This work was funded by the DFG (SFB 779 TPB8, Kr1879/3-1 MRK), DIP grant (MRK), EU FP7 MC-ITN NPlast (MRK), Center for Behavioral Brain Sciences (CBBS, Sahsen-Anhalt), (AK and SB), MM is a recipient of European Molecular Biology Organization (EMBO) Long-Term Fellowship (EMBO ALTF 884-2011) and Marie-Curie IEF.

从成年大鼠脑1。制备急性海马脑片<ol><li>麻醉大鼠异氟醚。注意：执行使用封闭exicator的过程中，不要吸入异氟醚。确保动物完全麻醉。 </li><li>杀头的大鼠，迅速 ​​隔离大脑，并沉浸在precarbonated（95％O 2/5％CO 2的混合气体）冰冷Gey中的溶液（组成MM：130氯化钠，4.9氯化钾，1.5氯化钙2·2H 2 O ，0.3硫酸镁4·2H 2 O，11氯化镁2·6H 2 O，0.23 KH 2 PO 4，0.8的Na 2 HPO 4·7H 2 O，5个葡萄糖·H 2 O，25 HEPES，22 碳酸氢钠 ，pH值7.32） 30分钟1,2,10,16。 </li><li>取下内嗅皮层，小脑和组成部分。与正中矢状切分出皮质半球，然后将每个半球倒在其内侧表面。此后使50-70°切割（50-70°横向）沿着每个半球13,14的背缘。 </li><li>胶水每个半球的切片系统的切片平台上的新鲜切割面。该平台应涵盖precarbogenated冰冷Gey中的解决方案。 </li><li>切350微米50-70°横切片由前至后一边用调整，以减少Z轴振荡àvibratome。海马结构，subicular和内嗅皮质，以及它们位于背侧海马的皮层将用于实验13,14的条带的一部分。 </li><li>转移海马为马蹄形，并浸没式孵化器和孵化至少2小时，在32℃，carbogenated人工脑脊液（学联含MM：110氯化钠，氯化钾2.5，2.5氯化钙2·2H 2 O，1.5硫酸镁4·2H 2 O，1.24 KH 2 PO 4，10葡萄糖·H 2O，27.4 碳酸氢钠 ，pH值7.3），1,2,10,16。 </li></ol> 2，定位电极的基线记录，和LTP的诱导<ol><li>海马切片传送到​​安装在显微镜下浸没式录音室。灌注（6-7毫升/分钟）与carbogenated ACSF中至少30分钟，在32℃下。 </li><li>准备玻璃毛细管微电极填充学联（尖阻力为3-5MΩ）。 </li><li>放置一个玻璃微电极填充ACSF中的CA1谢弗抵押纤维的刺激和在海马CA1辐射层为fEPSP记录1,2,10（ 图2）。电极之间的距离应为大约300微米。 </li><li>由谢弗抵押纤维与双相方波电流脉冲（200毫秒/极性）的刺激的范围3-4五唤起场兴奋性突触后电位（fEPSPs）加强</li><li>通过测量INP执行的最大刺激试验UT输出关系和定义的刺激强度作为获得最大fEPSP斜率值的40％，并保持它在整个实验常数。 </li><li>开始基线记录的最大刺激试验后至少15分钟。测量的响应来测试刺激整个实验过程中，每分钟。执行基线记录用低频刺激。 </li><li>记录基线为至少30分钟。 </li><li>对于晚期LTP的诱导采用高频100赫兹强直电刺激由三个1秒刺激的列车在100赫兹有5分钟intertrain间隔。为了增加在CA1区5微米荷包牡丹碱刺激的字段可以洗2分钟强直电刺激之前和最后强直电刺激后，应立即被淘汰。 </li></ol> 3，收集片的LTP和快速冷冻的诱导后<ol><li>停止LTP记录2分钟或强直性刺激的三列火车诱发晚期LTP后30分钟。 </li><lI&gt;取出电极，快速切片转移到precold金属平台上放置干冰。重复该过程控制切片保持在U型孵化器。注意：使用手套，同时用干冰工作。 </li><li>收集在1.5ml试管和存储每片在-80℃。 </li></ol>从海马CA1区4​​核的分离富集馏分<ol><li>取1.5毫升的Eppendorf管中冷冻切片从-80℃并保持在冰上。 </li><li>加入0.5ml的新鲜冷的TBS缓冲液含有蛋白酶（PI）和磷酸酶（PS）的抑制剂，进入管毫升。孵育2-3分钟，并传输到立体。注意：使用防护手套和实验室外套，同时采用PI和PS工作。 </li><li>解剖用两针的海马CA1锥体层区域。用一针，用于保持在体视显微镜和第二针用于切割CA1区下的切片。 </li><li>收集解剖CA1区域从5片（每组）到新的1.5毫升试管中含有50μl的裂解缓冲液（1×含以mM低渗裂解缓冲液：10 HEPES，1.5的MgCl 2，10的KCl，pH值7.9，用PI和PS）。请注意，此材料用量将足以运行2-3免疫印迹。 </li><li>均质化通过小心移液上下收集的组织。用200微升吸管。在冰上孵育的裂解液为5-7分钟以使细胞溶胀。 </li><li>取2微升的裂解液，滴在显微镜载玻片，并可视化明视场显微镜下的细胞的肿胀。与细胞的正常肿胀的细胞核显示为圆形完整结构。 </li><li>收集20微升样品到新的1.5 ml管的“匀浆分数”。添加8微升变性4×SDS上样缓冲液中。 </li><li>离心剩余的溶胞产物在11000 rpm离心1分钟。 </li><li>小心地从顶部（&#39;胞浆部分“）收集上清液，TRansfer到一个新的管中，加入20微升的4倍上样缓冲液。 </li><li>悬浮颗粒在60微升低渗缓冲液加入20μl4X采样缓冲区。该馏分被称为一个“核浓缩部分&#39;。 </li><li>匀浆，胞浆和核富含分可以储存在-20℃或-80℃，以后免疫。 </li></ol> 5，半定量免疫印迹的突触核蛋白质<ol><li>解冻的样品和在95℃下煮沸5分钟。 </li><li>采取从每个级分5微升，并确定由氨基黑测试或BCA测试中的蛋白质浓度。 </li><li>装载于SDS-PAGE电泳从匀浆等量的蛋白样品，细胞质和细胞核富集馏分。从控制和LTP切片样品应放置在同一凝胶的直接比较。 </li><li>执行标准的西方印迹程序（湿传递推荐）。 </li><li>探索与T膜他抗体的选择。随后，同样的印迹（或并行运行同一样品）可以探测与细胞质标记 - 神经元特异性enolase2（NSE2）和核标志 - 的NeuN和肌动蛋白抗体作为内参。 </li></ol> 6，数据分析<ol><li> LTP的诱导效率可以通过Clampfit软件进行分析。基线记录的平均坡度使用强直电刺激后的山坡上双因素方差分析，P比较&lt;0.05为差异有显著不同。该fEPSP斜率值在图中表示为均值±标准差</li><li>对于免疫印迹定量，无论是扫描的放射自显影胶片和分析蛋白条带通过的ImageJ或荧光带集成密度与LICOR系统。免疫反应性条带的数值应被归一化的负载和印迹控制。对于控制的比较和LTP的用户组nonparametrical采用Mann-Whitney U检验可能使用。 </li></ol><table border="0"CELLPADDING =“0”CELLSPACING =“0”&gt; <tbody><tr><td> 缓冲区的命名 </td><td> 试剂 </td><td> 浓度（mM） </td><td> 金额 </td><td> 评论/说明 </td></tr><tr><td> Gey中的溶液 （pH值：7.3〜7.4） </td><td>氯化钠</td><td> 130 </td><td> 7.6克</td><td>无菌过滤</td></tr><tr><td>千毫升</td><td>氯化钾</td><td> 4.9 </td><td> 0.37克</td><td></td></tr><tr><td></td><td>氯化钙2·2H 2 O </td><td> 1.5 </td><td> 0.22克</td><td></td></tr><tr><td></td><td>硫酸镁4·2H 2 O </td><td> 0.3 </td><td> 0.0739克</td><td></td></tr><tr><td></td><td>氯化镁2·6H 2 O </td><td> 11 </td><td> 2.24克</td><td></td></tr><tr><td></td><td> KH 2 PO 4的</td><td> 0.23 </td><td> 0.0313克</td><td></td></tr><tr><td></td><td>的Na 2 HPO 4·7H 2 O </td><td> 0.8 </td><td> 0.2145克</td><td></td></tr><tr><td></td><td>葡萄糖·H 2 O </td><td> 5 </td><td> 0.9909克</td><td></td></tr><tr><td></td><td> HEPES </td><td> 25 </td><td> 5.96克</td><td></td></tr><tr><td></td><td> 碳酸氢钠 </td><td> 22 </td><td> 1.85克</td><td></td></tr><tr><td> 学联 （pH值：7.3〜7.4） </td><td>氯化钠</td><td> 110 </td><td> 6.428克</td><td>无菌过滤</td></tr><tr><td>千毫升</td><td>氯化钾</td><td> 2.5 </td><td> 0.1865克</td><td></td></tr><tr><td></td><td>氯化钙2·2H 2 O </td><td> 2.5 </td><td> 0.368克</td><td></td></tr><tr><td></td><td>硫酸镁4·2H 2 O </td><td> 1.5 </td><td> 0.370克</tD&gt; <td></td></tr><tr><td></td><td> KH 2 PO 4的</td><td> 1.24 </td><td> 0.169克</td><td></td></tr><tr><td></td><td>葡萄糖·H 2 O </td><td> 10 </td><td> 1.9817克</td><td></td></tr><tr><td></td><td> 碳酸氢钠 </td><td> 27.4 </td><td> 2.3克</td><td></td></tr><tr><td> 1X低渗缓冲液 （pH值：7.9） </td><td> HEPES </td><td> 10 </td><td> 0.23克</td><td></td></tr><tr><td>百毫升</td><td>氯化镁2·6H 2 O </td><td> 1.5 </td><td> 0.0304克</td><td></td></tr><tr><td></td><td>氯化钾</td><td> 10 </td><td> 0.07455克</td><td></td></tr></tbody></table> 表1缓冲器。

<ol>
	<li>Behnisch, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+translocation+of+Jacob+in+hippocampal+neurons+after+stimuli+inducing+long-term+potentiation+but+not+long-term+depression.">Nuclear translocation of Jacob in hippocampal neurons after stimuli inducing long-term potentiation but not long-term depression.</a> PLoS One. 6 (2), e17276 (2011).</li><li>Cai, F., Frey, J. U., Sanna, P. P., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+degradation+by+the+proteasome+is+required+for+synaptic+tagging+and+the+heterosynaptic+stabilization+of+hippocampal+late-phase+long-term+potentiation.">Protein degradation by the proteasome is required for synaptic tagging and the heterosynaptic stabilization of hippocampal late-phase long-term potentiation.</a> Neuroscience. 169 (4), 1520-1526 (2010).</li><li>Chapman, C. A., Perez, Y., Lacaille, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+GABA(A)+inhibition+on+the+expression+of+long-term+potentiation+in+CA1+pyramidal+cells+are+dependent+on+tetanization+parameters.">Effects of GABA(A) inhibition on the expression of long-term potentiation in CA1 pyramidal cells are dependent on tetanization parameters.</a> Hippocampus. 8 (3), 289-298 (1998).</li><li>Ch'ng, T. H., Uzgil, B., Lin, P., Avliyakulov, N. K., O'Dell, T. J., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+transport+of+the+transcriptional+coactivator+CRTC1+from+synapse+to+nucleus.">Activity-dependent transport of the transcriptional coactivator CRTC1 from synapse to nucleus.</a> Cell. 150 (1), 207-221 (2012).</li><li>Dieterich, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caldendrin-Jacob:+a+protein+liaison+that+couples+NMDA+receptor+signalling+to+the+nucleus.">Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus.</a> PLoS Biol. 6 (2), e34 (2008).</li><li>Fainzilber, M., Budnik, V., Segal, R. A., Kreutz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+synapse+to+nucleus+and+back+again--communication+over+distance+within+neurons.">From synapse to nucleus and back again--communication over distance within neurons.</a> J. Neurosci. 31 (45), 16045-16048 (2011).</li><li>Hardingham, G. E., Bading, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+versus+extrasynaptic+NMDA+receptor+signalling:+implications+for+neurodegenerative+disorders.">Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders.</a> Nat. Rev. Neurosci. 11 (10), 682-696 (2010).</li><li>Ivanov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+role+of+synaptic+and+extrasynaptic+NMDA+receptors+in+regulation+of+the+extracellular+signal-regulated+kinases+(ERK)+activity+in+cultured+rat+hippocampal+neurons.">Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons.</a> J. Physiol. 57, 789-798 (2006).</li><li>Jordan, B. A., Kreutz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocytoplasmic+protein+shuttling:+the+direct+route+in+synapse-to-nucleus+signaling.">Nucleocytoplasmic protein shuttling: the direct route in synapse-to-nucleus signaling.</a> Trends Neurosci. 32 (7), 392-401 (2009).</li><li>Karpova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encoding+and+transducing+the+synaptic+or+extrasynaptic+origin+of+NMDA+receptor+signals+to+the+nucleus.">Encoding and transducing the synaptic or extrasynaptic origin of NMDA receptor signals to the nucleus.</a> Cell. 152 (5), 1119-1133 (2013).</li><li>Kim, M. J., Dunah, A. W., Wang, Y. T., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+roles+of+NR2A-+and+NR2B-containing+NMDA+receptors+in+Ras-ERK+signaling+and+AMPA+receptor+trafficking.">Differential roles of NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking.</a> Neuron. 46, 745-760 (2005).</li><li>Lai, K. O., Zhao, Y., Ch'ng, T. H., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importin-mediated+retrograde+transport+of+CREB2+from+distal+processes+to+the+nucleus+in+neurons.">Importin-mediated retrograde transport of CREB2 from distal processes to the nucleus in neurons.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (44), 17175-17180 (2008).</li><li>Leutgeb, J. K., Frey, J. U., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTP+in+cultured+hippocampal-entorhinal+cortex+slices+from+young+adult+(P25-30)+rats.">LTP in cultured hippocampal-entorhinal cortex slices from young adult (P25-30) rats.</a> J. Neurosci. Methods. 130 (1), 19-32 (2003).</li><li>Leutgeb, J. K., Frey, J. U., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+activity-dependent+cyclic+AMP-responsive+element-binding+protein+phosphorylation+during+long-lasting+long-term+potentiation+in+area+CA1+of+mature+rat+hippocampal-organotypic+cultures.">Single cell analysis of activity-dependent cyclic AMP-responsive element-binding protein phosphorylation during long-lasting long-term potentiation in area CA1 of mature rat hippocampal-organotypic cultures.</a> Neuroscience. 131 (3), 601-610 (2005).</li><li>Perlson, E., Hanz, S., Ben-Yaakov, K., Segal-Ruder, Y., Seger, R., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vimentin-dependent+spatial+translocation+of+an+activated+MAP+kinase+in+injured+nerve.">Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve.</a> Neuron. 45 (5), 715-726 (2005).</li><li>Xiang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+maintenance+of+mature+hippocampal+slices+in+vitro.">Long-term maintenance of mature hippocampal slices in vitro.</a> J. Neurosci. Methods. 98 (2), 145-154 (2000).</li><li>Zhao, M., Adams, J. P., Dudek, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern-dependent+role+of+NMDA+receptors+in+actin+potential+generation:+consequences+on+extracellular+signal-regulated+kinase+activation.">Pattern-dependent role of NMDA receptors in actin potential generation: consequences on extracellular signal-regulated kinase activation.</a> J. Neurosci. 25 (30), 7032-7039 (2005).</li></ol>

The authors have nothing to disclose.

在上述协议中所述的步骤提供指导如何准备从青年或成年大鼠海马急性切片，诱导和记录LTP，迅速解剖切片的刺激区域，并准备核浓缩部分的研究活动相关的蛋白质动力学。这种方法源于对彼此独立地使用几种不同的方法组合。我们优化的工作流程，并为初学者建立自己的实验后，LTP的诱导，研究蛋白质的亚细胞再分配提供足够的细节。作为一个例子，我们证明了LTP的晚期形式诱导S180的核积累磷...

突触N-甲基-D-天冬氨酸受体（NMDA受体）发挥在突触可塑性和细胞存活信号而激活突触外NMDA受体的可触发的神经变性和细胞死亡的关键作用。这些变化依赖于严密控制/受规管活动相关的基因表达，因此需要激活突触或树突和细胞核7之间不断的沟通。在MAP激酶ERK1 / 2的突触NMDA受体的信号的下游效应，并参与NMDA受体活化诱导的基因表达，而经由突触外NMDA受体信号没有或ERK1 / 2的活性8,11的抑制作用。 有迹象表明，已经显示出远端树突和细胞核之间穿梭蛋白的数量。许多这些蛋白质含有核定位信号，并正在积极沿着在动力蛋白和importin依赖性微管运输到核6,9。 InterestinglY，一些使者只是过境到细胞核针对特定突触的刺激。例如，环AMP应答元件结合蛋白2（CREB2）的逆行运输，通过化学股份有限公司但不LTP 12诱导。本地化的NMDA受体依赖的突触刺激驱动CREB调节转录共激活因子（CRTC1）进入细胞核，易位过程，其涉及在长期海马可塑性4。它最近的研究表明，该蛋白信使雅各布转位到后两个核，突触和突触外NMDA受体的激活和调节CREB依赖性基因转录的5。信号的突触和突触外原点编码雅各的翻译后修饰。突触活动引起雅各ERK1 / 2磷酸化依赖于在位置180（pJacobS 180）的一个关键丝氨酸这是一个必要的后续易位初级海马文化的核心。此外，我n个急性海马pJacobS 180转位到谢弗抵押LTP后核CA1区神经元而不是公司咯 。 pS180雅各导致的可塑性相关的基因表达增加，这基因表达反馈到突触功能。形成鲜明对比，雅各布该易位至细胞核后突触外NMDA受体活化不磷酸化在Ser180和可能具有不同的蛋白复合物有关在核中引起“CREB切断”和突触触头10回缩。 对突触核蛋白质的信使核进口的大多数已发表的研究中分离的神经细胞原代培养工作已经完成。因此，这将是有趣的，如果这样的调查结果可以复制使用海马生理更多的相关条件下神经元的连接和功能更好的保存。在这里，我们提出了一个优化的协议，以评估LTP-DE通过免疫印迹侧基的蛋白质信使核转。这个方法也适合于分析中的粗核级分的蛋白质的活性依赖的磷酸化。具体来说，目前的协议包括制备急性海马CA1区片，感应和记录的LTP。接着，CA1区用显微镜解剖，分离出刺激的区域。我们结合和修饰赵和他的同事17引入的更改提供CellLytic核抽提试剂盒的协议核隔离。优化过程包括解剖海马CA1区的低渗缓冲液裂解使细胞肿胀，核释放。细胞裂解和细胞核形态可以通过显微镜检查来确定。铀浓缩是一个短离心步骤来实现的。免疫印迹分析用抗体的NeuN和NSE2，核或细胞质级分的特异性标记物，表明该方法可以被用作快速和可重复的协议来隔离这些亚细胞组分，并研究样蛋白的磷酸化非常不稳定的翻译后修饰。此外，此方法是有利的，对于小的组织样本来自海马切片的CA1区解剖区域导出，并且可以组合使用，以海马切片的免疫组化分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Equipment</td>
		</tr>
		<tr>
			<td>CED 1401 AD/DA converter</td>
			<td>Cambridge Electronics Design, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/data_acquisition_system">Axon Digidata acquisition system</a></td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 10.0 Data acquisition and analysis software</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica microscope</td>
			<td>Leica TCS SP2&#65292;Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Standard microelectrode puller</td>
			<td>Sutter&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/stereomicroscope">Stereomicroscope</a></td>
			<td>Leica</td>
			<td>Leica S4E, Germany</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica VT1000S, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated pulse stimulator</td>
			<td>Model2100, A-M systmes, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, HERAEUS, FRSCO17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE system</td>
			<td>BIORAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>JULABO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field Microscope</td>
			<td>NIKON ECLIPSE TS100</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Electron Corporation, HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Luigs &#38; Neumann, SM-5, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting chamber and electric power supplier</td>
			<td>Hoefer Scientific Instruments, San Francisco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>Julabo, F12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, Heraeus, FRESCO 17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Submerged type Recording Chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-shape and submerged type incubator&#160;</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic culture dish&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Reagents</td>
		</tr>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.3957.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.6781.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.02382.0500</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgSO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>5886.05</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>AppliChem</td>
			<td>CAS-NO: 7791-18-6; EC-NO:2320946</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>MERCK</td>
			<td>12034.025</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.06574.1000</td>
			<td>extra pure</td>
		</tr>
		<tr>
			<td>Glucose&#183;H2O</td>
			<td>ROTH</td>
			<td>Art.-Nr.6887.1</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>ROTH</td>
			<td>Art.-Nr.9105.4</td>
			<td>PUFFERAN, &#8805;99.5%, p.a.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>MERCK</td>
			<td>1.06329.1000</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>Protease inhibitor Coctail</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphostop</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Tocris bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Isofluran</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Antibodies</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Species</td>
		</tr>
		<tr>
			<td>pJac-s180</td>
			<td>Biogenes/ purified</td>
			<td></td>
			<td>rabbit (dil. 1:100)</td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>Milipore</td>
			<td>MAB377</td>
			<td>mouse (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>NSE</td>
			<td>Cell Signaling</td>
			<td>D20H2</td>
			<td>rabbit (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>beta-actin</td>
			<td>Sigma</td>
			<td>A-5441</td>
			<td>mouse (dil. 1:5,000)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td>Species</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>DAKO</td>
			<td></td>
			<td>goat anti - mouse (1:5,000)</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>NEB</td>
			<td></td>
			<td>goat anti - rabbit (1:5,000)</td>
		</tr>
	</tbody>
</table>

isolation of ca1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins

学活性依赖的蛋白表达，细胞内转位，或磷酸化是必不可少的理解突触可塑性的潜在的细胞机制。长时程增强（LTP）和诱导的急性海马长时程抑制（LTD）被广泛地接受为学习和记忆的细胞模型。有迹象表明，使用活细胞成像和免疫组化的方法来可视化活动相关的蛋白质动力学大量的研究。然而，这些方法依赖于抗体的免疫细胞化学在单个神经元的荧光标记蛋白或过表达的适宜性。蛋白质的免疫印迹是一种替代方法提供的结果，独立确认。在制备由个别tetanized海马切片的亚细胞级分的第一限制因素是材料的低量。二，处理程序是至关重要的，因为升甚至很短，小的操作艾尔文片可能会引起一定的激活信号级联。在这里，我们描述，以便获得足够纯度的核浓缩部分的足够量的从大鼠脑急性海马切片的CA1区的优化工作流程。作为一个代表性的例子中，我们表明，突触核蛋白质的信使的ERK1 / 2磷酸化形式雅各积极进入细胞核后，诱导LTP，并且可以从CA1区神经元核浓缩部分进行检测。

之前我们已经表明，突触核蛋白质的信使雅各下诱导LTP，但不有限公司1个聚集在细胞核内。此外，刺激突触后转雅各需要激活蛋白激酶ERK1 / 2磷酸化雅各在Ser180（ 图1）。磷酸雅各易位至细胞核中的importin依赖性和磷酸化状态可以被保存在延长的时间周期由协会与中间丝αinternexin15（ 图1）。雅各的磷酸化状态是对的活性依赖性基因和细胞存活的表达很重要...

Watch this Scientific Journal Video about Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins at JoVE.c

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

我们提供了用于诱导的长时程增强，在海马CA1区和核富集级分的从片的tetanized区域的随后分离的详细协议。这种方法可以被用来确定活性依赖的核蛋白质导入在学习和记忆的细胞模型。

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

isolation-ca1-nuclear-enriched-fractions-from-hippocampal-slices-to

Research

JoVE Journal

Neuroscience

12.0K 次观看.  Leibniz Institute for Neurobiology. 我们提供了用于诱导的长时程增强，在海马CA1区和核富集级分的从片的tetanized区域的随后分离的详细协议。这种方法可以被用来确定活性依赖的核蛋白质导入在学习和记忆的细胞模型。 

视频: Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

科研

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

线粒体相关的ER膜（MAMS）和鞘糖脂丰富的微区（GEMS）：分离小鼠脑

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

使用急性海马脑片啮齿动物突触标记/捕获和交叉捕获的研究

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

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 ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

富氧运动器械中慢性硅探针的植入及海马位细胞的记录

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

一种改进的麻醉小鼠脑脊液采集方法

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

为电子生理记录准备急性人类河马切片

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...