain surface (or "brain movies") related to each experimental condition. Finally, we will highlight a few statistical techniques that enable us to make scientific inference across a subject population (i.e., perform group-level analysis) based on a common cortical coordinate space.

We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.

同时MEG / EEG和解剖约束的最小模估计皮质动态映射:听觉注意示例

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

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

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

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 ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

戊四氮诱导点燃小鼠模型

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.