这项工作是由瑞典医学研究理事会（K2009 - 75SX - 21028 - 01 - 3，K2008 - 61X - 20700 - 01 - 3）; FONCICYT 000000000091984; Jeansson的基础，Söderström Königska sjukhemmet，玛莎Lundqvist和Sigurd OCH艾尔莎Goljes Minne;和瑞典社会医学SLS - 95151。基因D.座教授，加州大学洛杉矶分校，感谢对稿件的宝贵意见。我们感谢为HCN通道阻滞剂博士迈克尔Andäng和海伦娜Johard博士，教授阿卜杜勒EL - Manira提供视频显微镜。 道德因素: 对所有实验动物是由瑞典卡罗林斯卡医学院和斯德哥尔摩的Norra Djurförsöksetiska Nämnd"所规定的准则和法规的规定执行。所有的动物实验的意图，以尽量减少任何可能的压力或不适的动物。

1。溶液的制备<ol><li>空气缓冲能力的培养液<ol><li>填写约800毫升无菌H 2 O（蒸压milliQ H 2 O）1升的瓶子。 </li><li>边搅拌，添加和混合下列物质:1容器的低血糖，无血清DMEM 2902无碳酸氢钠粉酚红（酚红与生物发光信号干扰）B27的补充50X，20毫升，4.7毫升7.5％的碳酸氢钠 3解决方案（或0.35克碳酸氢钠3），10毫升的HEPES 1M，2.5毫升PenStrep万U / mL和3.5克D -葡萄糖。让介质的搅拌直到完全溶解的成分。 最后培养基（1升）将包含: 1X的DMEM，1个B27的补充，4.2毫米碳酸氢钠3，10毫米的HEPES，25 U / mL青霉素，25 U / mL链霉素和19毫米D -葡萄糖。 </li><li>调整pH至7.2，使用氢氧化钠，以减少或HCL以提高pH值，并把音量用无菌H 2 O到1升</li><li>与H 2 O（减少渗透压）或D -葡萄糖（提高渗透压），调节渗透压。渗透压必须285-315 mOsm /公斤之间，但最佳范围是300-310 mOsm /公斤。不要稀释超过5％的中等。 </li><li>筛选培养基，使用康宁无菌真空过滤集（例如，2 × 500毫升孔径为0.22μm）在无菌罩。保持在4℃的介质，使用铝箔和保护。我们建议，中期3个月内使用。 </li></ol></li><li>汉克的平衡盐溶液（HBSS）缓冲区补充剂（切割片） <ol><li>填写〜600毫升消毒（灭菌milliQ）H 2 O与1升的玻璃瓶，并添加下列物质:100毫升的HBSS 10倍的股票，ml青霉素，链霉素10万单位/毫升，7.5％ 的碳酸氢钠 3溶液5 mL和10毫升HEPES 1M。 最后的切割解决方案将包括以下内容: 1X的HBSS，10毫米的HEPES，4.5毫米碳酸氢钠3，100 U / mL青霉素和100 U / ml链霉素。 </li><li>检查pH值，如有必要，调整pH至7.2，并把音量用无菌H 2 O到1升</li><li>检查渗透压，必须285-315 mOsm /公斤之间。 </li><li>冷的HBSS到4 ° C。 HBSS缓冲需要非常冷（4℃），在切片/切割过程中为了使新陈代谢和保持组织活力。 </li></ol></li></ol> 2。切割和培养切片前的准备工作<ol><li>组织培养在一个温暖的无CO 2的干燥室。为了避免晒出的文化需要密封油脂的菜盖玻片。为此，填补硅为基础的真空润滑脂的5毫升注射器。与小块的铝箔盖注射器的技巧和蒸压他们。此外，高压灭菌滤纸（在切片过程中使用）。 </li><li>前切片过程，适用于35 mm培养皿顶环表面灭菌真空润滑脂。准备一个单独的培养皿中，每个切片文化。 </li><li>开始切片过程中的所有非无菌文书之前，刀片，vibratome刀片，盖玻片及其他材料用于切片和文化的过程中，必须进行消毒。喷雾用70％乙醇的所有非无菌设备。发现至少30分钟前培养的紫外线照射紫外线仪器，并在无菌罩润滑的培养皿。 </li><li>同时，填写猎鹰管与空气缓冲的培养基（1.2毫升计数每一种文化; 8文化，例如10毫升培养基准备），添加刚刚解冻的荧光素（0.1 M的原液; Promega公司，麦迪逊，威斯康星州） （10μL，荧光素10毫升培养基;终浓度为0.1毫米）。 </li><li>对光敏感的荧光素是和股票的解决方案和荧光素介质中需要避光。荧光素介质放置在一个黑暗的36-37℃加热室的Falcon管。如果合适，还可以添加荧光素直接进入切削时间的培养皿。如果不添加荧光素，荧光素酶和荧光素，因此没有从组织的信号之间不会有的光反应。 </li><li>紫外线照射后，附着无菌刀片的vibratome。 </li></ol> 3。 SCN的切片程序下面的过程介绍，成人，一般为2-4个月左右，C57/BL6小鼠切片。请注意:在切割过程，以及动物在夜间灯光照射，差异可以重置的SCN阶段。为了避免这种情况，切割和培养应在光时间，最好是6-12之间的征途时，由于程序没有实质性的阶段转变发生12。如果在黑暗中进行采样的SCN，3.1和3.2都必须在红灯或执行夜间护目镜，以避免光致相移。 <ol><li>麻醉鼠标最好isofluorane在GLA（巴克斯特）SS室。当动物已经失去了其疼痛反射（请在爪子的指甲捏），但尚未停止了呼吸（保持尽可能长的氧气供应），迅速杀头一双剪刀或类似的头部。 请注意:在一些国家的CO 2曝光（高碳酸血症）仍允许作为麻醉方法，虽然据报道，以促进动物的焦虑。此外，颈椎脱位之前，可能需要斩首。 euthanizing动物时，请遵循当地的法例。 </li><li>取出用剪刀头的眼睛，以防止应变和视神经，它可以破坏的SCN的进一步激发。 </li><li>如果仍然连接，用剪刀除去最后的颈椎脊椎动物，消除皮肤（图1A），并用剪刀罚款一双（例如虹膜剪刀），沿线两侧的颅骨两侧切两个削减，从而使一个可移动的"盖子"。 </li><li>打开颅骨，与一个微型咬骨钳工具（精细解剖剪刀也可以使用鼠标），并删除所有的骨骼，直到嗅球中可以看出。向上的工作，从来没有压下来的大脑与工具，为了防止损害的腹侧SCN（图1b）。 </li><li>小心地切嗅球和使用精细的微观解剖春天剪刀半球之间的视神经。确保神经完全切断以来绵延视神经可导致损害在SCN和破裂片。 </li><li>倒挂转动头部，让完整的大脑下降到充满了容器（例如玻璃培养皿≈10厘米）（如果仍然连接到大脑，其他颅神经可能需要削减尾鳍视神经） 50-100毫升冷的HBSS，让大脑迅速冷却。两个视神经理想的情况下，应维持不变（图1C）。保持大脑的HBSS 30-60秒，以确保冷却大脑。 </li><li>用勺子或类似的地方冷冻大脑，无菌切割面，背水面上（例如玻璃培养皿盖天翻地覆）。为了准备一个冠状切开，用无菌刀片或大脑半球和小脑之间的解剖刀垂直切割，从而消除小脑。 </li><li>干属于vibroslicer / vibratome平台上，应用强力胶。 </li><li>拿起插入尖锐，弯钳在延髓部分的大脑（大脑半球没有小脑和无嗅球），并仔细擦干无菌滤纸上的HBSS，还拿着镊子的大脑。 （而不是插入一个镊子，无菌滤纸小块可用于附加和转移大脑）。 </li><li>修复喙尖向上，腹面最接近切割刀片的粘平台上半球。将属于vibratome（例如，英国从坎普登仪器）持有的平台，并立即填写与冷的HBSS。如果是正确的垂直切割，从而提供了一个很好的角度，包含双边SCN日冕削减半球应该站直了。 </li><li>为了达到目标SCN区域，开始切断较厚部分的半球（500-800微米），在高或最高vibratome的速度。移动的刀片，可以相当快，在开始前到达下丘脑，但应放慢视交叉变得可见时（高频振动叶片和动作迟缓的水平减少在切片的细胞​​损伤，从而增加了切片的可行性）。降低到100微米时视交叉变得更大（宽）和前联合变小的部分。为了获得一个中SCN的部分，自己"向下"（尾鳍方向）工作，直到两个SCN的原子核开始出现。放大镜可用于可视化的核所必需的。请注意:在小鼠大脑的SCN是位于视交叉的尾部相比，大鼠脑。 </li><li>如果已达到理想水平的SCN（SCN的会出现在这一点上更明确，圆形或杏仁状结构;图1D;约前囟- 0.46 - -0.70毫米鼠标13，前囟门的SCN的中心地区- 0.92 -1.40毫米大鼠14），切割的SCN节（图1E）。适宜切片厚度为250 ± 50微米鼠标;大鼠350 ± 50微米。 </li><li>用软刷，解除和转让从一个中等大小的培养皿充满冷的HBSS菜，摆下解剖显微镜或立体的SCN片盖。经放大检查，如果双边SCN是清晰可见。如果中间区域的SCN会选择（这不一定是唯一的"最佳"片的水平，但我们认为是最简单的方法，以规范切片的程序和减少的变化），它应该清楚地看到，至少在一旁的片节。如果切太喙，另一部分和SCN倍率下检查。 </li></ol> 4。器官型的SCN文化<ol><li>在HBSS中充满培养皿盖子，剖析了作为一个正方形（〜1.5毫米每边）用无菌手术刀在解剖显微镜下对组织的双边SCN。视交叉的一小片，将继续重视外植体，但没有其他的原子核应包括。削减尽可能接近而不删除的SCN组织。 </li><li>如果适合的计划实验，两国SCN可以被削减了一半，获得两个单方面的SCN。然后，可以使用一个单方面的SCN控制（图2a）。 </li><li>填写成≈35毫米培养皿1200μL荧光素介质和地方文化膜的Milli - CM 0.4微米，（Millipore公司，贝德福德，马萨诸塞）在液体的表面（2B）之上。培养基体积是至关重要的，文化膜应该坐下培养皿中的基础上的安全，而不是float或岩石在中等11。确保膜下有没有气泡。避免不必要的光线照射时，荧光素工作。 </li><li>外植体小而难有起色。因此使用1000μL移液器+提示吸进尖端的SCN外植体，按膜。在情况下，外植体是太大，轻松地安装到1000μL枪头（如大鼠的SCN和鼠标双边的SCN）提示可能用无菌切割工具，以创建一个更广泛的开放。丢弃膜移液器过度的HBSS。荧光素酶的录音，将只有一个SCN /碟（图2b），录音管检测从盘发出的所有光子和不区分来自不同组织的信号。 </li><li>与玻璃盖（≈40毫米，门泽尔格拉泽，德国）和真空润滑脂（道康宁公司，美国）11的密封碟。确保密封严密（图2C）。如果不是这样，更多的油脂密封。 </li><li>菜一个36-37 ° C晚间紧室，并立即启动光电倍增管记录。 </li></ol> 5。录制生物发光的荧光素酶活性测定小SCN组织的荧光素酶诱导生物发光信号检测和放大photonmultiplier管（PMT）检测器，内光紧室安装的组件。通常的光电倍增管置于培养皿8〜1-2厘米以上。光电倍增管记录设置可以定制或市售11。 <ol><li>根据PMT（光电倍增管产生的热量将删除盖玻片上的冷凝）的菜，关闭室。确保总商会是100％的SCN的组织所使用的光电倍增管的暗计数轻紧是非常低（小于20光子/分）。光电倍增管检测，即使是最弱的的漏光。 </li><li>启动数据采集软件（例如LumiCycle; Actimetrics公司，Wilmette，白细胞介素，美国），这是执行。集成光子计数超过1-10分钟的时间间隔，获得高分辨率的基因/蛋白表达。 </li><li>录制完成后，获得的生物钟基因/蛋白表达与相应的软件（原产地，OriginLab，北安普顿，马萨诸塞州，美国; ClockLab，Actimetrics; LumiCycle Actimetrics公司;计时，直到Roenneberg，慕尼黑大学，可以分析慕尼黑，德国），以确定阶段，周期（时间为一个周期）和幅度的节奏。峰值表达的基因或蛋白质主要是用来作为参考点，是在一个周期定义为最高的光子计数。这些数据可以平滑前阶段，周期和振幅的分析，特别是如果信号/噪声比是低的。有时基线的变化和需要进行分析之前，要减去。 </li></ol> 6。代表性的成果: 我们在座的振荡PER2:LUC的表达，作为一个阅读培养组织的生存能力和条件。在最佳条件下，如果该组织是活着，PER2::LUC的表达振荡与昼夜节律，如在图3所示。在SCN PER2是最大限度地表示，通常大约Zeitgeber时间12-13（12征途代表在12:12小时灯亮起:黑暗周期）。在规模较大的活组织，更高的光子计数而成。然而，organotypically培养组织的大小和厚度应保持小规模，以保持组织的可行的，最好不大于15 毫米2月11日，而不是厚度大于500 微米 7 。的SCN，如果解剖这里描述的，通常显示10.000-40.000/min之间的光子计数，如果该组织是一个纯合子PER2采样:LUC的动物。器官的SCN文化的振荡幅度通常非常高，与下面的周期相比，在第一个周期。它并不完全清楚，为什么第一个周期具有非常高的的amplitUDE。一种可能的解释是，相当一部分组织细胞可能死亡后不久，最初的切割和培养，因此不能利用荧光素后的第一个周期。切割过程也可能会导致过度的激励，这可能会在第一个周期的荧光素酶信号放大。 图3所示的SCN片发光的痕迹，并包含一个跟踪（红色）从最初没有健康的片获得。死或不健康的组织有低的光子计数基准。 （此外，死组织常常游离于的菜，不能在一块膜删除。） 益在本报告中描述的技术可以用于药理实验。图4显示了一丝的文化，我们之间的1和2天的治疗与HCN通道阻滞剂（ZD7288，10μM）。正如图中可以看出，此前公布的15阻滞剂显着减少PER2昼夜振荡幅度;然而，与正常培养液洗涤后的振荡回来，表明阻塞影响的分子钟，但组织是可行的和健康的。 <img src="/files/ftp_upload/2439/2439fig1.jpg" alt="图1" /> 图1。切片过程 一）安乐死断头鼠标头部与眼睛和皮肤中删除 B）头骨是一个微型咬骨钳工具删除。使用该工具时，必须向上的工作从来没有向下压的大脑与工具C）大脑中倒挂没有嗅球（腹朝上）。可以看到白色的两个完整的视神经视交叉。接近视交叉位于视交叉上核（SCN，用红色标记的边界）D）一个平台vibroslicer的SCN的水平，冠切脑 E）日冕的大脑部分（250微米厚）含有视交叉（OC），第三脑室（3V）和双边的视交叉上核（SCN）。 <img src="/files/ftp_upload/2439/2439fig2.jpg" alt="图2" /> 图2。器官型组织培养。 A）这两种单方面的SCN核（插入）从切片解剖B）文化膜，中，外植体，但没有真空润滑脂和玻璃盖培养皿培养皿（35毫米）。可以被看作是菜和膜之间的液体介质（1.2毫升）。文化膜被放置在一个单方面的SCN核。雪白的小组织部分是一块视交叉（插入）。 三）膜和SCN的组织培养皿中，用真空油脂和一个圆形的玻璃盖密封。 <img src="/files/ftp_upload/2439/2439fig3.jpg" alt="图3" /> 图3。从健康和非健康的SCN的组织培养，生物发光录音 。 PERIOD2生物发光录音的例子:荧光素酶表达的视交叉上核（PER2::LUC）（SCN）的获得片在12H举行的小鼠:12H:暗光周期。 PER2::LUC的蛋白质一昼夜（24小时）的变化，其中最大的蛋白质表达Zeitgeber时间12日至13日发生振荡。因此，基因节奏的阶段，是依赖于光线暗的时间表，在该动物是保持前牺牲。通常情况下，单方面SCN的解剖组织在协议中所述的生物发光显示〜10.000-40.000/minute之间的光子计数。该图显示了一个健康的（黑）SCN文化，一个非健康的SCN文化（红色）和一个SCN的文化，干（蓝色）后打开密封的培养皿中，在第4天，而不是重新密封的菜正确的痕迹（箭头表示）。 <img src="/files/ftp_upload/2439/2439fig4.jpg" alt="图4" /> 图4。生物发光录音期间和之后的药物暴露。 PER2:LUC的一种文化的表达了HCN通道阻滞剂（ZD7288，10μM）采取行动之前，期间和之后。第一个箭头指示时，阻塞的时间。第二个箭头表示冲刷，这是含空调控制介质介质更换药物。注意:经过2天的药物暴露，缺乏在4天的振荡和冲刷后的蛋白质节奏的快速复苏的减少幅度。

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+brain+slice+chamber+designs+for+investigations+of+oxygen+deprivation+in+vitro.">Comparison of brain slice chamber designs for investigations of oxygen deprivation in vitro.</a> J Neurosci Methods. 81, 103-111 (1998).</li><li>Burgoon, P. W., Boulant, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+properties+of+rat+suprachiasmatic+nucleus+neurons.">Temperature-sensitive properties of rat suprachiasmatic nucleus neurons.</a> Am J Physiol Regul Integr Comp Physiol. 281, 706-715 (2001).</li><li>Nishide, S. Y., Honma, S., Honma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+pacemaker+in+the+cultured+suprachiasmatic+nucleus+from+pup+mice+is+highly+sensitive+to+external+perturbation.">The circadian pacemaker in the cultured suprachiasmatic nucleus from pup mice is highly sensitive to external perturbation.</a> Eur J Neurosci. 27, 2686-2690 (2008).</li><li>Yoshikawa, T., Sellix, M., Pezuk, P., Menaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+the+ovarian+circadian+clock+is+regulated+by+gonadotropins.">Timing of the ovarian circadian clock is regulated by gonadotropins.</a> Endocrinology. 150, 4338-4347 (2009).</li><li>Kohn, R. A., Dunlap, T. 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与荧光素酶报告技术的优点和缺点相比之下体外方法，如RT - PCR技术，原位杂交和免疫印迹，需要在许多不同的时间点（给人一种根据采样频率一般2-4小时的时间分辨率低）的组织取样，以研究昼夜在基因和蛋白表达的变化，荧光素酶报告技术允许在同一准备多天的昼夜振荡的研究高分辨率（1-10分钟）。因此，使用动物的数量最小化，并详细研究的阶段和时期的节奏效果是可行的，通常是不可能使用传统的取样技术的时间分辨率低。然而，虽然节奏相对幅度测量是可能的，但应强调的是，记者的技术是不定量的，因此不能用来衡量翻译转录基因或蛋白质的量。 组织就可以开始录音后立即培养，一个很大的优势，直接研究分子的影响 ， 在体内的压力在体内的光致相移等，分析了器官的SCN文化，允许长期（周）成人大脑的药理操纵组织包含一个完整的成熟的突触的网络和荧光素酶报告技术，允许许多个星期的稳定的录音。因此，组织并不需要新生儿或产后早期为了获得健康的文化，并在慢性药物治疗急性切片可以执行。相反的质粒记者转染细胞培养中，那些没有导致基因组DNA纳入，荧光素酶的转基因动物模型（以及lenti病毒转染）是有利的，如果要研究遗传学改变。它应该在这方面还应该提到，发光成像是时下可能与高灵敏度CCD相机11，录音允许单一的SCN神经元（约6-10微米）和其他类型的细胞研究昼夜节律的主要实验室所采用，甚至在。最后，几个昼夜的调查，通常使用使用其他记者，如绿色荧光蛋白，以研究时钟基因/ 蛋白振荡16-19分子表达的监测。 层厚的方面这里推荐的SCN片（200-300微米）的厚度，可以减少或增加，如果需要的话。不过，虽然组织文化几天后合并膜，它是不建议超过500微米厚度的初步切割片，以维护该组织7可行性。切片厚度超过100微米是难以处理的机械和现实的原因。由于SCN组织是异质的切片厚度可能会影响荧光素酶的输出信号的相位，因为SCN（例如"核心"与"空壳"，背侧与腹侧地区）内不同地区的不同阶段的振荡20和不同的阶段后，重新同步转移21。切片的厚度也影响光子计数的基线，因为更多的组织发出的光子数量较多。分子振荡的幅度可能在这样间接受到影响外植体的大小。基于这些原因，控制和治疗的文化应始终是相同的尺寸，厚度和包含的SCN同一地区以振荡在相同的相位和幅度相同。两个单方面核，另一方面，振荡阶段，彼此只要类似的振幅vibratome削减水平和角度（这又是依赖于切小脑和两个半球之间的分离是垂直台面）。执行的SCN培养的调查可能会遇到的SCN文化不相相互振荡。实践和切片技术的标准化，即片总是削减rostro，尾鳍的SCN在同一水平，提高疗效。 关键步骤<ol><li>氧气供应是至关重要的。 因为大脑需要大量的氧气22，切片的过程，需要快速（接近3.1-3.10〜5-6分钟就越好，以保持组织的健康）。 </li><li> SCN是敏感的温度变化和中小型交流。 SCN是温度敏感和大的温度变化可以相移心脏起搏器，并导致实验文物23。如果中等或交换解决方案，或添加在一个正在进行的实验，例如，当应用一种药物后的天中文化的夫妇，中期或solutioñ需要预热到36-37℃（室相同的温度），然后将另外。涉及中小型交流的药理实验，重要的是要始终与预调节的细胞培养液中。加/洗出用新鲜的，无条件的中型可能相移分子SCN节奏24以及其它类型的细胞培养25。 </li><li>培养基成分。 它是重要的文化组织，在正确的pH值和渗透压。空气缓冲液含有大量的HEPES，其中有一个最佳的缓冲能力在pH值6.8-8.2的范围，也为适当缓冲pH值的变化，可能发生的细胞呼吸的结果。 HEPES，另一方面，与碳酸氢钠，这是在培养液中的少量相比的温度变化更敏感。碳酸氢钠在较低的pH值（5.1-7.1）范围26，因此在CO 2气氛适当的更大的缓冲能力。然而，增加空气缓冲液（DMEM培养液2902）碳酸氢钠量显着增加的渗透压不能加入不同时稀释介质，在不正确的氨基酸，维生素和离子组成之交结果。 编制中期的忠告 : <ol><li>混合介质时，要精确和细致的。 </li><li>使用新鲜或刚解冻的股票解决方案。 </li><li>在非常高渗透压的情况下是不值得的稀释。超过5-6％稀释介质，将最有可能无法正常工作。使用D -葡萄糖，增加渗透压，如果它太低。 </li></ol></li><li>由于准备时间​​相移 片准备的时间是至关重要的的。零或最小的阶段效果，得到如果切片的程序之间的征途6-12 12执行。所有切片解剖应在昼夜或昼夜周期相同阶段进行，以尽量减少错误由于阶段的准备工作之间的变化。 </li></ol>可能的修改<ol><li>对于急性的SCN电，与林格（人工脑脊髓液;学联）（95％O2，5％的CO 2）与氧饱和的缓冲前开始切片，切片。此外，氧气的需求不断增加缓冲区，在整个切割过程中，电气和突触活动是直接依赖氧气供应22。 </li><li>水平和矢状切片也可以成功地制备和培养的SCN。作者曾与水平切片的荧光素酶的录音经验，根据我们的经验给予了较低的幅度与冠状切割相比，昼夜振荡。 </li><li>的SCN约1毫米长rostro尾端，这里描述的切片技术是对在中部地区获得的SCN节的目标。然而，可取得一个以上的SCN节从小鼠和大鼠大脑，使生物钟基因/蛋白表达的SCN核延髓与尾鳍水平的调查。 </li><li>从年幼的动物，产后仔兔甚至胚胎切片文化，也可以准备。请注意，在非常年轻的幼崽的大脑和颅骨，非常柔软和敏感。此外，视神经是薄的，而不是充分发展。解剖幼鼠大脑，所以不被损坏，SCN区域时，一定要小心。例如，微咬骨钳工具不能用于打开鼠标幼崽的头骨，因为他们是太大。精细剪刀足以减少在年轻的幼崽的软组织。 </li><li>这里描述的玻璃盖为基础的文化技术也可以用于图像发光的CCD / EM - CCD的相机，也用在单一的SCN神经元。 </li><li>其他中枢神经系统的地区和从转基因PER2周边器官组织:LUC的动物可以很容易地获得，培养和分子振荡分析，作为PER2:LUC的表达继续振荡多天，在其他一些组织也和细胞类型10。这些外围组织，例如肝脏，并不需要为生存膜，而是将培养直接沐浴在中期 11 。请注意，由于相移，以切片，培养和交流媒介可能不遵循同样的原则为SCN。 </li></ol>意义在转基因小鼠和大鼠株（mPER2::LUC的; mPer1 - LUC 8，10，27）荧光素酶的活性反映蛋白质或基因表达节律，可以通过评估生物发光录音。荧光素酶生成的生物发光，给人一种弱信号，但背景发光接近于零，使得这种方法的优势。此外，由于荧光素酶的分子是不稳定的，并迅速退化，有没有pH值耳毒性，它可以出现在长期的兴奋照明 28 。两者合计，这些属性允许长期实验的荧光素酶报告技术的高度优势，在昼夜研究。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>B27 supplement 50x</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Cover glasses</td>
<td>&nbsp;</td>
<td>Menzel-Gl&auml;ser</td>
<td>40#1</td>
<td>&#8776; 40 mm</td>
</tr>
<tr>
<td>Culture membranes</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICMORG50</td>
<td>0.4 &mu;m</td>
</tr>
<tr>
<td>DMEM low glucose w/o phenol red</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2902</td>
<td>Powder for 1L</td>
</tr>
<tr>
<td>Filter paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1003 055</td>
<td>&#8776; 55 mm</td>
</tr>
<tr>
<td>Filtration set</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>431097</td>
<td>Pore size 0.22 &mu;m
Polyethersulfone</td>
</tr>
<tr>
<td>Forceps </td>
<td>&nbsp;</td>
<td>Allgaier Instrumente</td>
<td>0203-7-PS</td>
<td>Dumant #7</td>
</tr>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15630-056</td>
<td>100 ml</td>
</tr>
<tr>
<td>HBSS 10x</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco </td>
<td>14065-049</td>
<td>500 ml</td>
</tr>
<tr>
<td>NaOCH3 7.5%</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>25080-060</td>
<td>50 ml</td>
</tr>
<tr>
<td>Luciferin </td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1602</td>
<td>Beetle luciferin, potassium salt</td>
</tr>
<tr>
<td>Micro rongeur tool</td>
<td>&nbsp;</td>
<td>Allgaier Instrumente</td>
<td>332-097-140</td>
<td>&nbsp;</td>
<tr>
<td>PenStrep 10,000 U/ml</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15140-122</td>
<td>100 ml</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430165</td>
<td>&#8776; 35 mm</td>
</tr>
<tr>
<td>PMT</td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>H9319-11MOD</td>
<td>&nbsp;</td>
<tr>
<td>Disposable scalpels</td>
<td>&nbsp;</td>
<td>Paragon</td>
<td>P503</td>
<td>Size 11</td>
</tr>
<tr>
<td>Vacuum filter</td>
<td>&nbsp;</td>
<td>Fisher </td>
<td>09-761-5</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>50 g</td>
</tr>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Campden Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus

一个中央昼夜时钟（24小时）在生理和行为的协调生活节奏，居住在位于前下丘脑视交叉上核（SCN）的。时钟是由光线通过视网膜和视神经直接同步。昼夜振荡所产生的一些所谓的"时钟基因"，其蛋白产物，包括期间 （ 人事 ）基因相互作用的负反馈循环。核心时钟也依赖于细胞膜去极化，钙和1号营地 。的SCN显示在时钟基因的表达，代谢活动和自发的电活动的日常振荡。值得注意的是，这种内源性的循环活动仍然存在的 SCN 2-4成人组织切片。生物钟通过这种方式，可以很容易地在体外进行研究，使心脏起搏器功能的分子，电生理和代谢调查。 SCN是一个小的，定义良好的双边的结构，位于上方的视交叉5。在大鼠，它包含在每个细胞核〜8.000神经元，尺寸约947微米（长度，rostrocaudal轴）x 424微米（宽）x 390微米（高度）6 。剖析是必要的，在特定的大脑可以识别的SCN削减大脑切片的SCN。在这里，我们描述的SCN，这是小鼠和大鼠大脑类似的解剖和切片的过程。此外，我们展示了如何以文化的解剖组织上的膜7，山崎等人开发的一个SCN组织培养技术organotypically 8。最后，我们将演示如何转基因组织，可用于测量生物钟基因/蛋白质使用的动态荧光素酶报告技术，一个原本是用于昼夜测量Geusz等方法 9。的表达。我们这里使用转基因SCN组织敲在PERIOD2:Yoo 等产生的荧光素酶的小鼠 10 。小鼠包含一段时期的融合蛋白（PER），2和萤火虫酶的荧光素酶。 PER2表达PER2当翻译，即荧光素的荧光素酶底物的存在，可以作为生物荧光监测时，荧光素酶催化荧光素的氧化。发射光子的数量，积极相关的生产PER2蛋白质量，生物发光的节奏相匹配PER2蛋白质 在体内10节奏。 PER2表达的周期性变化，在这种方式可以连续监测，实时在许多天。我们组织培养和实时生物发光录音遵循的协议已经彻底山崎和高桥11。

Watch this Scientific Journal Video about Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus at JoVE.com

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

准备含有成年小鼠的下丘脑视交叉上核（SCN），和快速的方式来培养器官培养条件的SCN组织切片，程序报告。此外，使用动态的荧光素酶报告技术的振荡时钟基因蛋白表达的测量是描述。

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

slice-preparation-organotypic-tissue-culturing-luciferase-recording

Research

JoVE Journal

Neuroscience

35.1K 次观看.  Karolinska Institutet. 准备含有成年小鼠的下丘脑视交叉上核（SCN），和快速的方式来培养器官培养条件的SCN组织切片，程序报告。此外，使用动态的荧光素酶报告技术的振荡时钟基因蛋白表达的测量是描述。

视频: Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...