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><tbody><tr><td>3 M 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 &amp;micro;m x 2 cm, C18</td></tr><tr><td>Acclaim PepMap 100</td><td>Dionex/Thermo Scientific</td><td>164569</td><td>75 &amp;micro;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&amp;nbsp;&amp;nbsp;</td><td>Fluka</td><td>9830</td><td></td></tr><tr><td>Ammonium hydroxide&amp;nbsp;</td><td>Fluka&amp;nbsp;</td><td>44273</td><td></td></tr><tr><td>Ammonium persulfate&amp;nbsp; (APS)</td><td>AppliChem</td><td>A2941</td><td></td></tr><tr><td>Biofuge &lt;em&gt;pico&lt;/em&gt;</td><td>Heraeus GmbH</td><td>75003280</td><td></td></tr><tr><td>Blue R-250&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Pierce /Thermo Scientific</td><td>78420</td><td></td></tr><tr><td>HEPES Buffer solution&amp;nbsp;</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 &amp;nbsp;K-ControlTLC 4957&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Kaltenbach &amp;amp; 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&amp;nbsp;</td><td></td><td></td></tr><tr><td>Phosphatase Inhibitor Cocktail 3</td><td>Sigma-Aldrich</td><td>P0044</td><td></td></tr><tr><td>PhosphoRS 3.1</td><td>IMP/IMBA/GMI</td><td></td><td></td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>4906845001</td><td></td></tr><tr><td>Plunger/pestle made of PTFE</td><td>Sartorius AG</td><td>854 2651</td><td></td></tr><tr><td>PotterS homogenizer</td><td>Sartorius AG</td><td>853 3024</td><td></td></tr><tr><td>Protease Inhibitor complete mini</td><td>Roche</td><td>4693159001</td><td></td></tr><tr><td>Quantity One 4.5.1</td><td>BioRad</td><td></td><td></td></tr><tr><td>RapiGest</td><td>Waters</td><td>186002122</td><td></td></tr><tr><td>Shuttle box</td><td>Coulbourne Instruments</td><td></td><td></td></tr><tr><td>Sodium dodecylsulfate (SDS)</td><td>AppliChem</td><td>A1112</td><td></td></tr><tr><td>Sodium molybdate</td><td>Carl Roth GmbH</td><td>274.2</td><td></td></tr><tr><td>Sodium tartrate dihydrate</td><td>Sigma-Aldrich</td><td>228729</td><td></td></tr><tr><td>SONOREX RK 156 Ultrasonic Bath</td><td>BANDELIN electronic GmbH &amp;amp; Co. KG</td><td>305</td><td></td></tr><tr><td>Soundproof chamber</td><td>Industrial Acoustics Company</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Carl Roth GmbH</td><td>4621.2</td><td></td></tr><tr><td>Tetramethyl ethylene -1,2-diamine (TEMED)</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Thermomixer&lt;em&gt; basic&lt;/em&gt;</td><td>CallMedia</td><td>111000</td><td></td></tr><tr><td>&lt;a name=&quot;RANGE!A60&quot;&gt;Titansphere TiO 5&amp;micro;m&lt;/a&gt;</td><td>GL Sciences Inc. Japan</td><td>502075000</td><td></td></tr><tr><td>TLA 100.1 rotor</td><td>Beckman Coulter</td><td>343840</td><td></td></tr><tr><td>Trifluoro acetic acid&amp;nbsp; (TFA)</td><td>Sigma-Aldrich</td><td>T6508</td><td></td></tr><tr><td>Tris ( hydroxymethyl) aminomethane (TRIS)</td><td>AppliChem</td><td>A1086</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8532</td><td></td></tr><tr><td>Trypsin Gold</td><td>Promega</td><td>V5280</td><td></td></tr><tr><td>Ultimate 3000 Ultra HPLC&amp;nbsp;</td><td>Dionex/Thermo Scientific</td><td></td><td></td></tr><tr><td>Ultracentrifuge tube</td><td>Beckman Coulter</td><td>343776</td><td></td></tr><tr><td>Unijet II Refrigerated Aspirator</td><td>Uniequip Laborger&amp;auml;te- und Vertriebs GmbH</td><td></td><td></td></tr><tr><td>UNIVAPO 100 H Concentrator Centrifuge&amp;nbsp;</td><td>Uniequip Laborger&amp;auml;te- und Vertriebs GmbH</td><td></td><td></td></tr><tr><td>Urea</td><td>AppliChem</td><td>A1049</td><td></td></tr><tr><td>Water (high quality purifed)</td><td></td><td></td><td>Resistivity: &gt; 18.2 M&amp;Omega;*cm at 25 &amp;deg;C&amp;nbsp; Pyrogens: &amp;lt; 0.02 EU/ml&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TOC: &amp;lt; 10 ppb</td></tr><tr><td>Xcalibur 3.0.63</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>ZipTip&lt;sub&gt;C18&lt;/sub&gt; Pipette Tips</td><td>MILLIPORE</td><td>ZTC18S960</td><td></td></tr></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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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

Universidad de Cartagena UDEC

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

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

突触量化：基于一个免疫细胞化学检测，以量化突触数量

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

科研

神经科学

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

与耳蜗内毛细胞传入树突：多泡发布监测功能区突触的突触后录音

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

为远程沉默的神经活动在鼠害的方法在学习独立的阶段

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

行为学

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

小鼠大脑中的 LFP 记录用于认知研究和药物分析

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal populations. It serves as a crucial tool in cognitive research, particularly in brain regions like the hippocampus and prefrontal cortex. Dual LFP recordings between these areas are of particular interest as they allow the exploration of interregional signal communication. However, methods for performing these recordings are rarely described, and most commercial recording devices are either expensive or lack adaptability to accommodate specific experimental designs. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of antipsychotic drugs and potassium channel modulators on LFP properties in these areas. The technique enables the measurement of LFP properties, including power spectra within each brain region and coherence between the two. Additionally, a low-cost, custom-designed recording device has been developed for these experiments. In summary, this protocol provides a means to record signals with high signal-to-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

作者聚焦:使用双 LFP 记录方案研究药物对小鼠大脑信号的影响

This protocol outlines the use of a custom-designed recording device and electrodes to record local field potentials and investigate information flow in the mouse hippocampus and prefrontal cortex.

小鼠海马体和前额叶皮层中的双重细胞外记录

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

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此视频中的章节

突触量化：基于一个免疫细胞化学检测，以量化突触数量

与耳蜗内毛细胞传入树突：多泡发布监测功能区突触的突触后录音

为远程沉默的神经活动在鼠害的方法在学习独立的阶段

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

海马鼠脑切片的突触密度评价

免疫荧光定量小鼠脑突触蛋白的异种分布

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

小鼠海马体和前额叶皮层中的双重细胞外记录

小鼠海马体和前额叶皮层中的双重细胞外记录