This work was supported by Ministerio de Econom&#236;a y competitividad (BFU2012-37146) and Junta de Castilla y Le&#242;n (BIO103/VA45/11, VA145U13 and BIO/VA33/13), Spain. MCR was supported by Junta de Castilla y Le&#242;n (Spain) and the European Social Fund. We thank the late Dr. Philippe Br&#251;let (1947-2013) for the mitochondrial GFP Aequorin plasmid.

伦理声明:涉及动物主题程序已经在与欧洲的欧洲公约123 /理事会和86/609 / EEC协议批准巴利亚多利德大学动物住房设施的协议处理。 1.短期和长期的大鼠海马神经元的文化<ol><li>涂覆聚-D-赖氨酸的制备12毫米的玻璃盖玻片。 <ol><li>消毒直径12mm的玻璃盖玻片在乙醇中至少24小时。让他们在无菌条件下干燥。 </li><li>分布在封口膜在培养皿条盖玻片。覆盖每个盖玻片的表面上用200μl的1mg / ml的聚D-赖氨酸。允许治疗O / N。 </li><li>第二天用双蒸无菌水洗涤盖玻片，每15分钟对在无菌条件下90分钟。 </li><li>填写四井multidish板500微升的Neurobasal培养基，每孔。神经基础培养基应加以补充10％胎牛血清，2％B27，1μg/ ml的庆大霉素和2mM L-谷氨酰胺。 </li><li>放置盖玻片在multidish板。维持它们在湿度37ºC和5％CO 2培养箱中直至使用。 </li></ol></li><li>从分离新生大鼠海马神经元。 <ol><li>制备1.8毫升木瓜蛋白酶溶液（直接购买）在小瓶中（20微升/毫升）在无菌条件下:为了获得20微升的最终浓度/ ml，在1.8毫升Hank氏加0.6％BSA的添加约40微升木瓜蛋白酶（微升应计算根据供应商的条件）。汉克的0.6％BSA中制备通过混合85 ml的HBSS培养基用15ml的4％牛血清白蛋白（BSA，制备求解4克的BSA在100ml的HBSS）。在无菌罩准备吧。 </li><li>孵育在汉克的加0.6％BSA的木瓜蛋白酶使用前30分钟，在37ºC。使用在使用前0.22μm滤器过滤溶液。 </li><li>加入贝科的准备联队的F-12培养基改进的900毫升双蒸无菌水Eagle培养基粉（每升13.5克）。然后加入6克HEPES和336毫克碳酸氢钠和用NaOH 4 N.添加无菌水，以获得一个1升溶液，并用0.20微米的聚醚砜过滤它将pH调节至7.42（PES）瓶顶过滤器。在无菌罩携带这一过程。最后饱和无菌条件下，使用前用CO 2的溶液。存储解决方案，在4ºC，并使用它们冷冻。 </li><li>安乐死新生（P0）幼鼠被斩首，并在无菌HBS中洗了头。打开用无菌剪刀的帮助下头骨。提取大脑用抹刀和联队的F-12培养基快速清洗。 </li><li>使一对角切割用手术刀在每个半球（图1B）的帮助下，它携带到含有含的F-12培养基的皮氏培养皿。 </li><li>放弃脑膜仔细解剖钳的帮助，并在放大镜下。 </li> &lt;LI&gt;确定海马特征凹的位置上使用放大镜皮层。然后，通过从一个边境精心拉动和从它的位置用眼科剪取出分离皮层海马。 </li><li>转移海马组织到一个4孔板填充用无菌汉克的介质缺乏的Ca 2+和Mg 2+加0.6％BSA和清洗海马组织。如果不取出介质切割使用相同的眼科剪小块组织（约2×2毫米）。 </li><li>转移海马件含有1.8 ml的预过滤的木瓜蛋白酶溶液的小瓶。然后培养他们在37ºC偶尔，轻轻摇动。 15分钟后，加入90微升DNA酶I溶液（50μg/ ml的终浓度）孵育另外15分钟。 </li><li>转移组织到10ml的离心管中，并用新鲜的Neurobasal培养基洗片段。通过5毫升的塑料PIP通过组织碎片获取细胞悬液ETTE。 </li><li>离心细胞悬浮液，在160×g离心5分钟。使用塑料无菌巴斯德吸管除去上清液，并小心地中止沉淀用1毫升在1毫升的Neurobasal培养基的自动移液管。 </li><li>测量使用Neubauer计数室中的细胞密度。把玻璃盖玻片在纽鲍尔室。添加10微升的细胞悬浮液。确保细胞悬浮液进入腔室均匀且使得无气泡。算在显微镜下的细胞数，并执行相应的计算，以获得合适的降40-80微升含有30×10 3细胞 。细胞的总数量将取决于所使用的动物数量。 </li></ol></li><li>短期和海马神经元的长期培养。 <ol><li>在含有500微升的Neurobasal培养基的四个井multidish板在下降了约50微升至每个前制备并保持在培养箱，围绕板30×10 3细胞含有一种聚-D-赖氨酸包被的，直径12mm玻璃盖玻片的multidish板的井。 </li><li>维持原代海马细胞湿度37ºC和5％的二氧化碳培养箱中培养实验之前， 在体外 2-5天（DIV，短期的，年轻的培养）或&gt; 15 DIV（长期，老年文化），而不改变企业文化媒体。 </li></ol></li></ol>胞质钙2.荧光成像2+浓度 <ol><li>准备荧光成像测试解决方案。 <ol><li>制备含有一个外部的HEPES缓冲盐水（HBS）溶液，以mM:氯化钠145，氯化钾5，MgCl 2的1， 氯化钙 2 1，葡萄糖10，钠- HEPES 10（pH值为7.42）。 </li><li>制备含有外部和 Mg 2+，HEPES缓冲的盐水（HBS）溶液，以mM:氯化钠146，氯化钾5， 氯化钙 2 1，葡萄糖10和HEPES 10（pH值为7.42）。 </li><li>解决的NMDA镁2+，HEPES缓冲的盐水（HBS）在一个FINA用10μM的甘氨酸为100μM升浓度和补充它。 </li><li>通过求解（以mM计）制备含有氯化钾代替NaCl的HBS溶液:氯化钾145，MgCl 2的1， 氯化钙 2 1，葡萄糖10和HEPES 10（pH值为7.42）。 </li></ol></li><li>海马细胞荧光钙探针fura2 / AM的装载。 <ol><li>以含盖玻片细胞培养掉孵化器，并与哈佛商学院中在室温通过将它们转移到含有500微升哈佛商学院的每孔一个新的四井multidish板把它们洗干净。 </li><li>孵育细胞fura2 / AM4μM的在黑暗的地方（在相同的HBS介质制备）60分钟在RT（25℃）。 </li><li> 60分钟后，洗净盖玻片新鲜HBS网上平台。 </li></ol></li><li>胞质的记录荧光图像的[Ca 2+]在培养的细胞。 <ol><li>打开灯，显微镜，灌注系统，荧光相机和电脑。 </li><li>放置在盖玻片的恒温平台在倒置显微镜阶段开12个毫米的玻璃盖玻片，并选择使用40X物镜（油，NA:1.3）的显微镜字段。连续灌注细胞在没有或有试验物质存在预温（37℃）哈佛商学院的网上平台。保持流量在约5-10毫升/分钟的速率。 注意:对于活细胞细胞灌注系统安装在用于打开12个毫米的玻璃盖玻片的恒温平台。 8线重力驱动的灌注系统配备了一个阀控制器被用于灌注的解决方案。真空泵负责去除多余的网上平台。解决方案使用的是在管加热系统加热。 </li><li>捕获背景图像的快门关闭在两个激发波长。 </li><li>外延照亮细胞轮流在340和380纳米。在520nm处每5-10秒与荧光摄像头，这是由一个呋喃-2分色镜过滤记录光发射。 </li><li>当记录周期​​结束，保存完整的序列Òf图像在520nm在计算机中以供进一步分析发射。 </li></ol></li><li>分析记录荧光图像。 <ol><li>打开实验文件。使用aquacosmos软件，点击"率"，然后选择所需的比例范围内。计算通过在所产生的图像的像素比例的像素，以便获得比图像的序列。 </li><li>通过调整"背景消除"扣除背景。按"开始计算"。 </li><li>按"所有时间序列"按钮，删除感兴趣的古区（投资回报）​​。对于单个细胞定量分析，建立利益或投资回报相对应的单个神经元的新区域。在每个像素中对应于每个感兴趣区域，每个图像都平均比率值，以获得的用于各个感兴趣区（细胞）比荧光值的记录。 </li><li>要绘制个人录音输出对应于每个投资回报率的比率荧光值到图形PROGR上午。 </li><li>使相应的计算使用合适的数据分析估计比值的荧光的上升的大小响应于每个刺激和制图软件。 </li></ol></li></ol>线粒体钙3.生物发光成像2+浓度 <ol><li>转染培养海马神经元与mitGAmut质粒。 注:将细胞首先用含有一个线粒体靶向序列和突变的水母发光蛋白缺乏融合到绿色荧光蛋白（GFP）一个的 Ca 2+结合位点的mitGAmut质粒。这种结构检测大上升中线粒体的Ca 2+摄取 ，并允许转染的细胞通过GFP荧光的识别。质粒被开发和由P.Brûlet和同事18（CNRS，巴黎）友情提供。 <ol><li>制备的Neurobasal TF，含有的Neurobasal培养基，缺乏胎牛血清，B27，gentamic在和2mM L-谷氨酰胺。 </li><li>准备50微升的Neurobasal TF加在小瓶（溶液A）2.5微升转染试剂。制备50微升的Neurobasal TF的加4微克mitGAmut质粒在小瓶中（溶液B）。加入轻轻溶液B在溶液A允许在20分钟内搅拌，无晃动。 </li><li>含转移海马神经元含200微升新鲜的Neurobasal TF新multidish板盖玻片。 </li><li>一滴一滴添加100微升溶液A + B的过每盖玻片。温育30分钟。删除网上平台。洗一次细胞用新鲜的Neurobasal TF。返回盖玻片到原来的神经基础培养基。 </li><li>培养神经元的另外24小时，在37ºC和5％的CO 2染后，以允许有效表达和探头的定位。 </li></ol></li><li>准备生物发光成像的测试解决方案。 <ol><li>制备含NMDA 100微米或145毫米氯化钾为HBS解决方案上述（步骤2.1）。制备含有10mM 的 Ca 2+加毛地黄皂苷100μM的HBS溶液。 </li></ol></li><li>重组线粒体有针对性的水母发光蛋白与腔肠素的n。 <ol><li>转印盖玻片含转染的海马神经元到4孔平板含有200微升的HBS。 </li><li>加入4微升腔肠N（200微米），以200微升HBS的为4微米的终浓度，轻轻混匀。孵育2小时，在室温（25℃）并在黑暗中。 </li></ol></li><li>线粒体记录生物发光图像[ 钙 ]。 <ol><li>打开显微镜，灯，灌注系统，生物发光照相机和计算机。 </li><li>转移重构盖玻片一个恒温（37℃）灌注腔，并在5-10毫升/分钟的速率与预先温热的HBS溶液连续灌注。 </li><li>使用40倍物镜（油，NA:1.3），选择含有细胞表达的一个微观领域如图所示，通过使用FITC滤光片的绿色荧光蛋白（GFP）的表达相关的荧光发射poaequorins。典型的字段可包含1-2转染的细胞。捕获GFP荧光图像，使用CCD照相机。 </li><li>关闭显微镜的光。删除含有二向色箱设在光路。关闭激发光和关闭暗框完全黑暗。 </li><li>细胞灌注5-10毫升/分钟，HBS中有或无的测试解决方案预先加热至37ºC。 </li><li>捕获光子发射图像，每10秒，用图像处理器处理的光子计数照相机。 </li><li>在每个实验结束时，透化以释放所有其余的光子发射细胞用0.1毫洋地黄皂苷在10mM的CaCl 2在HBS。 注意:这些光子的排放必须相加，以便估计的总光子的排放量，在钙离子浓度的校准所需的值&lt;。/ LI&gt; <li>在电脑与相关联的绿色荧光图像存储生物发光图像的光子计数成像前抓获。 </li></ol></li><li>分析生物发光图像反映线粒体钙离子浓度的。 <ol><li>使用特定的软件量化光子排​​放。光子排放可利用布利尼等15描述的算法转换为线粒体游离钙离子浓度（[ 钙 ] MIT）。 </li><li>打开实验和GFP荧光图像。感兴趣（投资回报）​​与特定的软件的帮助下通过绘制根据在实验开始捕获的荧光图像的转染细胞的形状选择的区域。 </li><li>每次粘贴图像上相同的投资回报。在每个ROI总光子发射计算与特定的软件获得每个细胞在每个时间点的发光发射值（L）。 </li><li>添加了所有的光子EMISS离子在生物发光图像，包括毛地黄皂苷透化，使用特定的软件，以获得含有所有光子排放生物发光图像后所得到的那些。 </li><li>光子排放的利益进行计算特定软件的每个区域的出口值。要做到这一点，在为了计算生物发光值对每个ROI在每次点击"图形"。选择"总价值"和"使用当前的投资回报率的所有图像。按"计算"。保存"txt.file"和导出数据到工作表。对于每一个投资回报率，的[Ca 2+] MIT是使用以下算法计算: 的[Ca 2+]（M）= [R +（R·K TR） - 1] / [K 的R - （R·K R）]; 哪里， R = [L /（L 总 λ）] 1 / n的 L是在测量中的每个感兴趣区域的时间发射的发光。 l总为总发光加成存在于组织中当时在测量时间为每个感兴趣的区域中的计数后剩余。 λ，K TR，K R和n是常数，其值取决于水母发光蛋白结合（野生型或突变）和coelenterazin使用（野生型或n型），以及在记录温度16。对于这里使用的组合（突变AEQ和coelenterzine n）时，在37ºC，我们使用了以下值:K R = 8.47×10 7，K TR = 165.6×10 3中，n = 1204和λ= 0138。 </li></ol></li><li>使相应的计算使用合适的数据的分析来估计在生物发光的上升的大小响应于每个刺激和制图软件。 </li></ol>

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N., O'Keefe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Place+navigation+impaired+in+rats+with+hippocampal+lesions.">Place navigation impaired in rats with hippocampal lesions.</a> Nature. 297 (5868), 681-683 (1982).</li><li>Geinisman, Y., Detoledo-Morrell, L., Morrell, F., Heller, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+markers+of+age-related+memory+dysfunction:+behavioral,+electrophysiological+and+morphological+perspectives.">Hippocampal markers of age-related memory dysfunction: behavioral, electrophysiological and morphological perspectives.</a> Progress in Neurobiology. 45 (3), 223-252 (1995).</li><li>Sodero, A. O., Weissmann, C., Ledesma, M. D., Dotti, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+stress+from+excitatory+neurotransmission+contributes+to+cholesterol+loss+in+hippocampal+neurons+aging+in+vitro.">Cellular stress from excitatory neurotransmission contributes to cholesterol loss in hippocampal neurons aging in vitro.</a> Neurobiology of Aging. 32 (6), 1043-1053 (2011).</li><li>Brini, 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=Transfected+aequorin+in+the+measurement+of+cytosolic+Ca2++concentration+([Ca2+]c).+A+critical+evaluation.">Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation.</a> The Journal of Biological Chemistry. 270 (17), 9896-9903 (1995).</li><li>Villalobos, C., Alonso, M. T., Garc&#236;a-Sancho, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+calcium+oscillations+inside+intracellular+organelles.">Bioluminescence imaging of calcium oscillations inside intracellular organelles.</a> Methods in Molecular Biology. 574, 203-214 (2009).</li><li>Villalobos, 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=Redistribution+of+Ca2++among+cytosol+and+organella+during+stimulation+of+bovine+chromaffin+cells.">Redistribution of Ca2+ among cytosol and organella during stimulation of bovine chromaffin cells.</a> FASEB Journal. 16, 343-353 (2002).</li><li>Rogers, K. L., 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=Visualization+of+local+Ca2++dynamics+with+genetically+encoded+bioluminescent+reporters.">Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters.</a> The European Journal of Neuroscience. 21 (3), 597-610 (2005).</li><li>Barreto-Chang, O. L., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+cortical+neurons+using+Fura-2+AM.">Calcium imaging of cortical neurons using Fura-2 AM.</a> Journal of Visualized Experiments. (23), 1067 (2009).</li></ol>

None of the authors have competing interests or conflicting interests.

细胞内钙的大脑老化的重塑2+稳态已涉及到认知的损失，易感性增加缺血性损伤，兴奋性毒性和神经退行性疾病。为了验证这种假设在体外 ， 钙成像程序可用。不幸的是，旧的海马神经元的活培养是不可靠的。最近，人们已经观察到大鼠海马神经元老化体内包括ROS的积聚，形成脂褐素颗粒，异色灶，活化的pJNK和p53 / p21的通路，胆固醇损失的典型标志的存在许多，和?...

兴奋性毒性是有助于神经元损伤和细胞死亡的神经系统损伤，如局部缺血的最重要机制之一，并且在某些神经变性疾病如阿尔茨海默氏病1。这种类型的神经毒性的主要是对Ca介导的谷氨酸演技2+ -permeable，离子型NMDA受体（NMDA受体）2。曝光培养神经元对谷氨酸的可导致兴奋性中毒3，这会导致神经细胞凋亡4。我们和其他人先前认为神经易受NMDA诱导的凋亡可能与发育的体外和老化5-8变化。 它已被广泛接受的增加胞质无Ca 2+浓度（[Ca2 +浓度cyt的 ）导致细胞的活化。但是，如果此温升过高和/或持续的话，它可以触发细胞死亡9。此外，已经提出该兴奋性中毒，需要线粒体的Ca 2+摄取10中，由于治疗神经元对谷氨酸诱导的细胞死亡11线粒体解偶联剂保护神经元。如果线粒体占用过多的钙离子，线粒体通透性转换孔的开放，可能会出现，导致释放细胞色素C等促凋亡因子，并诱导细胞凋亡。我们最近已表明，这种线粒体的Ca 2+摄取直接相关的年龄依赖性易感性兴奋性毒性，通过直接测量NMDA诱导的线粒体的Ca 2+摄取在单个海马神经元5，其被报告在本文中的方法。海马，涉及生理过程，如学习，记忆等认知过程12，极易受到衰老和神经退行性疾病13。已经提出的是，数周体外后，培养海马神经元表现出了一些老化神经元14的典型特征。因此，长期培养海马神经元可以提供一个全面的模型来研究衰老增强兴奋性毒性的钙离子介导的机制。 提出的方法的总的目标是，因此，调查在细胞内Ca 2+体内稳态或Ca 2+重塑大脑老化实质性的变化包括由NMDA受体激动剂在一个长期培养海马神经元激发的差的 Ca 2+响应。该方法包括大鼠海马神经元的培养物的详细描述和胞浆和线粒体的 Ca 2+浓度的单个神经元的监测荧光和生物发光成像，分别。细胞内钙离子在培养的神经元荧光成像是一个标准的程序。然而，该方法是用于子不太可靠细胞钙测量，包括线粒体钙 。原因包括缺乏合成探针的适当的定位和不恰当的亲和性钙离子浓度可在线粒体从低μM级改连到毫水平的。以蛋白质作为例如水母发光蛋白的使用钙离子探针，已经允许靶向亚细胞器和使用使用不同的coelenterazines或突变的探针缺乏具体的钙离子结合位点15衍生物不同的钙亲和力。以这种方式，表达线粒体靶向水母发光蛋白的生物发光成像可允许的线粒体的 Ca 2+浓度在单个神经元的监测。然而，这个过程可能需要使用光子计数摄像机或超灵敏CCD相机对生物发光成像16-18。这种方法可能会产生新的结果，应该在更establ确认ished大脑衰老模型作为，例如，从旧的动物的大脑切片。

<table>
	<tbody>
		<tr>
			<td>Neurobasal Culture Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS medium</td>
			<td>Gibco</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 medium</td>
			<td>Gibco</td>
			<td>31330-038</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (from bovine pancreas)</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum</td>
			<td>Lonza</td>
			<td>DE14-801E</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-032</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm glass coverslips</td>
			<td>Labolan</td>
			<td>0111520/20012</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003127</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well multidish plaques</td>
			<td>Nunc</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>Petry dishes</td>
			<td>JD Catalan s.l.</td>
			<td>2120044T</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes</td>
			<td>Fisher Scientific</td>
			<td>431030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura2-AM</td>
			<td>Life Technologies</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine n</td>
			<td>Biotium</td>
			<td>BT-10115-2250 uG</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D5628</td>
			<td></td>
		</tr>
		<tr>
			<td>NMDA</td>
			<td>Sigma&#160;</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>50046</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axiovert S100 TV microscope</td>
			<td>Carl Zeiss Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xcite ilumination system</td>
			<td>EXFO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ORCA ER fluorescence camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VIM photon counting CCD camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VC-8 valve controller</td>
			<td>Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SH-27B heating system&#160;</td>
			<td>Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aquacosmos Software</td>
			<td>Hamamatsu Photonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence and bioluminescence imaging of subcellular ca2+ in aged hippocampal neurons

易感性相关的神经退行性疾病和局部缺血神经元细胞死亡的中老年大脑是极其增加，但负责的机制都是很知名。兴奋性毒性，认为有助于诱导既损伤神经元损害的方法，通过激活谷氨酸受体，促进钙离子内流和线粒体的 Ca 2+超载的介导。在细胞内钙离子稳态细胞内钙离子稳态或改造的显着变化可能有利于老神经细胞神经元损伤。在老化研究的 Ca 2+重塑我们已经使用活细胞成像在大鼠海马神经元的，类似于在老化体内神经元的某些方面的长期培养物。为这个目的，海马细胞，在第一个地方，新鲜从新生大鼠海马分散并涂布于POLI-D-赖氨酸包被的玻璃盖玻片。然后培养物保持在控制的媒体的数天或数瓦特eeks调查年轻人和老年人的神经元，分别。第二，培养的神经元中装入fura2并进行使用数字荧光比率成像细胞内的Ca 2+浓度的测量。第三，培养的神经元被转染质粒表达低亲和力水母发光蛋白和绿色荧光蛋白靶向线粒体串联。 24小时后，水母发光蛋白在细胞内重构与腔肠素和神经元受到生物发光成像监测线粒体Ca 2+浓度的。这三个步骤的过程允许细胞质和线粒体的 Ca 2+响应的监测，以相关的刺激如例如谷氨酸受体激动剂NMDA和比较是否这些和其它响应由老化的影响。此过程可产生新的见解，如何老化影响细胞溶质和线粒体的 Ca 2+响应所选的刺激以及所选药物旨在防止神经元细胞的测试死亡年龄相关的疾病。

在这里，我们描述一个简单的方法来评估的 Ca 2+重塑和NMDA对胞浆和线粒体的影响的[Ca 2+]老年神经元。 图1示出了该过程的示意图，用于隔离和从新生大鼠中培养的海马神经元。在开始之前，我们需要准备无菌，D - 聚赖氨酸涂层的玻璃盖玻片并找到他们在一个4孔盘。接着，新生大鼠被打死，大脑中删除。分离海马后，组织精心分散使用木瓜蛋白酶。分离的细胞洗涤并...

Watch this Scientific Journal Video about Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons at JoVE.com

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

fluorescence-bioluminescence-imaging-subcellular-ca2-aged-hippocampal

Research

JoVE Journal

Neuroscience

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

8.3K 次观看.  Consejo Superior de Investigaciones Cientìficas. Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging. 

视频: Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

科研

神经科学

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

神经调节和线粒体运输:在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

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

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

从产前小鼠海马神经元的分离和培养

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

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

彗星试验测定DNA损伤的海马神经元

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

在肠神经系统的原位 钙成像

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described here identifies the basic building blocks of this circuitry. The in-depth studies of cortical regions will ultimately provide other scientists with the circuit components needed for an understanding of how the brain acquires, processes and stores information and what goes wrong in disease, while the electrophysiological and morphological data are widely used by computational neuroscientists in the construction of model networks that explore information processing. The protocol outlined here describes how biocytin-filled cells recorded in the CA2 region of the hippocampus are recovered and then reconstructed in&#160;3D. Additionally, the protocol describes the demonstration of calcium binding protein or peptide content in recorded interneurons.

The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.

填充海马 ca2 间神经元的生物环素恢复与三维重建

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described ...

Methods Article

亚细胞钙荧光和生物发光成像