Name: organotypic slice cultures for studies of postnatal neurogenesis
Uploaded: 2015-03-04T13:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

注:所有动物的程序符合比较医学系的多伦多大学的动物健康和福利的指导方针。 1.准备海马脑片<ol><li>消毒使用干高压釜以下仪器在125℃:手术刀手柄（＃3）（2），标准模式钳，大（1），小解剖剪（倾斜到另一侧）（1），微勺（勺子和平坦抹刀端）（1），微抹刀（圆形和圆角锥形端）（2）中，细画笔（1），火抛光的巴斯德吸液管（2），纱布正方形，2×2英寸（5）。 </li><li>当无菌的，把工具插入一个无菌容器中，并保持盖直至使用。立即解剖前，浸在70％的乙醇溶液的仪器。 </li><li>通过加入1ml培养基/孔，并存储该板在培养箱中在35℃的开始清扫程序之前准备一个6孔培养板培养插管次5％的CO 2。 </li></ol> 2.安排解剖工具在无菌层流罩<ol><li>喷层流罩，用70％的乙醇，并从醇除去灭菌解剖仪器。允许仪表干燥而搁置在无菌培养皿，以避免醇与解剖大脑组织之间的接触。 </li><li>存款5-7毫升的2个大型无菌培养皿消毒冰冷的解剖解决方案。一盘会放松并清洁头（脏），另一种用于冷却和冲洗舀出脑（清洁）。放置无菌滤纸的培养皿盖子解剖出脑之一。 </li><li>放置一个小的，无菌滤纸的小的培养皿盖子解剖出海马中的一个。存款3-5毫升消毒冰冷的解剖解决方案2小，无菌培养皿。一盘将举办挖出海马，一会的海马部分分离时持有的部分解剖显微镜下。 </li><li>由胶带一块无菌滤纸到切割阶段和安装无菌刀片准备组织斩波。湿用无菌解剖溶液的滤纸上。 </li><li>喷雾清洁生物袋用酒精，并将其放置在层流超净工作台。 </li></ol> 3.海马解剖<ol><li>喷P7只SD大鼠幼崽，用70％的乙醇的层流净化台的外面，并迅速用层流长凳里面大无菌手术剪刀斩首动物。让头部落入冰冷解剖溶液中的培养皿1。 </li><li>在培养皿中，冲洗过的血液，并迅速转移到头部灭菌滤纸，腹侧下来。 </li><li>使用手术刀，沿背表面在矢状面切割以暴露下面的头骨。切穿皮肤，但不是底层骨，这是软的，很容易穿透大鼠吨的他的年龄。抛开这种"脏"手术刀，也不对脑组织使用。 </li><li>用小剪刀剥离（角度到另一侧）和钳，切开颅骨沿颅骨矢状缝到前囟门，在那里的冠状缝被矢状缝垂直相交的头骨解剖点。使用镊子从头骨的中线拉头骨襟翼起来了。 </li><li>放置在脑的下侧，脑干下方的微勺，轻轻抬起脑出颅骨。提起大脑暴露视神经和嗅球脑基底表面。切开这些结构用小剪刀从头骨完全分离的大脑。删除和完整的大脑转移到含有冰冷的解剖解决方案等大型培养皿。 </li><li>使用微勺，大脑转移到含有无菌滤纸的小培养皿盖。用无菌巴斯德吸管，冲洗大脑用几滴DIS的secting的解决方案，以保持组织的湿润。 </li><li>使用"干净"的手术刀切开两半球分开。转让左半球背到大培养皿微勺子上下放置半球PIA一边为后续使用冰冷的解剖解决方案。 </li><li>查看面对内侧右半球和识别边缘穹窿，白质沿内侧缘海马的一个突出带。用无菌解剖刀，使通过穹窿矢状切，但照顾因为手术刀尖端仅0.5厘米将足以切割穹窿。 </li><li>使用2微抹刀，通过将右手抹刀上脑干和起吊上覆皮质与左手刮刀移除右半球第一海马。轻轻抬起皮质揭示海马侧脑室内侧面。白色曲线，伞端，现在应该是可见的。 </li><li>抹刀的曲率对准看台伞端的自命轻轻按下伞下铲。向左滑动锅铲，沿着喙，尾轴骑，然后抬起铲背的方向，除去海马。 </li><li>海马转移到第二小培养皿用冰冷的解剖溶液。重复左侧半球相同的步骤拆下左海马。 </li><li>用微型抹刀，仔细海马转移到组织菜刀阶段。排列它们相邻并彼此平行并垂直于斩波叶片的轴线。使用画笔来定位组织并在海马的顶部加几滴解剖溶液。 </li><li>削减400微米的切片组织没有停下来删除单个片（通常他们会不会坚持到刀片）。整个海马已被切断后，用画笔来的部分轻轻地转移到第二小的培养皿剥离的解决方案。 </li><li>根据广告issecting显微镜，仔细分离使用amicro，铲和画笔浮动片。 </li><li>取出预先准备好的培养板从培养箱并置于层流罩培养插管。 </li><li>使用火抛光的巴斯德吸管，吸取4-5片放入吸管和转移切片文化插入膜的顶面。接着，调整用画笔的定位和离开的各个部分，并培养插入物的边界之间的空间。 </li><li>用无菌巴斯德吸管，去除膜的顶面多余的解剖解决方案。 注:卸下解决方案避免将组织切片放入吸管。另外，使用常规的移液管（P200或P1000）用灭菌枪头慢慢消除解决方案。 </li><li>放置在培养板用含血清的培养基中，并在海马切片放回培养箱中在35℃和5％的CO 2。 注意:如果实验通话从多个动物产生的文化，彻底清洁夹层之间的层流洁净工作台。返回仪器酒精溶液，并更换所有培养皿，滤纸和之前第二清扫消毒刀片。 </li></ol> 4.饲养和维护器官切片<ol><li>饲料文化在无菌层流洁净工作台。 </li><li>执行部分培养后2天解剖的第一馈送。使用消毒的玻璃吸管吸出旧的培养基。 </li><li>使用无菌5毫升血清吸液管来加入1 ml新鲜，无菌的，含有血清的培养基的孔中。 </li><li>轻轻地取代文化插入和照顾，以消除可能已经膜表面之下形成的气泡。 </li><li>第一进给后，介质改变每隔一天。 </li></ol> 5.孵化组织切片与胸苷类似物标签​​新生神经元<ol><li>为了探讨成熟和整合齿状颗粒细胞在海马，孵化器官切片与胸苷类似物，如溴脱氧尿苷（尿嘧啶）或Chlorodeoxyuridine（CldU）。在这里，我们使用，因为在生理盐水中的溶解度较高的CldU。 </li><li> 3DIV后，对于10微克/ ml的终浓度加入1微升10毫克/毫升CldU原液在盐水到1ml培养基。如果BrdU的被使用，校正分子量的差异的浓度。与CldU添加培养基培养孔，并培育组织与CldU含培养基2小时，在35℃。 </li><li>以下2小时温育在35℃，除去含CldU培养基并用常规给送介质替换以恢复正常喂养时间表（上面概述的）。 </li></ol> 6.组织固定和存储<ol><li>建立的时间表用于施加处理和固定组织样品开始培养实验（图2B）之前。 </li> &lt;li&gt;在预定天后解剖，准备以下在实验室通风柜:含有10-50毫升烧杯4％多聚甲醛（PFA）在磷酸盐缓冲盐水（PBS）;空烧杯丢弃培养基;小镊子;和1000毫升吸管与一次性吸头。 </li><li>去除孵化室和转移培养板（S）到通风橱。倾斜单井板镶钳。接下来，用吸管去除培养基，并转移到处置烧杯中。倾斜培养板以一定角度，以帮助确保所有培养基被除去。 </li><li>散场时间删除介质一种文化板块。当媒体已被删除，加入1 ml 4％PFA的每培养孔和密封孔板封口膜。重复许多培养板作为必要和转移板到冰箱，在4℃下放置24小时。 </li><li> 24小时后，准备在通风橱下:10-50毫升烧杯含有0.1％叠氮化钠的PBS中;空烧杯丢弃PFA（遵守安全注意事项丢弃有毒物质时）;小镊子;和吸管一次性吸头千毫升。 </li><li>按照6.4中概述用于添加煤灰的方法，但添加1ml的PBS中与叠氮化钠代替。一旦完全转移，密封孔板供将来使用通过包装边缘封口膜，并在4℃下储存在冰箱中。 </li></ol> 7.组织切片的免疫组化<ol><li>执行组织使用振动切片。以下一系列步骤有助于最大限度地从器官型培养可用组织切片的产率。 注:紧随海马解剖和切片的电镀，所述组织的厚度为约400微米。然而，2-3周，在孵育室后，组织切片会开始变平，从而产生最后一节的150-300微米厚。 </li><li>准备好以下项目第培养组织:一个＃11手术刀B拉德和处理，含冰冷PBS，微解剖钳，和一个清洁的vibratome切割阶段装入组织的玻璃培养皿。 </li><li>使用解剖刀小心沿圆形刀片膜的周边切割，以便它可以从塑料插入件壳体上卸下。离开培养切片和手术刀之间的充足的空间。 </li><li>该分离插入膜转移到含PBS的培养皿。漂洗在PBS后，用钳子向膜转移到的vibratome安装阶段。 </li><li>接下来，用手术刀来消除周围的培养切片多余膜切掉多余的材料，以创造清洁的边缘。这个步骤将确保该膜是平坦的，可以很容易地粘附在切割表面。 </li><li>放置1-2滴粘合剂上的vibratome切割阶段和散布在偶数层用22克针。散布粘合剂为矩形形状具有平行于的vibratome的切割刀片的长边。执行此步骤快，以防止干燥的粘合剂。 </li><li>使用镊子包含海马切片修整膜转移至所述切割阶段，并轻轻地定位于胶膜，并确保没有气泡。 </li><li>作为强力胶干燥，用粘膜回的PBS含培养皿转移的vibratome阶段。制备的vibratome刀片和一个48孔平板含有叠氮化钠来存储组织切片。 </li><li>使用的vibratome生成器官切片组织和转移的30微米的部分，以一个48孔板含有叠氮化钠进行存储和随后的免疫组织化学染色。 </li><li>参阅预先存在用于免疫组化染色26,29协议。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+hippocampal+slice+cultures.">Neurogenesis in hippocampal slice cultures.</a> Molecular And Cellular Neurosciences. 26 (2), 241-250 (2004).</li><li>Kamada, M., 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=Intrinsic+and+spontaneous+neurogenesis+in+the+postnatal+slice+culture+of+rat+hippocampus.">Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.</a> The European Journal Of Neuroscience. 20 (10), 2499-2508 (2004).</li><li>Kim, H., Kim, E., Park, M., Lee, E., Namkoong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+culture+from+the+adult+mouse+brain:+a+versatile+tool+for+translational+neuropsychopharmacology.">Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 41, 36-43 (2013).</li><li>Legradi, A., Varszegi, S., Szigeti, C., Gulya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+hippocampal+slices+as+in+vitro+models+for+neurodegeneration:+Studies+on+cell+viability+and+apoptotic+processes.">Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes.</a> Brain Research Bulletin. 84 (1), 39-44 (2011).</li><li>Sadgrove, M. P., Laskowski, A., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+granule+layer+cell+count,+cell+density,+and+single-pulse+BrdU+incorporation+in+rat+organotypic+hippocampal+slice+cultures+with+respect+to+culture+medium,+septotemporal+position,+and+time+in+vitro.">Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.</a> The Journal of Comparative Neurology. 497 (3), 397-415 (2006).</li><li>Mielke, J. G., 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=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. 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=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., 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=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, 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=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Duque, A., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+effects+of+bromodeoxyuridine+and+[3H]thymidine+incorporation+into+DNA+on+cell+proliferation,+position,+and+fate.">Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 31 (42), 15205-15217 (2011).</li><li>Namba, T., Mochizuki, H., Onodera, M., Namiki, H., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+in+hippocampal+slice+cultures:+early+in+vitro+labeling+of+neural+precursor+cells+leads+to+efficient+neuronal+production.">Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.</a> Journal of Neuroscience Research. 85 (8), 1704-1712 (2007).</li></ol>

The authors declare that they have no competing financial interests.

以下CldU（或尿嘧啶）施用，施用药剂的时间轴可以被选择为目标时特定发育窗户新生儿DGCS。例如，一个假想的代理可以在第二星期后CldU注射，这是建议用未成熟的神经元是在发育阶段的GABA被去极化的年龄相一致时被应用。使用该协议的将来的研究可以适应的药理剂和暴露于"量体裁衣"的方法对感兴趣的具体的实验问题的窗口。 用于确定切片文化是产后神经研究的一个有效?...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

<table><tbody><tr><td>5-chloro-2&#039;-deoxyuridine (CldU)</td><td>MP Biomedicals</td><td>105478</td><td>Hazardous, Carcinogenic</td></tr><tr><td>Cell culture inserts, 30 mm diameter, 0.4 &amp;micro;m pore size</td><td>Thermo scientific&amp;nbsp;</td><td>140660</td><td>Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td></tr><tr><td>Conical Centrifuge tubes (sterile)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Dissector scissors (angled to side)</td><td>Fine Science Tools&amp;nbsp;</td><td>14082-09</td><td></td></tr><tr><td>Minimum essential medium (MEM)</td><td>Gibco</td><td>11095; liquid</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Eclipse Ni-U fluorescent microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Glue for tissue</td><td>Krazy Glue</td><td>KG585</td><td>Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS) (500 ml)</td><td>Gibco</td><td>14025-092</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Horse Serum Heat Inactivated (500 ml)</td><td>Gibco</td><td>16050-122</td><td>Make 50 ml aliquots and store at -20 &amp;deg;C</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clarke</td><td>TW 31KYPBX</td><td></td></tr><tr><td>Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td><td></td><td></td><td></td></tr><tr><td>Petri Dish (100 mm x 15 mm) and (60 mm x 15 mm)</td><td>Fisher Brand</td><td>FB0875712 and FB0875713A</td><td></td></tr><tr><td>Scalpel blades #11</td><td>Fine Science Tools</td><td>10011-00</td><td></td></tr><tr><td>Scalpel handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Serological Pipettes</td><td>Sorfa Medical Plastic Co.</td><td>P8050</td><td></td></tr><tr><td>Standard Pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Sterile vacuum filter</td><td>Thermo-Scientific</td><td>565-0020</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14054-13</td><td></td></tr><tr><td>Syringe driven filter unit</td><td>Millipore-Millex</td><td>SLGP033RS</td><td></td></tr><tr><td>Tissue chopper with moveable stage</td><td>Stoelting&amp;nbsp;</td><td>51425</td><td></td></tr><tr><td>Fine tip paintbrush</td><td></td><td></td><td></td></tr></tbody></table>

organotypic slice cultures for studies of postnatal neurogenesis

在这里，我们描述了使用器官切片培养技术研究海马神经发生产后的啮齿类动物的大脑的技术。该方法保持了海马的特征地形的形态，同时允许直接应用的药剂给显影海马齿状回。此外，切片培养物可维持4周，因此，允许一个研究新生儿颗粒神经元的成熟过程。切片培养允许有效药理操纵海马切片的同时排除复杂的变量，例如与海马的深解剖位置，以及血脑屏障的不确定性。由于这些原因，我们试图专门优化器官切片文化为产后神经研究。

确定是否器官培养物将适合于成年神经研究需要它们满足两个主要标准:1），该片段保持海马切片的特性形态学特征后10-21天内在体外 （DIV），和2），初生DGCS可以量化采用常用于成年神经研究标准的免疫组化技术。关于第一个标准， 图1A和1B突出保存海马形态。特征，如齿状回（DG），CA1和CA3地区是容易辨认。 对于第二准则， 图1C（</stro...

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

在这里，我们描述了学习的海马神经发生产后使用的器官切片培养技术的技术。此方法允许在体外操纵成年神经发生，并允许直接应用的药剂的培养海马。

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

Research

JoVE Journal

Developmental Biology

12.2K 次观看.  University of Toronto. 在这里，我们描述了学习的海马神经发生产后使用的器官切片培养技术的技术。此方法允许在体外操纵成年神经发生，并允许直接应用的药剂的培养海马。 

视频: Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

器官型E18大鼠脑切片文化

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

科研

生物学

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

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 ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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 ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

来自胚胎GFP-表达小鼠的Ex Vivo Oculo运动切片培养,用于对Oculo运动神经外生长进行时移成像

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

器官细胞生存在器官细胞切片培养

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

器官切片文化为产后神经再生的研究

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