笔者想感谢的MRC-LMB车间生产的增强型基因枪枪管。我们还要感谢大卫·R·汉普森博士在多伦多大学使用的设备和资源。

利用动物和动物组织中应严格遵守当地下的规则和法规的伦理委员会的批准。本研究中坚持的MRC-LMB的动物实验指南中获得的所有组织。 1，准备材料，文化传媒<ol><li>准备用微孔水所有的解决方案;仅使用分析纯试剂。准备并储存所有试剂在室温下（除非另有说明）。 </li><li>使500毫升HEPES缓冲的盐水中（10mM HEPES，120mM氯化钠pH 7.2）中的。通过0.2微米的无菌过滤装置进行过滤。保存在4℃。 </li><li>制成500毫升磷酸盐缓冲盐水（PBS;的10mM的Na 2 HPO 4，2mM的KH 2 PO 4，137 mM氯化钠，2.7 mM的氯化钾，pH 7.4）中。通过高压灭菌消毒。 </li><li> 100 N2ス：通过补充的DMEM（Dulbecco改良的Eagle培养基），用25mM HEPES，10％胎牛血清，30mM的葡萄糖，1制备的神经介质pplement，青霉素，链霉素1100单位/毫升; pH值为7.2。用0.2微米的无菌过滤装置进行过滤。保存在4℃。 </li><li>使聚乙烯吡咯烷酮（PVP）储备溶液用20毫克的PVP在1ml 100％乙醇。分装在1ml的量，冷冻并储存在-20℃的这一点。工作溶液是0.05毫克/毫升，因此加10微升的PVP原液每4毫升的100％乙醇。 </li><li>准备一个0.05M的亚精胺原液在微孔水;将pH调节至7.2。 </li><li>使在DMEM 2％的琼脂糖溶液（2克电泳级琼脂糖在100毫升的DMEM中的无菌螺旋盖瓶）。微波炉，直到琼脂糖完全溶解。储存在4℃冰箱数周，如果有必要的。 </li></ol> 2，准备器官切片的<ol><li>安乐死C57黑色6小鼠所需的年龄，用CO 2窒息后斩首。采用横向切割头骨开始在枕骨大孔，并在结束消除大脑嗅叶。轻轻提起颅骨从背面露出的脑。 </li><li>嗅叶和额叶皮层，并且与小脑延髓之间切断。轻轻提起大脑延髓部分，切断视神经。最后，取出大脑，并放置在培养皿中填充有在冰上冰冷的HEPES缓冲盐水。 </li><li>使用解剖显微镜下，用细镊子剥去软;小心地取出周围脑血管和脑膜。 </li><li>将新鲜的大脑进入一个小的收件人模具和覆盖它与琼脂糖溶液冷却到略高于室温。然后迅速地通过将其放置在冰上冷却模具至4℃。 注：处理片尽可能快地避免生存能力的损失。 </li><li>当琼脂糖设定（5 - 10分钟），除去从模具的琼脂糖包埋的脑（修剪如果必要的话），然后将其粘到vibroslicer平台。把这个变成含无菌冰冷的PBS vibroslicer室。迪被用70％乙醇，以尽量减少后续污染前sinfect的vibroslicer室。 </li><li>剪下使用定制vibroslicer或商业等同大脑。该模型的概念是设计一个组织切片，可以产生大振幅和高频率的运动水平，以在叶片的边缘，同时最小化垂直振动。 </li><li>使用90赫兹的振荡频率，以1.5的幅度 - 3.2毫米，并且所述切割刀片定位在15°到水平平面的角度;设置在安装块保持的琼脂糖包埋脑移动至1.7毫米/朝向叶片分钟。收集部（150微米）到腔室含有冰冷的培养基（DMEM青霉素/链霉素+10％FCS）。组织切片看切割和操纵作为阿瑟诺和奥布莱恩13补充视频。 </li><li>轻轻将切片放入细胞培养插件（0.4微米，直径30毫米）的6多井托盘与文化MEDIA上的插入件的外部，并孵育在湿润的培养箱中在37℃下用5％的CO 2。 注：为确保正常组织活力，是非常温和的，而传输和处理的切片。另外，过多的流体将防止器官切片附着在过滤器插入件的膜。如果发生这种情况，去除多余的液体，或再次板上。 </li><li>虽然器官切片可以是几个星期培养，电镀最佳转染率后继续转不晚于4天。 注意：为了避免在长期的培养条件下，用阿糖胞苷或无 ​​血清培养基2胶质增生。 </li></ol> 3，制备DNA包被的微型和纳米弹丸<ol><li>准备采用40纳米直径的金颗粒的纳米弹。简言之，加入50微升的0.05M的亚精胺和在1毫克/毫升（pEYFP-N1）至10mg的金颗粒的10微升的DNA。 <ol><li>添加钙和亚精胺的溶液混合在DNA的金微TURE颗粒制剂，以便于DNA分子的结合，以帮助到金纳米粒子。 注：每金​​载波毫克（DLR）使用的DNA的量，范围应的金每1〜5微克DNA的1毫克。此外，金载体​​颗粒，将每盒（MLQ）下拍摄的量，应本身的范围从0.1到0.5毫克的金。 DLR的和MLQ应尝试和根据所研究的特定的系统，以及使用的颗粒和基因枪的种类上的每个用户进行了优化。 </li></ol></li><li>混合使用涡旋，同时缓慢加入50微升的1M的CaCl 2中的10级分- 15微升。以下5分钟，偶尔涡旋，离心分离机以1000×g离心30秒，然后除去上清液。悬浮金颗粒沉淀物3.5毫升，0.075M的PVP。 </li><li>得出这样的悬挂成管（2.36毫米的直径）使用注射器。放置在管道准备站的管道。允许金颗粒沉淀并通过抽吸用注射器除去上清液。旋转管均匀分布的金颗粒，其随后在恒定的氮气流量在5升每分钟进行干燥。 </li><li>要创建DNA的子弹，用切管器1厘米的长度切断油管。或者立即插入到基​​因枪盒或保持干燥的（4℃）直到需要。 </li></ol> 4，基因枪转染对器官切片<ol><li>执行的无菌层流净化罩中进行如下操作。 </li><li>插入一个9伏电池和一个空墨盒架到赫利俄斯基因枪。 </li><li>附加基因枪氦罐氦气软管。消防2 - 3空白（空槽）在50psi加压氦气软管和喷枪的水库。 </li><li>加载包含所述DNA包被的金墨盒插入墨盒座和支架加载到基因枪。 </li><li>拧下基因枪间隔和高度经修饰的基因枪管的基因枪。 </li><li>用无菌镊子，将包含器官切片放入无菌塑料培养皿过滤器插件。 </li><li>从器官切片取出介质。 </li><li>设置在50psi的气压。 注：眼睛保护是必要的，保护耳朵为宜。 </li><li>通过使用测量到10mm的安装放置在适当的距离上的基因枪。仔细瞄准镜筒的中心以上的基因递送所期望的区域。 </li><li>烧成后，用新鲜培养基，5％CO 2培养箱更换刀片放回盘，并将它们返回到正常生长条件下， 即 37℃。 </li><li>一旦完成，关闭氦气罐，释放基因枪的压力和氦气罐分离基因枪。 </li><li>取出墨盒座和抛弃盒和清洁基因枪。 </li><li>检查组织的形态和成功渗透visualizing使用倒置显微镜切片。如果使用的微粒，均匀地分散在黄金;应该看到该切片上的金颗粒没有密集的区域。由于它们的小尺寸纳米颗粒不能被看作是单个单元通过常规的显微技术。 </li><li>在适当的时间间隔后（通常1 - 2天），固定于4％多聚甲醛（PFA）进行显微镜观察切片。 注：固定在PFA并不总是需要的。活细胞成像避免这一步。 </li></ol> 5，固定和脑片的可视化<ol><li>基因枪转染后，在PBS中2分钟，两次冲洗切片。 </li><li>孵化在新鲜出炉的，冰冷的4％PFA的PBS 20分钟修复片。 </li><li>为2分钟，在PBS中洗两次切片。 </li><li>轻轻切圆膜支持使用手术刀，不影响安装在他们的片。使用镊子放在载玻片每个膜支撑/片。休息organotyp集成电路片上载玻片上的膜的顶部上。 </li><li>加安装媒体的一滴在片的顶部，并直接与器官切片接触轻轻加盖玻片，没有任何力。 </li><li>固定盖玻片有一层薄薄的指甲油了。 </li><li>查看该切片上的适当的显微镜。 </li></ol> 6，激光共聚焦应用<ol><li>通过使用一个堂堂正正的激光扫描共聚焦显微镜用60X / 1.4数值孔径（NA）油浸物镜可视化的切片。 注：共焦显微镜的最成问题的特点是针孔大小（设置为1 AU），它能够隔离和收集焦点平面，从而消除了失焦“阴霾”常看到的荧光样本。细细节往往被这个霾遮蔽，不能在一个非共显微镜检测。 </li><li>氩激光器设置为488纳米的激发波长和优化用于收集青霉的itted光在500至520纳米波段。通常情况下，收集的图像，在1024×1024像素，并与堆叠的z轴节在0.5微米的间隔为包括分析每一个神经细胞的整个宽度。 <ol><li>以观察细胞核的形态，计数器染色这些细胞用DAPI（激发波长设定在420之间收集405 nm，发射光 - 4 40纳米）。最后，用于检查红色荧光，设置激光在561纳米激发和565 - 620纳米的发射。 </li></ol></li><li>通常情况下，获得通过使用4倍变焦扫描，涵盖57.6 平方微米的面积图像;图像尺寸均在1,024×1,024像素的区域。的z轴节在0.5微米的间隔记录栈和5的突起 - 8帧的总和。 </li></ol>

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	<li>Klein, R. M., Wolf, E. D., Wu, R., Sanford, 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=High-velocity+microprojectiles+for+delivering+nucleic+acids+into+living+cells.">High-velocity microprojectiles for delivering nucleic acids into living cells.</a> Biotechnology. 24, 384-386 (1987).</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-Psychopharmacolog., &amp; Biological Psychiatry. 41, 36-43 (2013).</li><li>Morin-Brureau, M., De Bock, F., Lerner-Natoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slices:+a+model+to+study+the+neurovascular+unit+micro-environment+in+epilepsies.">Organotypic brain slices: a model to study the neurovascular unit micro-environment in epilepsies.</a> Fluids and Barriers of the CNS. 10, 11 (2013).</li><li>Millet, L. J., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over+a+century+of+neuron+culture:+from+the+hanging+drop+to+microfluidic+devices.">Over a century of neuron culture: from the hanging drop to microfluidic devices.</a> The Yale Journal of Biology and Medicine. 85, 501-521 (2012).</li><li>Cho, 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=Spatiotemporal+evidence+of+apoptosis-mediated+ischemic+injury+in+organotypic+hippocampal+slice+cultures.">Spatiotemporal evidence of apoptosis-mediated ischemic injury in organotypic hippocampal slice cultures.</a> Neurochemistry International. 45, 117-127 (2004).</li><li>Daviaud, N., 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=Modeling+nigrostriatal+degeneration+in+organotypic+cultures,+a+new+ex+vivo+model+of+Parkinson's+disease.">Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease.</a> Neuroscience. 256, 10-22 (2013).</li><li>Drexler, B., Hentschke, H., Antkowiak, B., Grasshoff, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cultures+as+tools+for+testing+neuroactive+drugs+-+link+between+in-vitro+and+in-vivo+experiments.">Organotypic cultures as tools for testing neuroactive drugs - link between in-vitro and in-vivo experiments.</a> Current Medicinal Chemistry. 17, 4538-4550 (2010).</li><li>Oishi, Y., Baratta, J., Robertson, R. T., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+factors+regulating+axon+growth+between+the+cortex+and+spinal+cord+in+organotypic+co-cultures:+effects+of+age+and+neurotrophic+factors.">Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: effects of age and neurotrophic factors.</a> J Neurotrauma. 21, 339-356 (2004).</li><li>Baratta, J., Marienhagen, J. W., Ha, D., Yu, J., Robertson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+innervation+of+cerebral+cortex+in+organotypic+slice+cultures:+sustained+basal+forebrain+and+transient+striatal+cholinergic+projections.">Cholinergic innervation of cerebral cortex in organotypic slice cultures: sustained basal forebrain and transient striatal cholinergic projections.</a> Neuroscience. 72, 1117-1132 (1996).</li><li>Barateiro, A., Domingues, H. S., Fernandes, A., Relvas, J. B., Brites, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Cerebellar+Slice+Cultures+Exposed+to+Bilirubin+Evidence+Reactive+Gliosis,+Excitotoxicity+and+Impaired+Myelinogenesis+that+Is+Prevented+by+AMPA+and+TNF-alpha+Inhibitors.">Rat Cerebellar Slice Cultures Exposed to Bilirubin Evidence Reactive Gliosis, Excitotoxicity and Impaired Myelinogenesis that Is Prevented by AMPA and TNF-alpha Inhibitors.</a> Molecular Neurobiology. 49 (1), 424-439 (2013).</li><li>Franke, H., Schelhorn, N., Illes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+neurons+develop+axonal+projections+to+their+target+areas+in+organotypic+co-cultures+of+the+ventral+mesencephalon+and+the+striatum/prefrontal+cortex.">Dopaminergic neurons develop axonal projections to their target areas in organotypic co-cultures of the ventral mesencephalon and the striatum/prefrontal cortex.</a> Neurochemistry International. 42, 431-439 (2003).</li><li>Mingorance, 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=Regulation+of+Nogo+and+Nogo+receptor+during+the+development+of+the+entorhino-hippocampal+pathway+and+after+adult+hippocampal+lesions.">Regulation of Nogo and Nogo receptor during the development of the entorhino-hippocampal pathway and after adult hippocampal lesions.</a> Molecular and Cellular Neurosciences. 26, 34-49 (2004).</li><li>Arsenault, J., O'Brien, 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=Optimized+heterologous+transfection+of+viable+adult+organotypic+brain+slices+using+an+enhanced+gene+gun.">Optimized heterologous transfection of viable adult organotypic brain slices using an enhanced gene gun.</a> BMC Research Notes. 6, 544 (2013).</li><li>De Simoni, A., Griesinger, C. B., Edwards, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+rat+CA1+neurones+in+acute+versus+organotypic+slices:+role+of+experience+in+synaptic+morphology+and+activity.">Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity.</a> J Physiol. 550, 135-147 (2003).</li><li>Laywell, E. D., 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=Neuron-to-astrocyte+transition:+phenotypic+fluidity+and+the+formation+of+hybrid+asterons+in+differentiating+neurospheres.">Neuron-to-astrocyte transition: phenotypic fluidity and the formation of hybrid asterons in differentiating neurospheres.</a> J Comp Neurol. 493, 321-333 (2005).</li><li>Walder, S., Zhang, F., Ferretti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+neural+stem+cell+markers+suggests+the+occurrence+of+dedifferentiation+in+regenerating+spinal+cord.">Up-regulation of neural stem cell markers suggests the occurrence of dedifferentiation in regenerating spinal cord.</a> Dev Genes Evol. 213, 625-630 (2003).</li><li>Brien, J. A., Holt, M., Whiteside, G., Lummis, S. C., Hastings, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+to+the+hand-held+Gene+Gun:+improvements+for+in+vitro+biolistic+transfection+of+organotypic+neuronal+tissue.">Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue.</a> J Neurosci Methods. 112, 57-64 (2001).</li><li>Brien, J., Unwin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+spines+on+the+dendrites+of+Purkinje+cells.">Organization of spines on the dendrites of Purkinje cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 1575-1580 (2006).</li><li>Brien, J. A., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-biolistics:+a+method+of+biolistic+transfection+of+cells+and+tissues+using+a+gene+gun+with+novel+nanometer-sized+projectiles.">Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles.</a> BMC Biotechnology. 11 (66), 1472-6750 (2011).</li><li>Brien, J. A., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diolistic+labeling+of+neuronal+cultures+and+intact+tissue+using+a+hand-held+gene+gun.">Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun.</a> Nature Protocols. 1, 1517-1521 (2006).</li><li>Brien, J. A., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diolistics:+incorporating+fluorescent+dyes+into+biological+samples+using+a+gene+gun.">Diolistics: incorporating fluorescent dyes into biological samples using a gene gun.</a> Trends in Biotechnology. 25, 530-534 (2007).</li><li>Han, X., 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=Validation+of+an+LDH+assay+for+assessing+nanoparticle+toxicity.">Validation of an LDH assay for assessing nanoparticle toxicity.</a> Toxicology. 287, 99-104 (2011).</li><li>Lein, E. 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=Genome-wide+atlas+of+gene+expression+in+the+adult+mouse+brain.">Genome-wide atlas of gene expression in the adult mouse brain.</a> Nature. 445, 168-176 (2007).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> Journal of Visualized Experiments. 12, (2008).</li><li>Su, T., Paradiso, B., Long, Y. S., Liao, W. P., Simonato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cell+damage+in+organotypic+hippocampal+slice+culture+from+adult+mouse:+a+potential+model+system+to+study+neuroprotection.">Evaluation of cell damage in organotypic hippocampal slice culture from adult mouse: a potential model system to study neuroprotection.</a> Brain Research. 1385, 68-76 (2011).</li><li>Mewes, A., Franke, H., Singer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+of+adult+transgenic+P301S+mice--a+model+for+tauopathy+studies.">Organotypic brain slice cultures of adult transgenic P301S mice--a model for tauopathy studies.</a> PloS One. 7, 45017 (2012).</li><li>Ito, T., Ibe, K., Uchino, T., Ohshima, H., Otsuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+DNA/Gold+Nanoparticle+Encapsulated+in+Calcium+Phosphate.">Preparation of DNA/Gold Nanoparticle Encapsulated in Calcium Phosphate.</a> Journal of Drug Delivery. 2011, 647631 (2011).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Appl Phys Lett. 87, 014103 (2005).</li><li>Meijering, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron+tracing+in+perspective.">Neuron tracing in perspective.</a> Cytometry. Part A : the Journal of the International Society for Analytical Cytology. 77, 693-704 (2010).</li><li>Sasaki, T., Matsuki, N., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+axon-attached+recording+with+fluorescent+patch-clamp+pipettes+in+brain+slices.">Targeted axon-attached recording with fluorescent patch-clamp pipettes in brain slices.</a> Nature Protocols. 7, 1228-1234 (2012).</li><li>Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+'DiOlistic'+labeling+of+the+nervous+system+using+lipophilic+dye+combinations.">Multicolor 'DiOlistic' labeling of the nervous system using lipophilic dye combinations.</a> Neuron. 27, 219-225 (2000).</li><li>Livet, J., 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=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450, 56-62 (2007).</li><li>Gholizadeh, S., Tharmalingam, S., Macaldaz, M. E., Hampson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+the+central+nervous+system+after+intracerebroventricular+injection+of+adeno-associated+viral+vectors+in+neonatal+and+juvenile+mice.">Transduction of the central nervous system after intracerebroventricular injection of adeno-associated viral vectors in neonatal and juvenile mice.</a> Human Gene Therapy Methods. 24, 205-213 (2013).</li><li>Arsenault, J., 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=Botulinum+protease-cleaved+SNARE+fragments+induce+cytotoxicity+in+neuroblastoma+cells.">Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells.</a> Journal of Neurochemistry. 129, 781-791 (2014).</li><li>Arsenault, J., 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=Stapling+of+the+botulinum+type+A+protease+to+growth+factors+and+neuropeptides+allows+selective+targeting+of+neuroendocrine+cells.">Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells.</a> Journal of Neurochemistry. 126, 223-233 (2013).</li></ol>

Dr. John A. O'Brien holds patents for the improved gene gun barrel. US patent number 10/380,452 and European patent Number 01974517.3.

本协议描述的方法通过使用增强基因枪提供染料和遗传物质进入成人器官切片。本质上，染料的种类和各种可能的基因使该方法多方面和适合于回答广泛的生物学问题。用，在这种情况下，以特定的大脑区域中的区域选择性递送方法，打开用于实验作为局部区域的异源基因的调制的新途径的生物学相关架构内尚未容易可行之前。我们先前已经确定的，该方法能对成人器官切片，其中成熟的突触被完全成形的，因为它显示出改善的细胞存活和异源基因表达的许多周13被使用。用的新的和改进的培养条件为哺乳动物大脑以及其它组织的出现，使用区域选择性遗传调制的，将成为各种各样bioche的日益有用MICAL分析。 正如我们将要在这里和前面突出显示由他人24所提到的，许多不同的因素可容易地影响基因枪递送的准确性和可靠性。首先，与染料或DNA的金颗粒的制备方法，必须有足够的平衡，以防止聚集，并促进暗盒货物驱逐。钙和亚精胺中的DNA的金微粒溶液可以帮助DNA分子结合于金纳米颗粒。穿透细胞减压能力的纳米颗粒被预先决定的17。其次，基因枪必须安装尽可能平稳和正确的角度和距离。一个可靠的压力表也是必要的重复性，以维持组织的完整性，并妥善加快金颗粒其预定的目标。事实上，距离，方向，和设备的稳定性也是一个重要的因素为吨argeting和精确的区域选择性交付。作为这些大脑结构是非常小的，在触发角的微小变​​化可以很容易地使整个过程更不可靠。最后，编写和维护的可行器官切片已经误人子弟用了几十年的困难对于不同的动物，大脑区域，年龄和共培养物还丰富，可靠的协议是目前可用的2,4,25,26。这些程序应该是用户提交的优化组织，以基因枪过程之前。这将会更容易地允许用户无偏确定该生物射弹粒子的影响，所述异源基因的上自己的文化的影响。为此，利用荧光基因如EYFP和EGFP的既可以使一个新的用户，以测试靶向精度和也遵循通过异源基因表达的细胞的存活率随着时间13持续的时期。许多可靠的关键步骤且可重复使用该协议在以下三段突出显示。 对于高效的转染，所述DNA浓度必须适当。浓度过低会妨碍转屈服而浓度是太高会导致纳米颗粒的附聚。黄金的聚集体可以显着降低转染效率，导致不均匀的分布，并且可以导致增加的细胞毒性和氧化胁迫。与涂覆效率的问题很可能是由于亚精胺溶液期满（应改为每2〜3个月）。对于适当的基因枪传播，标签必须是偶数。金纳米颗粒/ DNA悬浮液的非均相标记可能是由于PVP的溶液。它也应更换，每2至3个月。黄金纳米粒子的涂层，可以看到在琼脂糖凝胶电泳更高MW波段27。 每个生物SYST根据调查青霉以及使用可能需要在气体压力的微小变化，达到转染和基因枪传播的优化水平各个不同的仪器。关键参数是剥离从塑料盒中的微米或纳米载体和推动它们进入组织所需的氦脉冲的压力。高气体压力会影响细胞存活，因为它会造成整个目标冲击波，破坏或从基质17中分离的组织。针对基因枪管和具有完全垂直于组织的装置是用于精确基因枪递送重要。所选择的区域应当放置在枪管的直接中心在从外开口恰恰10mm以下。这将导致一个直径3毫米的震中周围的金颗粒递送。基因枪枪管应在每次使用后70％的乙醇进行清洗。为了防止大颗粒聚集体组织，桶中还含有尼龙网的SHOULD定期更换以保持最佳性能。为了更换筛网拧开盖子然后将盖和O形环之间的新的尼龙网。 2天震中周围染后 - 荧光标记的细胞应该至少1是可见的。没有或标签错位可导致制剂基因枪安装的不足和不稳定。使用共聚焦显微镜细胞的观察结果取决于许多因素：在激发/发射光的波长，针孔大小，物镜的NA，组件中的光路的折射率，所述组织，并且对齐的深度仪器。这些因素应为每个系统在调查进行优化。 在基因枪法的最新进展进行了利用这种方法的成功。在本研究中使用的基因枪管，以便减少所需的压力以及漏斗生物射弹已被修改散射图案融入了更多的限制和具体的区域17,19。其他桶改装试图限制基因枪传播，减少流出压力28但这种定制设计的基因枪枪管由奥布莱恩博士设计是安装在标准的Helios基因枪单个组件。接着，亚微米的金颗粒被用来减少我们先前已经确定的微米颗粒的浸润引起的细胞损伤，并最终持续异源基因表达的丧失。这些变化的生物射弹方法改进了方法13的整体可行性和可重复性。片上制备其他诸如深入的故障排除在切片过程中，使用的DMEM琼脂糖基质以保护大脑，和许多其他有用的进步，器官型脑片的制备此前报道13使我们能够探索利用成人片了已经开发成熟突触网络。 在我们的研究中，我们主要使用的染料和荧光异源基因以可视化细胞的形态作为转染效率的读出，并有能力将图像的小鼠脑组织的神经元的形态学特征。该信号所定义的范围内随机配送的信噪比使我们的母语环境中完全分离单个细胞的树突状海港。无数的努力被用来研究这些形态特征，绘制“connectomics”或标记单个细胞的各种分析，这种方法可以很容易地与现有的协议，如电生理和神经突起的测量29,30组合。显然，荧光异源基因的组合可以使一个更强大的划分单细胞荧光蛋白的随机分布，他们的组合将确定各个单元31,32的颜色。使用细胞特异性启动子，如突触蛋白和胶质原纤维酸性蛋白的异源基因的外显子的上游，也可以限制为表达对亚群33。事实上，无休止各种遗传物质，也可以在一个区域选择性地通过使用相同的过程金纳米颗粒递送。的总转染区域因此可以用传统的方法，如解剖并提交该区域的免疫印迹分析，异源基因的局部效果进行分析。 在器官切片程序的改进也将允许更广泛地使用脑组织的长期试验，以及允许使用其它组织和共培养的，并能促进转染程序。脂和聚合物的转染之前已经报道了影响膜稳态，内化，蛋白渗透率34和经常表现出非常低的效率，转染德rminally分化的神经元。小区特定货物的定位也适合仅向细胞中表达所需的内在化的细胞表面受体，并且虽然这种方法可以是高度选择性的，它可以缺乏靶向区域选择性35。病毒载体否则往往困难和昂贵的生产，需要严格的法规，并且对之际显示的安全性顾虑。基因枪递送因而具有无与伦比的能力，可行的组织中，以区域选择性转染神经元和其他类型的细胞。作为金是非在用基因枪法有毒，进一步进展将铺路生物医学用途，例如经皮药物递送，非侵入性的体内基因递送，以及局部的非病毒基因疗法。

最初的生物射弹技术中，点灯-的短语“生物弹道学”，建立了粒子介导的基因转移到植物细胞1。细胞转化的这种物理方法加速微 - 或纳米颗粒以高速来克服不渗透细胞膜的物理屏障，以提供货物，如DNA或染料。因为它不依赖于特定的配体 - 受体和/或在细胞表面的膜的生化性质，粒子介导的基因转移可以容易地适用于各种生物系统中，如器官型脑切片。 利用器官切片比其他优势， 在体外平台，因为他们认为很多解剖和生化性质是相关的体内生物学2-4。切片大多保存从那里起源和preser当地的建筑特色已经神经化学活性和突触连接。使用脑切片用于基础研究的，并在药剂的努力，已伴随增加，可能的生物技术操作来测量和监测大脑在体内的神经生物学行为，如上下文3,5-7数。使用器官切片为基础的检测系统的主要优点是它提供了容易的实验控制，并且允许细胞外环境的精确操作。 富有成效，器官切片培养系统已经建立了从各种脑区域，例如，但不限于，皮质，脊髓和小脑8-10。此外，一些共同培养已经证实，这允许在远端的脑区，以及神经元和神经病理细胞11,12间的连接通讯的评估。许多协议已经建立送uccessfully文化的器官切片，并能保持长期的活力和最近的许多研究，现在利用膜界面的方法和各种修改它13。这个原理通过将片上的多孔膜过滤器保持器官切片在培养基和培养器的加湿空气之间的界面。因此，媒体可以提供足够的营养物质通过毛细血管运动喂器官切片。通常切片已准备从出生后早期的动物（3 - 9天之久; P3 - 9）。然而，从这些切片的脑组织显示细胞可塑性的高水平和有机械应力的固有电阻，这是有益的，以获得可行的培养物，但成熟的突触和神经解剖学电路没有完全开发在体内直到2至3周龄14。例如，以前的观察结果表明，从P0得到的海马切片 - 1新生儿，虽然高度VIAB乐以下准备，逐渐失去了一些形态特征。本质上，它们被证明不适合于长期培养表明未成熟细胞更可能去分化相比从老年动物15,16器官型培养。由于这个原因，我们的方法进行了优化，成人器官大脑切片在其成熟和建筑发展已达到其终端阶段13,17-21。然而，这种方法也适用于新生儿和少年器官切片。 一旦可行器官切片已经生产含有切片的整个板可以被带到该基因枪安装并提交给区域选择性输送和转染。正确安装的基因枪（如在图1中描述的）的，面向90°的距离为10毫米的正上方的片（从开口至所述组织），允许快速基因枪递送的40纳米的去LD颗粒涂层货物。这些货物如染料和荧光DNA载体，以及任何感兴趣的基因，可容易地输送到活切片自如。这种方法从而说明了此协议需要交付，在区域选择性的方式，货物涂层的金纳米粒子转化为可行的器官大脑切片。基因枪枪管和最佳的安装可以看出，在图1中 ，有关改进枪管和纳米弹道的详细信息，请参阅奥布莱恩等17。 随后精炼还适用于用户优化的条件，如金每个目标区域传递适量的，其定义为微载体装载量（MLQ），并以确定的DNA的每金毫克装载量，定义为DNA加载比率（DLR）。此前沉淀的DNA在金颗粒和其装入Tefzel管，它包括实际基因枪“墨盒9;，它是必要的，以计算DNA和金的需要为每个转染，可以组织和条件之间略有不同的量（比率应保持在1毫克黄金和1微克DNA到1:5）。至关重要的是，制备MLQ的适当比例，以DLR另有DNA包被的金颗粒会粘附在一起，形成比预期的结块，这可能会降低基因枪传播的整体均匀性，以及增加组织损伤和细胞毒性更大。 这种方法显示了基因枪递送的最新改进，这已经过测试，确定为适合新生儿，青少年和成人的器官大脑切片。此外，通过利用基因枪枪管的降低基因枪传播，用户现在能够选择性地转染准时区域中的脑。下列适当的温育时间，所述荧光蛋白的表达，从而达到其24和48之间的最大速率小时，可通过荧光和共聚焦显微镜观察。这些荧光团允许范围内的生物相关结构的形态学分析和单个细胞的定位。然而，器官切片由其它异源基因的区域选择性调制，也可以通过适合于这些制剂中的任何其他测试来监测。可能的工作策略的局部基因递送示于图2。

<table border="0" cellpadding="0" cellspacing="0" width="656">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Name of Material/ Equipment</td>
			<td style="width:141px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:173px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Helios gene gun system</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2431</td>
			<td>Gene gun and tube prep station</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">HEPES&#160;</td>
			<td style="width:141px;">Sigma</td>
			<td>83264-100ML-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaCl</td>
			<td style="width:141px;">Sigma</td>
			<td>S7653-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">KCl</td>
			<td style="width:141px;">Sigma</td>
			<td>P9333-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">Na2HPO4</td>
			<td style="width:141px;">Sigma</td>
			<td>S7907-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:141px;">Sigma</td>
			<td>P9791-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">0.2 &#181;m sterile filter unit</td>
			<td style="width:141px;">Millipore</td>
			<td>SCGPU05RE</td>
			<td>to filter media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMEM</td>
			<td style="width:141px;">Gibco</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fetal calf serum</td>
			<td style="width:141px;">Gibco</td>
			<td>10270-098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">D-Glucose</td>
			<td style="width:141px;">Sigma</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">N2</td>
			<td style="width:141px;">Life Technologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Penicillin/Streptomycin</td>
			<td style="width:141px;">Sigma</td>
			<td>p4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvinylpyrrolidone</td>
			<td style="width:141px;">Sigma</td>
			<td>856568-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Ethanol&#160;</td>
			<td style="width:141px;">Sigma</td>
			<td>277649-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Spermidine</td>
			<td style="width:141px;">Sigma</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Agarose</td>
			<td style="width:141px;">Bio Rad</td>
			<td>161-3100EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Vibroslicer</td>
			<td style="width:141px;">Laica</td>
			<td style="width:119px;">VT1200</td>
			<td>We used a custom design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gene gun mount</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>Built in house at U of T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Humidified cell culture incubator</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gold nanoparticles</td>
			<td style="width:141px;">cytodiagnostics</td>
			<td style="width:119px;">G-40-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pEYFP-N1</td>
			<td style="width:141px;">Clontech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubing cutter</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tefzel tubing</td>
			<td style="width:141px;">Bio Rad</td>
			<td>165-2441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Barrel</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>Custom made by the LMB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture insert</td>
			<td style="width:141px;">Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Paraformaldehyde</td>
			<td style="width:141px;">Sigma</td>
			<td>76240 Fluka</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dissecting microscope</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>for convenience</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Confocal microscope</td>
			<td style="width:141px;"></td>
			<td style="width:119px;"></td>
			<td>user defined for usage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glass slide</td>
			<td style="width:141px;">VWR</td>
			<td>2588-CA48323-185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microcover glass</td>
			<td style="width:141px;">VWR</td>
			<td colspan="2">2448-CA48366-067-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Vectashield mounting media</td>
			<td style="width:141px;">Vector laboratories</td>
			<td>H-1400&#160;&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

regioselective biolistic targeting in organotypic brain slices using a modified gene gun

DNA转染是非常宝贵的生物科学和最新进展，以器官型脑片的准备，各种异源基因的影响因此可以很容易地调查，同时保持体内生物学的许多方面。已经有越来越多的关注，转染终末分化的神经元的传统转染方法一直困难重重，如低收益率和存活率显著损失。基因枪转染可以规避许多的这些困难，但只有最近，这种技术被修改，使得它适合于在哺乳动物组织中。 新修改的加速器腔增强了基因枪的射击精度，并增加了它的深度渗透，同时还允许使用较低的气体压力（50磅），而转染效率的损失，以及允许PA的聚焦区域选择性扩散rticles在3毫米。此外，这种技术是直线前进和速度比乏味显微注射来执行。瞬态和稳定表达是可能的纳米粒子轰击，其中附加型表达可以在24小时内被检测到，并在细胞存活被证明是优于或至少等于，常规的方法。该技术具有但是1关键优点：它允许转染到在单个约束半径之内本地化从而使用户能够将解剖分离的异源基因的影响。在这里，我们提出了一个深入的协议，准备可行的成人器官切片，并提交他们使用了一种改进的基因枪区域选择性的转染。

器官切片生存能力可以乳酸脱氢酶活性，碘化丙啶标记和染色的dUTP进行监测正如以前报道的13,22。显然，切片的可行性和完整性是针对遗传交付货物的长期持续表达很重要。下列基因枪递送到器官切片，在荧光的异源基因的表达通过共聚焦显微镜监测。基因枪传播的使用改性桶的气密性可以看出，在图3A图3B显示了转染的海马区，使用的DNA包被的金粒子的代表性图像， 图4示出了转染的成体小鼠器官切片的代表性图像。如在图4A中 ，浦肯野神经元具有显着的树突状海港的浦肯野层的界面和减量的分子层中可以看出ebellum示出以下瞬时基因枪转染用质粒pEGFP-N1的明显标记。 图4B中示出了这些树突其中棘可观察到较高的放大率。 图4C示出了以下基因枪递送pDSredFP涂布的金纳米粒子在海马的CA1区发现了一个锥体细胞。 染料代替荧光编码的DNA的货物，也可以用类似的方式来可视化的器官切片的形态特征。 DIO，该标签质膜，可以更迅速地划定各个细胞的形态，也可以配合使用递送至相同的区域的DNA涂覆的生物射弹以确认regioselection。如在图5A中可以看到，在海马沾上DIO神经元也显示出长枝晶与众多棘。的箭头表示由突触终扣的存在下确定相比于dentrit轴突集成电路刺的其它突起。 图5B示出了CA1区，突出用DAPI复染剂众多海马突起标记细胞核。 图5C示出了这些类型的并行树突与众多棘的高倍放大的另一个例子。 很显然，这些照片反映了下列细胞标记的形态特征。没有排除被施加以提供任何数量的涂覆在金纳米颗粒的外源基因相同的方法。以允许成功转染的视觉确认，单个质粒可以被构造成共同表达感兴趣两个基因，并在同一载体上的荧光报告蛋白。 <img alt="图1" src="/files/ftp_upload/52148/52148fig1highres.jpg" /> 图1基因枪枪管和安装。（一）提高<html>由奥布莱恩博士在MRC-LMB，有一个限制基因枪传播，通过降低所需的压力，粒子加速减少组织损伤的开发ð桶。（二）优化基因枪的侧视图安装了亨德森博士开发和安德拉斯·纳吉博士在多伦多大学。安装保持基因枪在精确90˚垂直于器官切片，同时允许用户精确地控制基因枪递送的距离。为了获得最佳的基因枪条件枪管孔应直接放置在所感兴趣的区域在从器官切片10毫米的距离。基因枪安装的（C）的后视图。感兴趣的被转染的区域需要被直接放置在枪管孔的下方。针对可与染料（diolistic）或荧光蛋白转染（基因枪）来确认。ANK“&gt;请点击这里查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52148/52148fig2highres.jpg" /> 图2。器官切片制备和区域选择性靶向。（A）的DMEM嵌入式成年小鼠的大脑准备好切片（B）模具被粘到vibroslicer与刀片准备切割片安装（C）区域选择性异源基因表达策略基因枪递送到冠状器官大脑切片。从艾伦脑图谱解剖参考图谱23获得图片制作图片。左图显示了有针对性的，例如，两个不同的大脑区域。到限制的基因表达（1）向左侧半球的皮层区。到限制的异源基因的表达（2）至脑的下丘脑区域。中间的面板显示THA吨的两种异源基因的表达（图3和4），可以分离和两个半球的海马中的相同器官切片进行比较，基本上消除了来自使用不同的部分和/或给予的可能性，以解决来自的效果远端串扰任何偏压这些基因。右图显示转基因递送的可能重叠表达的第一个基因（5）精确的皮层区域，同时又表达基因（6）在一个完全不同但重叠的皮质区。这允许单独分析，也各外源基因在同一个片内组合的遗传调节的效果。 <a href="https://www.jove.com/files/ftp_upload/52148/52148fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52148/52148fig3highres.jpg" /> 图3枪法散射图形上看到FILT呃纸和低倍率的图像（A）中的改进的桶被解雇在滤纸上，确定金颗粒的扩散。左侧示出了改进的筒的分配，而右侧表示正常桶。黑棒：1厘米。从奥布莱恩图像拍摄等人 17（B）低放大倍数图像表示内生物射弹蔓延6周龄小鼠的海马区中的3毫米随机转染图案。白条：100微米<a href="https://www.jove.com/files/ftp_upload/52148/52148fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52148/52148fig4highres.jpg" /> 图4枪法交付荧光蛋白编码的DNA导入6周龄的小鼠器官切片的（A）无脊椎动物ð共聚焦图像的的pEGFP-N1涂金颗粒转浦肯野细胞的小脑固定器官切片的。图片摄于40倍的放大倍率。黑棒：30微米（b）为 60倍的另一个浦肯野细胞的树突状海港突出的刺。黑棒：5微米（c）所示 ，从海马区采取60倍的放大倍率4个固定片DSredFP转锥体细胞的共聚焦图像。黑棒：10微米<a href="https://www.jove.com/files/ftp_upload/52148/52148fig4large.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52148/52148fig5highres.jpg" /> 图5 Diolistic递送膜标记荧光染料到器官切片（A）示出了海马神经元升的实时成像在放大20倍abeled与DIO。的树突棘可清楚地观察到，以及所确定的不存在树突棘和由白色箭头指示突触终扣的存在轴突。白条：标有DIO和counterstained用DAPI的海马区的30微米（二）发音的染色。白条：海马CA1区发现了树突棘的30微米（C）的放大倍数（60倍）。白条：5微米<a href="https://www.jove.com/files/ftp_upload/52148/52148fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun at JoVE.com

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

区域选择性基因枪瞄准的器官脑片用改进的基因枪

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

regioselective-biolistic-targeting-organotypic-brain-slices-using

Research

JoVE Journal

Neuroscience

16.2K 次观看.  University of Toronto. Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices. 

视频: Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

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

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

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 ...

科研

神经科学

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

利用基于病毒的Cre重组马赛克产后小鼠脑开发基因功能分析

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

突触前多巴胺纹状体脑片的快速扫描循环伏安动态

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

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 ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

腺相关病毒和共聚焦显微镜对器官脑切片胶质细胞器的可视化和实时成像

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...