Name: fluorescence and bioluminescence imaging of subcellular ca2+ in aged hippocampal neurons
Uploaded: 2015-12-01T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons at JoVE.com

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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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，和长期培养改变的Ca 2+通道的密度和NMDA受体14。因此，在大鼠海马神经元的年轻人和老年人的文化钙成像实验可以提供钙重塑大脑老化的新见解。一个条件，是培养hippoca关键mpal神经元的长期是双重的。首先，在一个非常低的密度如上所述直接制版的细胞中，这是很重要的。第二，将细胞培养于补充Neurobasal介质而不改变介质。这种方法允许胶质但避免其过度生长的存在。的示意图，例如培养示于图1。 荧光成像合成钙离子探针允许细胞内的监测钙离子浓度在单个神经元19。使用在年轻人和老年人的神经元这种方法允许变化与年龄​​文化响应的监测。例如图2显示了典型的亮场和的增加[ 钙 ] CYT从老年文化的年轻神经元和神经元NMDA引起的钙离子的图像。请注意，在老年神经元响应是比在年轻的神经元大得多。但是，此过程是不可靠的[ 钙]亚细胞器如，例如，线粒体，其中的[Ca 2+]可以从下面的微米变化到高于毫水平之内测量。在这些情况下，蛋白基，线粒体定位的探针已被开发为，例如，水母发光蛋白。该探头是特别有趣，因为其对钙的亲和力，可微调监测很低或很高的[Ca 2+]像线粒体内达到休息的那些与刺激的条件下，分别。具体而言，有可能选择与野生型的水母发光蛋白或突变的水母发光蛋白不含Ca 2+结合位点以修改的亲和力。当不同coelenterazines（野生型，H，N）的组合使用不同aequorins 17微调实现。然而，尽管使用荧光的钙成像是普遍，监测生物发光并不简单，并且可能需要光子计数照相机。幸运的是，水母发光蛋白探针，如这里使用的之一，已得到改进共表达GFP的18使它们更稳定，能释放出更多的光子发射，并允许简单的识别转染的细胞由相关联的GFP荧光。成功在生物发光成像中的神经元不仅取决于所用的光子计数照相机的效率，还对线粒体靶向探针的转染效率。在海马神经元的情况下，一个简单的化学转染可能工作提供了一个高灵敏度的光子计数摄像头可用。 图明场，绿色荧光的年轻和老年生物发光图像的培养海马神经元2所示的例子。还显示了增加线粒体钙离子浓度由NMDA记录在转染的海马神经元诱导。注意，NMDA的效果大得多老年培养的神经元比年轻的神经元。在其他情况下，transfec的效率化可以增加通过使用病毒衍生的载体16,18。可替代地，转基因小鼠携带蛋白质基，亚细胞的 Ca 2+探针的开发是为未来的方法的一个新兴的选项，或许甚至在体内 。

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&#039;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&amp;nbsp;</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&amp;nbsp;</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。分离的细胞洗涤并铺于涂覆盖玻片。那么细胞是2-5或DIV&gt; 15 DIV得到年轻人还是老年人的文化，分 ​​别培养，并用于钙成像实验。 利用上述策略，就可以从同一样品老少神经元来测试，例如，是否NMDA诱导的[Ca 2+]在年轻文化前进上胞质差动作用水库比旧的培养物。 图2表明NMDA100μM的诱导胞浆较大增加的[Ca 2+]老年培养物比在年轻培养物。同样，NMDA诱导细胞死亡的旧的神经元，但不是在年轻的神经元5。在其中的 Ca 2+响应是非常高的情况下，类似的实验，可以进行与钙离子的探针与钙离子更少的亲和力，以避免染料饱和类似物，例如fura4F 5。以相同的方式，但也可以学习是否胞质的静息水平的[Ca 2+]是不同的。此外，该协议与定量免疫用抗体特异性NMDA受体亚单位的结合可能会允许在提供NMDA受体亚单位表达的差异CORRELATE变化反应的特异性抗体，可用5。的[Ca 2+]也可用于评估其他有关paramete胞质的测量RS包括钙离子存储内容，休息渗透性钙离子， 钙离子清除率和青年和老年的神经元之间的可能差别。 以类似的方式，但也可以检测线粒体这样的刺激的效果的[Ca 2+]。 图3显示了转染的线粒体靶向水母发光蛋白的海马神经元的生物发光成像的一个例子。刺激后释放光子的排放量是上升的线粒体的功能， 钙离子浓度来实现。请注意，在线粒体的NMDA诱导的上升钙离子浓度大得多老年的神经元比年轻的海马神经元，在那里NMDA几乎增加了光子排放。这些结果可能有助于解释为什么NMDA诱导细胞凋亡只在老年的神经元，但不是在海马细胞5青年文化。以相同的方式，有可能测试对于线粒体额外差异钙青年和老年的神经元使用专门设计的测试协议，例如与处理， 钙退出线粒体，线粒体钙摄取从细胞内贮存引起的钙释放透的神经元和线粒体钙吸收。此外，这种方法可用于检测影响线粒体的 Ca 2+超载，可以是用于神经保护对抗兴奋性毒性兴趣药物。 <img alt="图1" src="/files/ftp_upload/53330/53330fig1.jpg" /> 图1:步骤原代大鼠海马神经元的分离（A）的聚-D-赖氨酸，12个毫米的玻璃涂覆盖玻片制备在培养皿中，最后转移至4孔盘。大鼠脑（B）的背视图。虚线表示所在的切应。 （c）获得的uspension初级海马神经元细胞。除去海马后，得到的细胞悬浮液。然后细胞铺在含有聚-D-赖氨酸涂覆的盖玻片的4孔培养皿。 （ 四）明场图像显示了文化的原代大鼠海马神经元。律师代表10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53330/53330fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53330/53330fig2.jpg" /> 图2:NMDA会增加细胞内钙离子浓度的海马神经元的短期和长期培养的海马神经元加载fura2 / AM，并进行荧光成像。图片显示短期（A）和长期的代表性明场和伪彩色图像（比F340 / F380）（B </st荣&gt;）前（基础），并刺激与100μMNMDA后，海马神经元。温暖的色彩体现升高细胞内钙离子浓度（伪刻度显示在底部）。跟踪显示代表性，单细胞胞浆中记录的[Ca 2+]针对100μMNMDA短期（A）和长期（B）培养海马神经元。需要注意的是细胞内钙离子浓度增加是更大的长期高于短期培养的神经元。律师代表10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53330/53330fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53330/53330fig3.jpg" /> 图3:NMDA诱导线粒体钙超载海马神经元海马neur项转染的低亲和力，线粒体靶向水母发光蛋白融合到绿色荧光蛋白，孵育4μM腔肠素，并进行线粒体的[Ca 2+]生物发光成像。图片显示荧光（GFP），积累了光子发射（水母发光蛋白生物发光）代表短期（A）和长期（B）培养海马神经元的图像。发光强度是编码在伪（每像素1至16光子排放）。录音显示光子排放量（线粒体钙离子浓度）在短期（A）和长期（B）培养海马神经元的释放。 NMDA 100μM增加线粒体钙离子浓度在老年的神经元，但不是在年轻的培养海马神经元。律师代表10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53330/53330fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。&lt;</a>/ A&gt;

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

Universidad San Sebastian

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

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

科研

神经科学

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

使用荧光寿命成像在老化C.Elegans中淀粉样蛋白结构的表征

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

生物化学

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

行为学

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

发育生物学

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

亚细胞钙荧光和生物发光成像

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