这项工作得到加拿大自然科学和工程研究理事会以及加拿大卫生研究院的资助。

此协议基于先前描述的过程的修改 18 、 24 、 25 , 26 , 27 . 本议定书由麦吉尔大学动物保育委员会批准. 
 1. 实验准备 注意: 可以准备和维护以下库存解决方案直到使用. 
<ol> <li> 解剖解决方案 <ol> <li> 溶解3.62 克氯化钠 (氯化钠), 0.2 克氯化钾 (氯化钾), 0.069 克磷酸磷酸钠 (NaH 2 PO 4 和 #8729; H 2 O) 和 1.306 g 的 d-(+)-葡萄糖在500毫升的蒸馏 H 2 O。然后在溶液中加入12.5 毫升的 HEPES 缓冲液、450和 #181; 酚红溶液的 L 和20毫升的 BSA 分数 V。使用 1 N 氢氧化钠 (氢氧化钠) 调整到 pH 值 7.4. </li>
		<li> 用0.22 和 #181 进行灭菌, 在4和 #176 进行过滤和存储;此解决方案将保持稳定长达一周至10天. </li>
	</ol> </li> <li> 胰蛋白酶 <ol> <li> 在0.9% 氯化钠的浓度为25克/升的胰蛋白酶中制备一个储存溶液。将解决方案存储在-20 和 #176; C. </li>
	</ol> </li> <li> 胰蛋白酶抑制剂 <ol> <li> 通过在100毫升的 1 mM 中溶解1克鸡蛋白胰蛋白酶抑制剂, 准备一份浓度为10毫克/毫升的股票. 将此解决方案存储在-20 和 #176; C. </li>
	</ol> </li> <li> dnasei <ol> <li> 在10毫升的无菌水中溶解100毫克的 dnasei 1, 以生成10毫克/毫升的库存。将解决方案存储在-20 和 #176; C. </li>
	</ol> </li> <li> MgSO 4 <ol> <li> 添加6.02 克硫酸镁 (MgSO 4 ) 到50毫升蒸馏水 (H 2 O) 以生成1米的库存。将解决方案存储在4和 #176; C. </li>
	</ol> </li> <li> CaCl 2 <ol> <li> 溶解0.735 克氯化钙 (CaCl 2 和 #8729; 2H 2 o) 在50毫升蒸馏 H 2 o 到库存浓度 100 mM。存储解决方案在4和 #176; C. </li>
	</ol> </li> <li> 氯化钾 <ol> <li> 在50毫升的蒸馏 H 2 O 中溶解3.7275 克氯化钾, 并将 1 m 存储解决方案的库存集中在 4 & #176; C. </li>
	</ol> </li> <li> d-(+)-葡萄糖 <ol> 将1.802 克的 d-(+)-葡萄糖溶解在50毫升的蒸馏 H <li> 2 O 中, 以库存浓度为 100 mM。存储解决方案在4和 #176; C. </li>
	</ol> </li> <li> 单元格区域性媒体 <ol> <li> 准备单元格区域性媒体, 如下所示: 5 毫升热灭活透析胎牛血清, 500 和 #181; 200 毫米 l-谷氨酰胺, 100 和 #181; 50 毫克/毫升庆大霉素和1毫升1.0 米氯化钾应加到45毫升 过滤灭菌鹰和 #39 的最小基本培养基 (电子记忆), 补充1.125 克/升 d-葡萄糖, 产生50毫升的小脑颗粒神经元培养基。在4和 #176 存储媒体; C. </li>
	</ol> </li> <li> 胞嘧啶β-d-Arabino Furanoside <ol> <li> 在10毫升的生长培养基中溶解48毫克的胞嘧啶β-d-Arabino Furanoside, 其浓度为20毫米。存储解决方案在-20 和 #176; C. </li>
	</ol> </li> <li> 聚 d-赖氨酸 <ol> <li> 生成2毫克/毫升的聚 d-赖氨酸, 添加10毫升的无菌 H 2 O 到20毫克的聚 d-赖氨酸。聚 d-赖氨酸应贮存在-80 和 #176; C 在100和 #181; 等分. </li>
	</ol> </li> <li> 注意: 在解剖前应准备以下解决方案, 并保持在4和 #176; C 直到使用. </li>
	<li> MgSO 4 补充的解剖解决方案 <ol> <li> 添加300和 #181; 库存 MgSO 4 到250毫升的解剖解决方案. </li>
	</ol> </li> <li> 胰蛋白酶剥离解决方案 <ol> <li> 添加100和 #181; l 库存胰蛋白酶和12和 #181; 库存 MgSO 4 到10毫升的解剖溶液. </li>
	</ol> </li> <li> 胰蛋白酶抑制剂解决方案 1 <ol> <li> 添加164和 #181; 股票胰蛋白酶抑制剂, 250 和 #181; 库存 dnasei 1 和12和 #181; l 库存 MgSO 4 到10毫升的解剖解决方案. </li>
	</ol> </li> <li> 胰蛋白酶抑制剂解决方案 2 <ol> <li> 添加1040和 #181; 股票胰蛋白酶抑制剂, 750 和 #181; l 库存 dnasei 1 和12和 #181; l 库存 MgSO4 到10毫升的解剖溶液. </li>
	</ol> </li> <li> CaCl 2 补充的解剖解决方案 <ol> <li> 添加10和 #181; 库存 CaCl 2 和25和 #181; 库存 MgSO 4 到10毫升的解剖解决方案. </li>
	</ol> </li> <li> 聚 d-赖氨酸涂布培养皿 <ol> <li> 以覆盖培养皿, 稀释100和 #181; L 在40毫升的无菌 H 2 O 中储存聚 d-赖氨酸。对于35毫米发音培养皿, 加入2毫升稀释聚 d-赖氨酸溶液。对于4井板, 增加300和 #181; L 稀释聚 d-赖氨酸. </li>
		<li> 让聚 d-赖氨酸在吸气和洗涤前先坐1小时, 用4毫升或600和 #181; L (分别为 35 mm 培养皿或4个井板) 无菌 h 2 O。然后抽吸 H 2 O, 让盘子的空气干燥1小时. </li>
	</ol> </li> </ol> 2。小脑的脑提取与分离 <ol> <li> 使用斩首剪刀斩首6-7 天的老老鼠幼崽。在无菌组织培养罩中进行大脑解剖. </li>
	<li> 要卸下大脑, 请使用一副镊子抓住头部, 然后使用显微切割剪刀在头部前部切开皮肤, 如 图 1 所示。然后用一把新剪刀把头骨骨切开, 以减少污染的风险。用镊子将颅骨覆盖在脑部, 用镊子或刮刀将大脑梳理出来. 
	<ol> <li> 根据需要, 通过视神经切开以促进整个大脑的发育. </li>
	</ol> </li> <li> 将大脑放在与 MgSO 4 一起补充的解剖解决方案中。把溶液和大脑放在冰上. </li>
	<li> 以下脑切除术, 解剖显微镜下, 解剖了小脑从大脑, 而仍然在 MgSO 4 补充解剖解决方案, 并删除脑膜使用细钳。将小脑转向腹侧, 并确保脉络丛的切除. </li>
	<li> 将小脑池放入包含1毫升 MgSO 4 补充剥离解决方案的 35 mm 盘中. </li>
	<li> 将组织切成小块并转移到50毫升管中, 其中包含30毫升的 MgSO 4 缓冲剥离解决方案. </li>
</ol> 3。小鼠小脑颗粒神经元的分离和培养 <ol> <li> 离心50毫升管包含切碎的脑组织5分钟在 644 x g 和4和 #176; C. </li>
	<li> 清除上清液并添加10毫升的胰蛋白酶解剖溶液。然后在37和 #176 高速摇动管15分钟; C. </li>
	<li> 添加10毫升胰蛋白酶抑制剂溶液1到管和岩石轻轻地2分钟. </li>
	<li> 离心管5分钟在 644 x g 和4和 #176; C. </li>
	<li> 移除上清液并添加2毫升胰蛋白酶抑制剂溶液 2, 然后转移到15毫升管. </li>
	<li> 研制15毫升管中的组织, 直到溶液变得混浊为止。然后, 让解决5分钟的 </li>
	<li> 去除清上清液并将上清物转移至含有1毫升 CaCl 的新管 2 补充解剖溶液. </li>
	<li> 添加另一毫升的胰蛋白酶抑制剂溶液2到底部的管包含颗粒从步骤3.6。再次研制, 并让解决5分钟. 移除上清, 并将其添加到包含步骤3.7 中上清的管 (即重复步骤 3.6-3.7)。重复这个过程, 直到大多数组织是机械分离的. </li>
	<li> 添加0.3 毫升的 CaCl 2 补充解剖溶液的上清收集每毫升上清. </li>
	<li> 混合管的内容, 然后离心5分钟, 在 644 x g. </li>
	<li> 移除上清液, 并将新介质添加10毫升的颗粒和混合. </li>
	然后, <li> 单元格可以被计算并稀释到 1.5 x 10 6 单元/毫升的浓度。请注意, 一般1000万细胞预计从每个解剖的大脑, 取决于 trypsinization 时间和解剖效率。以前制作的聚 d-赖氨酸板上的平板细胞。对于4井板, 板 0.5 mL, 每井提供 7.5 x 10 5 单元格。对于35毫米的盘子, 4 毫升的盘子, 每个盘子给 6 x 10 6 的细胞. </li>
	<li> 24 小时后, 添加胞嘧啶-#946;-arabino furanoside (分析), 以减少胶质污染。每毫升的媒体0.5 和 #181; L 20 mM 分析是必需的。添加分析不需要媒体更改。如果要维持7-8 天的细胞, 在3天重复这种治疗. </li>
	<li> 维护 5% CO 中的区域性 2 37 和 #176 的孵化器; C 和100和 #181 的饲料; 100 mM 葡萄糖每2天的2天添加到培养基中。不需要进行完整的媒体更改. </li>
</ol> 4。慢包装、纯化和滴定 注意: 慢的包装、浓缩、纯化和滴定的协议以前已经详细描述过, 而不使用套件 28 , 包括用于滴定的替代方法, 如流式细胞仪 29 。在这里, 我们简要介绍的协议, 用于生产慢研究神经元损伤使用快速病毒纯化解决方案和 qPCR 慢滴定试剂盒. 
<ol> <li> 在 10 cm 培养皿中 Hek293T 细胞, Dulbecco 和 #39 的改性鹰和 #39 的最小必需培养基 (DMEM), 辅以 10% FBS。为了获得高的病毒效价, 每种转染至少要有 5 Hek293T 细胞。确保在转染细胞的一天是70% 汇合。在转染前三小时更换介质. </li>
	<li> 对于每个转染, 准备两个管。在管1中加入1.5 毫升的血清培养基和41和 #181; 转染试剂 L, 拌匀。在管 2, 增加1.5 毫升的记忆减少血清培养基和6和 #181; 转移质粒, 6 和 #181; g pCMV dR8.2 载体 (包装质粒), 3 和 #181; g pCMV-VSV-g 载体 (包络质粒), 和35和 #181; p3000 增强剂的 L。混合好, 并结合管1和 2, 漩涡和孵化15分钟. </li>
	<li> 从 Hek293T 板中取出50% 的介质, 并将 DNA 脂复合物添加到细胞中。孵育6小时, 在37和 #176; C, 5% CO 2 。取出培养基, 更换5毫升新鲜 DMEM, 一夜孵化. </li>
	<li> 在24小时 post-transfection, 从每个板块收集第一批病毒上清液。将病毒介质保持在4和 #176; C, 并在每个 Hek293T 细胞中加入5毫升的新鲜培养基。夜间孵化. </li>
	<li> 48 h post-transfection, 收集第二批病毒上清液, 将其与第一批和离心机结合, 病毒上清液为10分钟, 600 x g. </li>
	<li> 用45和 #181 的过滤器过滤清液; m 孔径大小. </li>
	<li> 使用快速病毒净化解决方案净化病毒。每45毫升的病毒上清, 添加5毫升的慢结合溶液, 混合和离心10分钟和 #62; 5000 x g 和4和 #176; C. </li>
	<li> 小心地除去上清, 以免扰乱颗粒. </li>
	<li> 添加20-40 和 #181; 中等和 re-suspend 的 L 颗粒。分-80 和 #176; C. </li>
	<li> 要获得慢效价, 使用 qPCR 慢滴定试剂盒。在500和 #181 中稀释2和 #181; 纯化病毒的 l; PBS 的 l。将稀释病毒的2和 #181 l 添加到18和 #181; 病毒裂解缓冲液 l (在工具箱中提供). </li>
	<li> 在 triplicates 中设置三不同的 RT q PCR 反应, 并将其标记为病毒裂解酶、阳性对照 1 (STD1) 和阳性对照 2 (STD2)。对于每个反应相加, 12.5、#181; l 2x qPCR MasterMix、2.5 和 #181; 或病毒裂解液或 STD1 (在试剂盒中提供) 或 STD2 (在试剂盒中提供) 和10和 #181; 试剂混合 (在试剂盒中提供), 0.2 毫升 PCR 管. </li>
	<li> 将 PCR 程序设置为:20 分钟, 在42和 #176; c 用于反转转录, 10 分钟在95和 #176; c 为酵素活化, 40 周期十五年代在95和 #176; c 为变性和 1 min 在60和 #176; c 用于退火/扩展. </li>
	<li> 使用此公式计算病毒滴度: 
	病毒裂解液滴度 = 5 x 10 7 /2 3 (酰-Ct1)/(Ct2-Ct1) . 
	酰 = 未知样品的平均 Ct 值, Ct1 = STD1 的平均值, Ct2 = 平均值 STD2. 
	注意: CGNs 可以是最好的转与慢或感染的腺取决于所研究的伤害模式。在培养基中添加慢2-3 到细胞培养液, 用于研究 NMDA 诱导的信号在7天的文化。这导致超过80% 转导, 是适当的追求与这些神经元的生化分析 ( 图 2 )。在5天的培养基中添加腺 (在莫伊 50) 和研究 NMDA 诱导的信号在7-8 天用于其他技术, 如成像。这种治疗结果在不到10% 的神经元感染, 使其理想的成像和共存的蛋白质。腺在电镀时可用于研究 DNA 损伤诱导的细胞死亡, 其方法是在2-3 天加入喜树碱或氧化应激增加过氧化氢。荧光蛋白的存在 (GFP, RFP, CFP 或 YFP) 标记的基因的兴趣可以用来估计百分比转导和潜在的毒性。在建议的莫伊, 我们看到最小的毒性和最大的转导 ( 图 3 ). </li>
</ol> 5。神经元损伤的建模 <ol> <li> 在体外诱导 nmda 诱导的神经元兴奋, 7 天后 , CGNs 100 和 #181; m NMDA 和 10 #181; m. 甘氨酸为 1 h. 从平行培养培养基中取代所有培养基治疗.这种浓度导致50% 细胞死亡24小时后治疗 ( 图 3C )。线粒体分裂是明显的在 6-8 h 岗位治疗 (参见 Jahani-手语 et al. 26 , 27 ). </li> <li> 诱导 ROS 诱导的细胞死亡 (氧化应激), 治疗神经ns 与75-100 和 #181; M 过氧化氢 (H 2 O 2 ), 并从并行区域性切换到条件媒体在5分钟接触到 H 2 o 2 之后。注意, 由于 h 2 O 2 的不稳定性, 将浓度优化为在 24 h 治疗后的培养中诱导50-70% 细胞死亡的水平. </li>
	<li> 诱导 DNA 损伤诱导的细胞死亡, 治疗 CGNs 与10和 #181; M 喜树碱。这种浓度导致超过50% 细胞死亡 24 h. 线粒体碎裂和早期凋亡信号发生2-3 小时后, 在这个浓度的喜树碱添加 (见 Jahani-手语 et al. 18 ) </li> <li> 在 CGNs 中诱导低钾诱导的神经细胞凋亡, 将含有25毫米 k + 的介质转换为具有 5 mm k + 7 天的体外 . </li>

<ol>
	<li>Goldowitz, D., Hamre, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cells+and+molecules+that+make+a+cerebellum.">The cells and molecules that make a cerebellum.</a> Trends in Neurosciences. 21 (9), 375-382 (1998).</li><li>Contestabile, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+granule+cells+as+a+model+to+study+mechanisms+of+neuronal+apoptosis+or+survival+in+vivo+and+in+vitro.">Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survival in vivo and in vitro.</a> Cerebellum. 1 (1), 41-55 (2002).</li><li>Bilimoria, P. M., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultures+of+cerebellar+granule+neurons.">Cultures of cerebellar granule neurons.</a> CSH Protoc. , (2008).</li><li>Kramer, D., Minichiello, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+primary+cerebellar+granule+cells.">Cell culture of primary cerebellar granule cells.</a> Methods Mol Biol. 633, 233-239 (2010).</li><li>Burgoyne, R. D., Cambray-Deakin, M. A. <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+neurobiology+of+neuronal+development:+the+cerebellar+granule+cell.">The cellular neurobiology of neuronal development: the cerebellar granule cell.</a> Brain Res. 472 (1), 77-101 (1988).</li><li>Hatten, M. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neural+patterning+and+specification+in+the+developing+cerebellum.">Mechanisms of neural patterning and specification in the developing cerebellum.</a> Annu Rev Neurosci. 18, 385-408 (1995).</li><li>Evans, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+signalling+in+cerebellar+plasticity.">Synaptic signalling in cerebellar plasticity.</a> Biol Cell. 99 (7), 363-378 (2007).</li><li>Hunt, D. L., Castillo, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+of+NMDA+receptors:+mechanisms+and+functional+implications.">Synaptic plasticity of NMDA receptors: mechanisms and functional implications.</a> Curr Opin Neurobiol. 22 (3), 496-508 (2012).</li><li>Arundine, M., Tymianski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+calcium-dependent+neurodegeneration+in+excitotoxicity.">Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity.</a> Cell Calcium. 34 (4-5), 325-337 (2003).</li><li>Szydlowska, K., Tymianski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium,+ischemia+and+excitotoxicity.">Calcium, ischemia and excitotoxicity.</a> Cell Calcium. 47 (2), 122-129 (2010).</li><li>Jahani-Asl, A., Germain, M., Slack, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+joining+forces+to+thwart+cell+death.">Mitochondria: joining forces to thwart cell death.</a> Biochim Biophys Acta. 1802 (1), 162-166 (2010).</li><li>Rego, A. C., Oliveira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+reactive+oxygen+species+in+excitotoxicity+and+apoptosis:+implications+for+the+pathogenesis+of+neurodegenerative+diseases.">Mitochondrial dysfunction and reactive oxygen species in excitotoxicity and apoptosis: implications for the pathogenesis of neurodegenerative diseases.</a> Neurochem Res. 28 (10), 1563-1574 (2003).</li><li>Wei, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proapoptotic+BAX+and+BAK:+a+requisite+gateway+to+mitochondrial+dysfunction+and+death.">Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.</a> Science. 292 (5517), 727-730 (2001).</li><li>Doyle, K. P., Simon, R. P., Stenzel-Poore, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+ischemic+brain+damage.">Mechanisms of ischemic brain damage.</a> Neuropharmacology. 55 (3), 310-318 (2008).</li><li>Coyle, J. T., Puttfarcken, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress,+glutamate,+and+neurodegenerative+disorders.">Oxidative stress, glutamate, and neurodegenerative disorders.</a> Science. 262 (5134), 689-695 (1993).</li><li>Cole, K., Perez-Polo, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+trauma+model:+in+search+of+Thanatos.">Neuronal trauma model: in search of Thanatos.</a> Int J Dev Neurosci. 22 (7), 485-496 (2004).</li><li>Cheung, E. C., McBride, H. M., Slack, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics+in+the+regulation+of+neuronal+cell+death.">Mitochondrial dynamics in the regulation of neuronal cell death.</a> Apoptosis. 12 (5), 979-992 (2007).</li><li>Jahani-Asl, 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=Mitofusin+2+protects+cerebellar+granule+neurons+against+injury-induced+cell+death.">Mitofusin 2 protects cerebellar granule neurons against injury-induced cell death.</a> J Biol Chem. 282 (33), 23788-23798 (2007).</li><li>Gallo, V., Kingsbury, A., Balazs, R., Jorgensen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+depolarization+in+the+survival+and+differentiation+of+cerebellar+granule+cells+in+culture.">The role of depolarization in the survival and differentiation of cerebellar granule cells in culture.</a> J Neurosci. 7 (7), 2203-2213 (1987).</li><li>Miller, T. 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=Bax+deletion+further+orders+the+cell+death+pathway+in+cerebellar+granule+cells+and+suggests+a+caspase-independent+pathway+to+cell+death.">Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.</a> J Cell Biol. 139 (1), 205-217 (1997).</li><li>Jekabsons, M. B., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+analysis+of+cerebellar+granule+neurons+undergoing+apoptosis+by+potassium/serum+deprivation.">Bioenergetic analysis of cerebellar granule neurons undergoing apoptosis by potassium/serum deprivation.</a> Cell Death Differ. 13 (9), 1595-1610 (2006).</li><li>Piccioli, P., 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=Inhibition+of+nuclear+factor-kappaB+activation+induces+apoptosis+in+cerebellar+granule+cells.">Inhibition of nuclear factor-kappaB activation induces apoptosis in cerebellar granule cells.</a> J Neurosci Res. 66 (6), 1064-1073 (2001).</li><li>D'Mello, S. R., Galli, C., Ciotti, T., Calissano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+apoptosis+in+cerebellar+granule+neurons+by+low+potassium:+inhibition+of+death+by+insulin-like+growth+factor+I+and+cAMP.">Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP.</a> Proc Natl Acad Sci U S A. 90 (23), 10989-10993 (1993).</li><li>Gallo, V., Ciotti, M. T., Coletti, A., Aloisi, F., Levi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+release+of+glutamate+from+cerebellar+granule+cells+differentiating+in+culture.">Selective release of glutamate from cerebellar granule cells differentiating in culture.</a> Proc Natl Acad Sci U S A. 79 (24), 7919-7923 (1982).</li><li>Messer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maintenance+and+identification+of+mouse+cerebellar+granule+cells+in+monolayer+culture.">The maintenance and identification of mouse cerebellar granule cells in monolayer culture.</a> Brain Res. 130 (1), 1-12 (1977).</li><li>Jahani-Asl, 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=CDK5+phosphorylates+DRP1+and+drives+mitochondrial+defects+in+NMDA-induced+neuronal+death.">CDK5 phosphorylates DRP1 and drives mitochondrial defects in NMDA-induced neuronal death.</a> Hum Mol Genet. 24 (16), 4573-4583 (2015).</li><li>Jahani-Asl, 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=The+mitochondrial+inner+membrane+GTPase,+optic+atrophy+1+(Opa1),+restores+mitochondrial+morphology+and+promotes+neuronal+survival+following+excitotoxicity.">The mitochondrial inner membrane GTPase, optic atrophy 1 (Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity.</a> J Biol Chem. 286 (6), 4772-4782 (2011).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. (32), (2009).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> J Vis Exp. (23), (2009).</li><li>Holubowska, A., Mukherjee, C., Vadhvani, M., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+cerebellar+granule+neurons+in+vitro+and+in+vivo+to+study+neuronal+morphology+and+migration.">Genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration.</a> J Vis Exp. (85), (2014).</li></ol>

作者没有什么可透露的。

在这里, 我们提供了一个简单的方法, 培养小鼠小脑颗粒神经元 (CGNs), 失去和增益的功能研究, 并建模不同的机制, 细胞死亡。有几个因素影响的重复性的结果使用这个程序, 需要密切监测。这些包括文化的纯净, 包括在文化中消除胶质细胞, 文化的汇合, 以及维持健康的细胞。在这些因素中引入变异性会对结果产生偏见, 并挑战重复性。下面我们将描述如何最小化影响结果的因素。
<p class="jove_conte...

小脑颗粒神经元 (CGNs) 具有良好的文化特征, 并已成为研究神经元死亡和发育的有效模型 1,2,3,4,56. CGN 培养的 n-甲基-d-天门冬氨酸 (nmda) 受体在体外的早期表达, 使其成为研究 NMDA 诱导信号的有吸引力的模型。这些受体的激活与 NMDA 结合膜去极化是用来模拟生理神经元活动的刺激, 并允许研究突触可塑性的机制7,8。相反, 这些受体的过度的 NMDA 配体可用于模型兴奋, 一个主要机制的神经元丧失的急性脑损伤和神经退行性疾病9。一个机制为诱导兴奋是通过 ATP 饥饿与减少的氧气, 如看见与急性神经元损伤。这导致膜的去极化和高水平的谷氨酸释放在突触。随后刺激的 NMDA 受体的高活性谷氨酸导致过量的 ca2 +通过这些受体的流入, 反过来又激活了包括 ca2 +激活的蛋白酶, 磷脂和酶, 导致严重的细胞成分和细胞死亡失控的退化。此外, 高胞内钙2 +导致氧自由基和线粒体损伤的生成10,11。
当 NMDA 诱导的神经元兴奋的大部分神经元丢失是由于钙的流入和 Bax/Bak 的独立, 其他机制的细胞死亡不能排除在此模型。兴奋的坏死和凋亡样细胞死亡的出现, 部分原因是由于高胞内 Ca2级级别12导致的活性氧 (ROS) 和 DNA 损伤的产生。DNA 损伤通过凋亡机制导致神经元死亡, 与凋亡细胞死亡的特征相关, 如染色质肿块和凋亡体的出现。诱导细胞凋亡是介导的细胞色素 c 从线粒体释放, 并已证明是依赖于 Bax/Bak 齐聚13。Bax/Bak 齐聚促进外线粒体膜的孔隙形成, 导致细胞色素 c 释放和亡调节剂的活化, 见轻度缺血性损伤14。
ROS 的产生是一个重要的问题, 在大脑中由于低内源性的抗氧化剂, 加上大的氧气需求的神经元功能的15。当暴露于缺血性事件时, 一氧化氮合酶上调, 产生一氧化氮和增加活性氧的种类14。氧自由基浓度的增加会导致 DNA 损伤, 并间接导致能量的饥饿。高水平的 DNA 双断裂是通过活化的聚 adp-核糖 polymerase-1 (PARP-1), 一个真核染色质结合蛋白负责催化转移的 ADP 核糖单位从和+, 一个过程积分到DNA 修复16。然而, 由于氧化应激造成的过度损害, PARP-1 活化可能导致能量饥饿, 由于增加的流失, 在和+, 一个必要的基质 ATP 生产通过氧化磷酸化。最终, 氧化应激将触发凋亡的 Bax/Bak 的依赖方式导致线粒体细胞色素 c 释放, 并已证明诱导线粒体重塑在 CGNs 17。
最后, CGN 培养基中氯化钾 (氯化钾) 浓度的变化可以用来模拟低钾/去极化介导的凋亡18,19,20。当暴露于低水平的 K+时, CGNs 经历了明显的生理变化, 导致线粒体呼吸和糖酵解减少, 这归因于细胞需求减少21, 以及减少核因子-nf-(NFκB), 它调节活动, 包括炎症和突触传递22。该模型对神经细胞发育过程中细胞死亡的研究具有特别的意义。低 K+环境更接近于生理条件, 并导致神经元发育过程中出现的细胞死亡特征23。
总之, CGNs 提供了一个长期的模型来研究神经元死亡和变性的基本分子机制。下面的协议将允许分离和培养 CGNs, 表达或抑制特定的基因通路使用病毒和诱导神经元死亡通过不同的机制, 代表神经元损伤和变性。

<table><tbody><tr><td>qPCR lentivitral titration kit&amp;nbsp;</td><td>ABM</td><td>#LV900</td><td></td></tr><tr><td>speedy virus purification solution&amp;nbsp;</td><td>ABM</td><td>#LV999</td><td></td></tr><tr><td>pCMV-dR8.2</td><td>Addgene</td><td>#8455</td><td></td></tr><tr><td>pCMV-VS.VG</td><td>Addgene</td><td>#8454</td><td></td></tr><tr><td>Distilled water&amp;nbsp;</td><td>Gibco</td><td>#15230162</td><td></td></tr><tr><td>200 mM L-Glutamine&amp;nbsp;</td><td>Gibco</td><td>#25030081</td><td></td></tr><tr><td>35 mm Nunc culture dishes</td><td>Gibco</td><td>#174913</td><td></td></tr><tr><td>PowerUP SYBR green master mix</td><td>life technologies</td><td>#A25742</td><td></td></tr><tr><td>BSA V Solution</td><td>Sigma Aldrich</td><td>#A-8412</td><td></td></tr><tr><td>CaCl2 &amp;bull; 2H2O</td><td>Sigma Aldrich</td><td>#C-7902&amp;nbsp;</td><td></td></tr><tr><td>Camptothecin</td><td>Sigma Aldrich</td><td>#C-9911</td><td></td></tr><tr><td>Chicken Egg White Trypsin Inhibitor&amp;nbsp;</td><td>Sigma Aldrich</td><td>#10109878001</td><td></td></tr><tr><td>Cytosine beta-D-Arabino Furanoside</td><td>Sigma Aldrich</td><td>#C-1768</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma Aldrich</td><td>#G-7528</td><td></td></tr><tr><td>DNase1&amp;nbsp;</td><td>Sigma Aldrich</td><td>#11284932001</td><td></td></tr><tr><td>Eagle-minimal essential medium</td><td>Sigma Aldrich</td><td>#M-2279</td><td></td></tr><tr><td>Glycine</td><td>Sigma Aldrich</td><td>#G-5417</td><td></td></tr><tr><td>Heat inactivated dialyzed Fetal Bovine Serum&amp;nbsp;</td><td>Sigma Aldrich</td><td>#F-0392</td><td></td></tr><tr><td>Hepes Buffer&amp;nbsp;</td><td>Sigma Aldrich</td><td>#H-0887</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma Aldrich</td><td>#216763</td><td></td></tr><tr><td>50 mg/mL Gentamycin&amp;nbsp;</td><td>Sigma Aldrich</td><td>#G-1397</td><td></td></tr><tr><td>MgSO4&amp;nbsp;</td><td>Sigma Aldrich</td><td>#M-2643</td><td></td></tr><tr><td>N-Methyl-D-aspartic acid</td><td>Sigma Aldrich</td><td>#M-3262</td><td></td></tr><tr><td>Phenol Red Solution&amp;nbsp;</td><td>Sigma Aldrich</td><td>#P-0290</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Sigma Aldrich</td><td>#T-4549</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher Scientific</td><td>L3000-008</td><td></td></tr><tr><td>p3000 enhancer reagent</td><td>Thermo Fisher Scientific</td><td>L3000-008</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>KCl&amp;nbsp;</td><td>VWR</td><td>#CABDH9258</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>VWR</td><td>#CABDH9286</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>VWR</td><td>#CABDH9298</td><td></td></tr><tr><td>Poly D-lysine&amp;nbsp;</td><td>VWR</td><td>#89134-858</td><td></td></tr><tr><td>DMEM</td><td>Wisent</td><td>#319-005-CL</td><td></td></tr><tr><td>FBS</td><td>Wisent</td><td>#080-450</td><td></td></tr></tbody></table>

modeling neuronal death and degeneration in mouse primary cerebellar granule neurons

小脑颗粒神经元 (CGNs) 是一种常用的神经元模型, 在小脑中形成了丰富的均质种群。根据其产后发育、丰度和可及性, CGNs 是研究神经元过程的理想模型, 包括神经元发育、神经元迁移和生理神经元活动刺激。此外, CGN 文化为研究不同的细胞死亡模式, 包括兴奋和细胞凋亡提供了一个很好的模型。在一个星期内的文化, CGNs 表达 n-甲基-d-天门冬氨酸 (NMDA) 受体, 一个特定的离子谷氨酸受体与许多关键功能的神经元健康和疾病。在 CGN 培养基中加入低浓度的 nmda 与膜去极化剂结合, 用于模拟生理性神经元活动刺激, 同时增加高浓度的 nmda 可用于模型兴奋神经元损伤。本文介绍了6天幼崽 CGNs 的分离培养方法以及腺和慢对 CGNs 的遗传操作。我们还提出了优化的协议, 如何刺激 NMDA 诱导的兴奋, 低诱导的凋亡, 氧化应激和 DNA 损伤后, 这些神经元的转导。

仔细解剖后, 完整的大脑应该被删除, 最小的伤害, 如图 1A-b所示。应努力减少对大脑的损伤, 特别是对小脑的损伤。损害小脑, 使更困难的识别和完全清除脑膜, 并增加了可能的污染的神经元文化。一旦脑膜被切除, 小脑可以从剩余的组织中解剖, 如图 1C所示, 并准备分离。
<p class="jove_content" fo:keep-together.within-page="1...

Watch this Scientific Journal Video about Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons at JoVE.com

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

本协议描述了一种简单的方法, 分离和培养的小鼠脑颗粒神经元 (CGNs) 从6-7 天的老幼崽, 有效的转导 CGNs 的损失和增益的功能研究, 并建模 NMDA 诱导神经元兴奋,低诱导的细胞死亡, DNA 损伤, 和氧化应激使用相同的培养模式。

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

modeling-neuronal-death-degeneration-mouse-primary-cerebellar-granule

Research

JoVE Journal

Neuroscience

8.0K 次观看.  Jewish General Hospital. 本协议描述了一种简单的方法, 分离和培养的小鼠脑颗粒神经元 (CGNs) 从6-7 天的老幼崽, 有效的转导 CGNs 的损失和增益的功能研究, 并建模 NMDA 诱导神经元兴奋,低诱导的细胞死亡, DNA 损伤, 和氧化应激使用相同的培养模式。

视频: Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

科研

发育生物学

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

生物学

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

神经科学

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

从胚胎小鼠小脑分离的神经元的主要培养

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

Modeling Neuronal Injury in Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates the generation of a neuronal injury model in mouse primary cerebellar granule neurons. It outlines the steps involved in inducing neuronal injury using NMDA, an excitatory neurotransmitter receptor agonist, and glycine, a co-agonist. NMDA and glycine bind to specific neuronal receptors, causing their overstimulation and resulting in a massive intracellular calcium influx, which leads to neuronal cell damage and death.

小鼠原代小脑颗粒神经元神经元损伤建模

1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.

	Dissection Solution
	
		Dissolve 3.62 g of ...

JoVE Encyclopedia of Experiments

Neuropathology

Trauma

Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video outlines the generation of a neuronal injury model using reactive oxygen species (ROS) in mouse primary cerebellar granule neurons. It details the process of inducing oxidative stress by adding high concentrations of hydrogen peroxide. ROS exposure causes DNA strand breaks and genomic instability. Additionally, the oxidation of cellular proteins impairs their functions, while lipid peroxidation compromises membrane integrity. This cascade leads to cytochrome c release and caspase activation, resulting in neuronal apoptosis.

小鼠小脑颗粒神经元活性氧诱导的神经元死亡建模

Neurodegenerative Diseases

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M., et al. <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

小鼠原代小脑颗粒神经元的分离和培养

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

摘要

探索更多视频

此视频中的章节

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

小脑颗粒神经元的遗传操作

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

从胚胎小鼠小脑分离的神经元的主要培养

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

利用体内出生后电穿孔研究小脑颗粒神经元形态和突触发育

小鼠原代小脑颗粒神经元神经元损伤建模

小鼠小脑颗粒神经元活性氧诱导的神经元死亡建模

小鼠原代小脑颗粒神经元的分离和培养

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

摘要

探索更多视频

此视频中的章节

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

小脑颗粒神经元的遗传操作

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

从胚胎小鼠小脑分离的神经元的主要培养

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

小鼠原代小脑颗粒神经元神经元损伤建模

小鼠小脑颗粒神经元活性氧诱导的神经元死亡建模

小鼠原代小脑颗粒神经元的分离和培养

利用体内出生后电穿孔研究小脑颗粒神经元形态和突触发育