（; U54GM104942 MNR），NIA（MNR; R15AG045812），阿尔茨海默氏症协会（MNR; NIRG-12-242187），西弗吉尼亚大学学院研究参议院拨款（MNR），西弗吉尼亚大学和格兰特PSCOR这项工作是由普通医学科学研究所的支持（MNR）。

1.涂层用酶或矩阵层的微电极阵列<ol><li> 制备蛋白质基质溶液 <ol><li>称出10毫克的牛血清白蛋白（BSA）和转移到1.5毫升离心管中。 </li><li>添加去离子水985μL含有BSA的微量离心管中。混合通过人工搅拌溶液（使用1000μL吸管〜3次重新移液，直到BSA溶解）。 注:请不要使用涡的解决方案，因为这样做可能会导入空气的混合溶液。另外，吸液管设定为体积&lt;1000μL（ 例如 700μL），以避免引入气泡。该溶液可以，如果必要被放置在微量。 </li><li>一旦BSA溶液溶解，加入5μL25％戊二醛溶液。 5倍 - 通过反转封闭管3混合溶液中。将得到的溶液在1％BSA与0.125％戊二醛。 </li><li>抛开蛋白基质SOLU和灰5分钟。该解决方案将出现在颜色浅黄色。 </li></ol></li><li> 制备所需的酶溶液（ 例如 ，谷氨酸盐氧化酶） <ol><li>通过添加过滤的DI水至冻干制备谷氨酸氧化酶溶液，纯化的酶（ 例如 ，添加到25个单位的谷氨酸氧化酶的50微升去离子水，得到0.5U /μL）。 注意:分装储液（ 例如 ，1微升），并在-20℃下，在适当大小的容器（ 例如 ，500个微升的微量离心管）的等分试样储存。等分试样可被存储在这些条件下长达6个月。 </li><li>添加BSA /戊二醛溶液含有谷氨酸氧化的离心管等分的4μL。混合通过人工搅拌的溶液（再吸移用10μL移液器。移液管应设置为体积&lt;5μL）。将得到的溶液在1％BSA，0.125％戊二醛和0.1U /μL谷氨酸氧化酶。使用该解决方案为立即涂层。 注意:谷氨酸氧化酶涂布液可以仅在15分钟内使用。虽然若干MEA可以与一个制备的溶液被涂覆，建议不超过谷氨酸氧化酶的可用时间窗口。可以在15分钟内进行涂覆的电极的数量而变化。一般地，4至6个电极可同时被涂覆。 </li></ol></li><li> 涂层MEA <ol><li>典型地，涂层的一对记录位点（S）上与所需的酶（谷氨酸氧化酶）一个MEA和一对不同的记录部位（一个或多个）（"前哨"位点（一个或多个））的涂有非活性蛋白基质。 </li><li>使用钝尖汉密尔顿microsyringes（例如，10微升）以涂覆的MEA与L-谷氨酸氧化酶或无活性蛋白基质溶液（BSA +戊二醛）。用于酶或无活性蛋白的基质涂层的步骤是相同的​​。 </li><li>使用前后清洁注射器（3个冲洗用DI水，冲洗3次用甲醇，5次漂洗用DI水）。 注:专用注射器用于施加酶（谷氨酸氧化酶）或蛋白质基质的涂层。 不要使用同一个注射器的不同的解决方案。建议的酶的涂层之前进行蛋白质基质的涂层。 </li><li>制定谷氨酸氧化酶或蛋白质基质用10μLHamilton注射器。轻轻按下柱塞在注射器尖端（使用解剖显微镜可视化）以分配的溶液中的小珠。 </li><li>使用解剖显微镜定位的MEA记录点，通过短暂地接触与溶液液滴/小珠，它代表一个层中的记录点的溶液应用到相应的记录点。三个层是足够实现该蛋白质 - 基质和酶的外套。厚的层可降低MEA的灵敏度。 </li><li>升高溶液滴直线上升和关闭录音的网站（ 即 syrinGE尖端不应跨越MEA表面刮去）。 </li><li>设定计时器1分钟。这是涂层应用到记录站点（多个）之间的最小时间要求。允许涂覆酶的MEA，在4℃下固化5-7天。 </li><li>记录应用大衣的数量，花了多长时间来应用涂料，电极数量和名称和日期在实验室笔记本。 </li></ol></li></ol> 2.电镀与间苯二胺为改善的选择性<ol><li> 制备间苯二胺二盐酸盐（MPD）溶液 注:板排斥层，的mPD，以防止多巴胺等大，干扰分子（ 例如 ，抗坏血酸）的氧化，从而提高了传感器，用于谷氨酸/ H 2 O 2过其他潜在-干扰分子的选择性。 <ol><li>在容量瓶中测量50毫升的0.05M磷酸盐缓冲盐水（PBS）和转移的0.05 50mL的的PBS至更大（150mL）中的锥形瓶中。 </li><li>使用连接到一个氮气罐（ 例如 ，使用管连接的喷射器的压力）的管路，附加一个移液管尖端（ 例如 P200尖端）到管道的开口端。放置用在PBS溶液中的附枪头管和覆盖用封口膜松散的溶液。打开氮气，轻轻泡液20分钟。 </li><li>权衡0.045克的mPD。 注意:主水平基准是一种潜在的致癌物质。的mPD称重应该使用包含在通风橱内的规模进行。使用主水平基准时，戴上手套和口罩。确保油烟机是功能和窗框被拉到合适的工作水平。可能已被污染的mPD立即清洁表面，以避免永久性染色。 </li><li>冒泡后，所有的MPD的转移到脱气PBS液。盖上开瓶封口膜，轻轻漩涡溶液溶解的mPD在溶液中。避免aggressivë混合或使用涡流的，因为这样做可能引入气体到溶液中。 </li><li>将溶液转移到一个50毫升烧杯中。搅拌棒，是没有必要的。 </li></ol></li><li> 电镀电极 <ol><li>获得参考电极。使用专用于只的mPD电镀参考电极。用于校准程序不同的参考电极。 </li><li>在烧杯中的参比电极保持器的塑料臂位置。检查在玻璃参考电极气泡（可能不能够看到）。轻轻拂去参考电极的远端处的尖端，以除去任何气泡。 </li><li>放置参考电极通过在塑料臂的开口和下部到含有PBS的烧杯中。确保参考电极不接触烧杯的底部。 </li><li>参考电极连接到探头（ 例如，2 PA / MV）和连接（ 例如 DIP连接器）的MEA为板的mPDd发送到探头。 </li><li>降低MEA放入烧杯溶液使得仅MEA尖端在溶液淹没。不超过所述黑色"泡沫"（黑色绝缘位于MEA尖端以上）液体浸没MEA提示。这样做可能会损坏MEA。 </li><li>运行"测试"校准验证电极通道和连接到探头的功能。 注:虽然硬件设置应体现设备正在使用，涉及到被分析物/干扰类型和浓度细节没有为"测试"校准输入。再编码模式应该是"电流分析法"和"V-施加"应该是-0.7V。验证的典型记录对于所有信道。 </li><li>通过选择"取消"在所有的记录点的连接进行验证取消。这是没有必要保存校准数据。 </li><li>选择桌面上的"电镀"图标。电镀要OL菜单会出现。确认被输入了正确的设置: PP振幅:0.25 偏移:-0.5 频率:0.05 持续时间（分钟）:20 </li><li>选择"暂停"按钮，开始电镀。已用时间字段应该开始倒计时。 </li><li>完成后（所有经过的时间），选择在弹出菜单中的"另一个MEA"开始的mPD电镀附加的MEA。更换待镀的mPD与MEA镀MEA和使用电镀工具重复电镀过程。 注:建议使用不超过2倍或1个小时的时间内将制备的mPD的解决方案。准备一个新的解决方案，准备2周以上的多边环境协定。 </li><li>选择"退出软件"来完成电镀。校准之前冲洗用DI水（以避免盐残基）和存储（24小时）的mPD镀电极尖端。 </li><li>从溶液中去除所述参考电极和放置到适当的S前用DI水冲洗torage溶液（ 例如 3M NaCl的）。确保参考电极尖端被浸没在溶液中。慢慢下玻璃参比电极到该溶液中，以避免损坏玻璃。 </li><li>处置在适当标记的有害废物容器中的mPD溶液。 </li></ol></li></ol> 3.校准为谷氨酸检测和选择性的MEA（图1）21 <ol><li>准备需要校准的解决方案。 注:请参阅表1。 </li><li>在恒定搅拌下进行校准，在37°C（磁性电池操作的搅拌器）。打开加热垫或循环水浴，将获得一个小的搅拌棒。慢慢搅拌，但速度不够快，当除了由（如下图），很快达到了新的高度。 </li><li>的探头（2 PA /毫伏）连接到记录控制系统，并插入MEA尖成40毫升搅拌的0.05M的PBS（使用50毫升的烧杯中）。 </li><li>连接Reference电极。弗里克结束，以确保有没有气泡。 </li><li>打开记录程序，并单击"校准"。确保设置正确（检查哨点选择，因为这可能与不同的电极改变），选择MEA号码并按下"开始"。 </li><li>允许5 - 10分钟以达到平衡;开始一旦基线已经稳定（&lt;0.004 NA /分钟变化）。 </li><li>选择"基线"，并添加500 20μL1mM抗坏血酸（干扰;终[250μM]）并选择"干扰物"（AA被认为是脑宽干扰）一旦电流达到了新的，稳定的平台期，如果任何。允许0.5 - 加入之间1分钟。如果MEA正常电镀，几乎没有变化应得到遵守。 </li><li>添加20mM的L-谷氨酸（最终[20μM]）的40μL，并且一旦电流达到一个新的，稳定的高原，标记1 次加法"分析物"。重复三次，总共3 glutamat的Ë补充。 </li><li>加入40微升DA。 不要选择 "分析物";选择"测试物质" - DA可能只包含DA方面的干扰。 </li><li>加入40微升过氧化物。 不要选择 "分析物";选择"测试物质"。 </li><li>一旦校准完成后单击"停止"按钮。 </li><li>为了确定MEA的功能，单击计算选项卡，并记录在实验室笔记本下面的参数。 <ol><li>在这些校准中使用的MEA的设计（3000微米2），使用MEA与≥0.004 NA /μM的斜率，但较低的斜坡可用于某些实验。如果有小于在酶包衣和未包衣站点之间斜率的10％的差异，然后利用这些的MEA用于诱发释放仅或清洁/丢弃该MEA。 </li><li>使用检测（LOD）的限制≤1.5微米。如果LOD大于1.5微米时，则使用这些多边环境协定的诱发release和只摄取。不要使用这些电极记录进补水平。 </li><li>使用的选择性超过&gt; 20个任意单位的干扰的期望的分析物（ 即 ，谷氨酸）。如果选择性小于20，则清洁/丢弃MEA。 </li><li>使用≥0.90的校准曲线的线性度（R 2）。如果线性度小于0.90，则清洁或丢弃MEA。 </li></ol></li><li>保存与MEA，日期＃文件，和人的校准缩写。 </li></ol> 4.组装的微注:微量移液器（毛细管玻璃）应具有的尖端具有10的内径 - 15微米。 <ol><li>将微量集中所有的四个铂记录点之间，并使用粘蜡和橡皮泥装50-100微米以上的MEA。 </li><li>加载微量，使用1mL注射器填充有谷氨酸或KCl溶液，用0.22μm的无菌注射器过滤接r和97姆·隆恩，28号移液管填料，回填微量。也就是说，插入针在微管的顶部和微量（朝MEA结束）的底部填充，确保没有气泡。 </li><li>附加微量到压力喷射系统在2 - 20psi下约1秒。 </li></ol> 5.板的微型参比电极在体内使用<ol><li>制备电镀液（50克NaCl / 90毫升的1摩尔HCl的100mL烧杯（镀浴））并切断浴电极（〜铂的10cm长（Pt）的金属丝（〜直径0.02&quot; ））。 </li><li>切断10 - 15cm长片Teflon涂层的银线（0.008&quot; 裸）的，并使用刀片从银线的端部的每一端刮掉约1cm聚四氟乙烯涂层的。 </li><li>焊接在一端具有金引脚和裸露的银导线的一端放入镀浴。 </li><li>使用DC适配器（仅使用具有〜9的输出端的DC适配器 - 15 VDC最大），夹紧"红"（+）上的金销侧线到制备的银线（参比电极）。夹住"黑"（ - ）导线铂浴阴极。 </li><li>插上DC适配器。寻找正确的电镀工艺 - 泡沫应该出现在浴电极;参考电极变成银/灰色。 注意:不要开关引线部分黑色（ - ）对红（+） -镀液会发生这种情况时和新鲜的电镀液需要进行毁了。一个坏的解决方案将在颜色变黄: 不要使用！ 注:电镀反应进行2-5分钟通常:AG 0 + Cl -的氯化银→+ E - </li><li>测试参考电极。 <ol><li>设置一个万用表到100 - 200 mV的DC设置。 </li><li>放置一个基准（即，已知的可正常工作的先前测试的电极）在3M NaCl的参比电极和测试对新镀参考电极。 注意:如果我们荷兰国际集团一个新的参考电极，浸泡在3M NaCl的使用前（12个小时推荐）。 </li><li>放置" 红 "（+）引线的参考电极的金销上进行测试。将" 黑 "（ - ）对基准电极引线。可接受的读出范围为±10毫伏。 0毫伏是跨越2个参考电极理想。 </li></ol></li></ol> 6.一般动物手术治疗MEA录音<ol><li> 准备手术 <ol><li>前一天晚上将手术工具消毒。 </li><li>从消毒液中取出工具，彻底冲洗后放入工具，在无菌手术垫。获得一个加热垫。 </li><li>校准两个电极和作为程序3和4所述连接微量。 </li><li>使参比电极如第5中描述。 </li><li>让200μM谷氨酸和70毫米氯化钾。请参阅表1。 </li><li>获取动物并记录手术床单它的重量。 </li><li>准备麻醉并打开加热垫。 </li></ol></li><li> 执行MEA手术 <ol><li>使用异氟烷（在氧4％的流量），麻醉动物在感应室，直到所述呼吸速率已显著下降，动物不到脚趾或尾巴捏响应。记录呼吸频率和体温，每15分钟。 </li><li>使用耳酒吧稳定头放置动物的立体定位装置。确保动物的安全性和头部不动。降低异氟烷流动到1.5 - 在氧为3％。确保加热垫被适当地定位在动物和下设定为37℃，以提供补充的热量。未能提供补充热量可能导致深低温和动物的过早死亡。 </li><li>用消毒棉签应用眼膏，并记录呼吸频率和体温。使用电池供电的修剪器，刮头顶。使用小型手术剪，除去耳朵附近的皮毛。 </li><li>在这一点上，戴上无菌手套手术。应用碘再用酒精（三次）到头皮。 </li><li>使用手术刀，做一个切口直降头皮的中间，用斗牛犬夹波及皮肤。以无菌棉签来吸收任何血液。使用过氧化氢以促进前囟和lambda的外观。 </li><li>检查头被适当地定位，通过测量D / V和前囟和lambda的M / L坐标。坐标的变化应该是零，如果头是在一个平面。调整头部和耳朵酒吧，既要保证一个平面。 </li><li>查找前囟和零坐标。使用所需的坐标，移动到左侧和右侧，并用永久性记号笔做一个记号。使用cauterizer /标记，使周围的标志的大广场有足够的空间来达到所希望的区域。 </li><li>使用无菌旋转刀具，钻头周围的方形标记。用消毒棉签吸收任何血液。彻底消毒钻头在每次使用前。 </li><li>附加MEA到探头和回填与所述第一期望的溶液移液管。请务必留在吸管差距不解决方案，使被驱逐的解决方案可以参考一下。连接管的玻璃微。 </li><li>打开氮气罐和压力喷射器（PSI = 5，时间= 0.6秒）。测试（按手动红色按钮），以确保吸管开始之前没有堵塞。 </li><li>校准微量。从0开始到刻线，并放置了一块蓝色胶带上的探头，以表明启动点。打开数字阅读器，并将其重置为零。下井1毫米（DV）和计数蜱该解决方案已在光罩移动的数目。 10个蜱应等于1毫米（1 mm相当于250 nL）的，所以1个滴答= 25纳升。 </li><li>放置在一个参比电极从MEA远程位置，使得它仍然在与大脑以完成电路液体接触。 </li><li>查找前囟门附带的MEA和零的数字阅读器。移动MEA到所需的坐标。慢慢放下MEA直到尖端接触大脑。零的DV协调，慢慢放下MEA进入大脑。 </li><li>在记录程序中，点击所需的校准的电极，然后点击"执行实验。"然后，系统将开始录制的目标分析物进补水平，而动物麻醉。 </li><li>放大到一个轴，这将在观察基线帮助和允许电极与基线20 - 45分钟。 </li><li>基线稳定之后，喷射器压力设定为0.6秒和5磅，然后按上的压力喷射器喷射的红色按钮。记录移动的喷射片（ 图2）上的时间和压力以及体积/蜱。做任何必要的票据（ 即较大峰值，稀释效应，堵塞的移液管，嘈杂基线/邻范围）。 </li><li>谷氨酸注射，等待3 - 注射之间5分钟。对于氯化钾注射等待1 - 注射之间2分钟。注射使用相同的压力和时间的至少3倍。更改时间和重复。尝试除非微量堵塞不改变的压力。 </li><li>如果微管堵塞，增加压力可达30磅。 </li><li>从期望的大脑区域的记录，重复使用不同的解决方案的大脑的两侧。记录在每一个新的区域的基线20分钟。 </li><li>采取与微管的MEA连接，用去离子水冲洗，并在PBS浸泡一夜，直到所有的血就没了。 </li><li>安乐死动物并保存脑在预标记的管和储存在-80℃冰箱或保持一侧，用于固定。安乐死的动物过量异氟醚（吸入）。用手术剪刀执行斩首并剖析了期望的区域。</li></ol></li></ol> 7.清洁涂层多边环境协定后使用<ol><li>不干净的多边环境协议，如果不使用。如果有棉绒或灰尘的表面上，清洁用甲醇和让干燥涂布前24个小时。 </li><li>放水浴（80℃）浸泡在MEA的尖端30分钟。小心地擦拭用棉花在电极的尖端涂药器以去除可能在电极尖端留下任何碎屑。不要让MEA湿的黑"绝缘部分"。 </li><li>使用三种不同的超声波仪，浸泡在（1）Citrisolv，商业溶剂和清除剂（2）异丙基MEA的尖端，以及（3）去离子水5分钟。小心地擦拭用棉花电极的尖端涂药器以去除可能在电极尖端留下任何碎屑。 </li><li>检查解剖显微镜下的提示，以确保该层已被成功删除。 </li></ol> 8.分析<ol><li>对数据进行分析，首先通过双击完成的实验，然后从文件下拉菜单中选择"导出数据"导出所需的实验。 </li><li>这个数据保存到所需的文件位置和开拓分析程序。单击分析程序"打开"，然后选择刚刚保存的文件。 </li><li>给节目几分钟上传的文件，然后将该事件标记标签下的实验。下输出，检查盒所需的参数。例如，基线，振幅，（曲线下面积）的峰面积，T 上升 ，和T 80。 </li><li>点击刷新，然后进行分析，将数据导出到电子表格中（这可能需要几分钟）。该程序会提示将文件保存到所需位置。一旦保存，该文件可以在电子表格进行编辑。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+the+glutamate+clearance+time+course+from+transporter+currents+in+CA1+hippocampal+astrocytes:+transmitter+uptake+gets+faster+during+development.">Deriving the glutamate clearance time course from transporter currents in CA1 hippocampal astrocytes: transmitter uptake gets faster during development.</a> J Neurosci. 25 (11), 2906-2916 (2005).</li><li>Greene, J. G., Borges, K., Dingledine, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+transcriptional+neuroanatomy+of+the+rat+hippocampus:+evidence+for+wide-ranging,+pathway-specific+heterogeneity+among+three+principal+cell+layers.">Quantitative transcriptional neuroanatomy of the rat hippocampus: evidence for wide-ranging, pathway-specific heterogeneity among three principal cell layers.</a> Hippocampus. 19 (3), 253-264 (2009).</li><li>Borland, L. M., Shi, G., Yang, H., Michael, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltammetric+study+of+extracellular+dopamine+near+microdialysis+probes+acutely+implanted+in+the+striatum+of+the+anesthetized+rat.">Voltammetric study of extracellular dopamine near microdialysis probes acutely implanted in the striatum of the anesthetized rat.</a> J Neurosci Methods. 146 (2), 149-158 (2005).</li><li>Jaquins-Gerstl, A., Michael, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+brain+penetration+injury+associated+with+microdialysis+and+voltammetry.">Comparison of the brain penetration injury associated with microdialysis and voltammetry.</a> J Neurosci Methods. 183 (2), 127-135 (2009).</li><li>Azbill, R. D., Mu, X., Springer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riluzole+increases+high-affinity+glutamate+uptake+in+rat+spinal+cord+synaptosomes.">Riluzole increases high-affinity glutamate uptake in rat spinal cord synaptosomes.</a> Brain Res. 871 (2), 175-180 (2000).</li><li>Gourley, S. L., Espitia, J. W., Sanacora, G., Taylor, 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=Antidepressant-like+properties+of+oral+riluzole+and+utility+of+incentive+disengagement+models+of+depression+in+mice.">Antidepressant-like properties of oral riluzole and utility of incentive disengagement models of depression in mice.</a> Psychopharmacology (Berl). 219 (3), 805-814 (2011).</li><li>Frizzo, M. E., Dall'Onder, L. P., Dalcin, K. B., Souza, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riluzole+enhances+glutamate+uptake+in+rat+astrocyte+cultures).">Riluzole enhances glutamate uptake in rat astrocyte cultures).</a> Cell Mol Neurobiol. 24 (1), 123-128 (2004).</li><li>Fumagalli, E., Funicello, M., Rauen, T., Gobbi, M., Mennini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riluzole+enhances+the+activity+of+glutamate+transporters+GLAST,+GLT1+and+EAAC1.">Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1.</a> Eur J Pharmacol. 578 (2-3), 171-176 (2008).</li><li>Day, B. K., Pomerleau, F., Burmeister, J. J., Huettl, P., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrode+array+studies+of+basal+and+potassium-evoked+release+of+L-glutamate+in+the+anesthetized+rat+brain.">Microelectrode array studies of basal and potassium-evoked release of L-glutamate in the anesthetized rat brain.</a> J Neurochem. 96 (6), 1626-1635 (2006).</li><li>Kane, R. L., Martinez-Lopez, I., DeJoseph, M. R., Vina, J. R., Hawkins, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na(+)-dependent+glutamate+transporters+EAAT1,+EAAT2,+and+EAAT3)+of+the+blood-brain+barrier.">Na(+)-dependent glutamate transporters EAAT1, EAAT2, and EAAT3) of the blood-brain barrier.</a> J Biol Chem. 274 (45), 31891-31895 (1999).</li><li>Ramsden, 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=Age-dependent+neurofibrillary+tangle+formation,+neuron+loss,+and+memory+impairment+in+a+mouse+model+of+human+tauopathy+(P301L).">Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human tauopathy (P301L).</a> J Neurosci. 25 (46), 10637-10647 (2005).</li><li>Oddo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triple-transgenic+model+of+Alzheimer's+disease+with+plaques+and+tangles:+intracellular+Abeta+and+synaptic+dysfunction.">Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction.</a> Neuron. 39 (3), 409-421 (2003).</li><li>Zang, D. W., 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=Magnetic+resonance+imaging+reveals+neuronal+degeneration+in+the+brainstem+of+the+superoxide+dismutase+1+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis.">Magnetic resonance imaging reveals neuronal degeneration in the brainstem of the superoxide dismutase 1 transgenic mouse model of amyotrophic lateral sclerosis.</a> Eur J Neurosci. 20 (7), 1745-1751 (2004).</li></ol>

GG是Quanteon，LLC，使得用于这项研究谷氨酸测量的FAST-16录音系统独资经营。 JEQ是Quanteon付费顾问。

所述MEA技术允许在体外和体内神经递质的释放和摄取的快速动力学的测量。因此，该技术产生了各种各样的数据输出包括补品神经递质水平，诱发神经递质释放，和神经递质的间隙。然而，由于采用多边环境协定是一个相对复杂的过程，有可能需要为成功使用进行优化许多因素。例如，在校准期间，人们可能注意到，不存在的信号波形（示波器屏幕），或者对刺激的响?...

在大脑中的神经递质测量改变为神经科学家研究中枢神经系统（CNS），其通常的特征在于神经递质失调的疾病的重要工具。虽然微透析在用高压液相色谱法（HPLC / EC）组合已经以测量细胞外神经递质水平1，2，变化的最广泛使用的方法3， 图4，微透析探针的空间和时间分辨率可能不是理想的神经递质，如谷氨酸，被在细胞外空间5,6紧密调节。因为在遗传学和成像的最新进展，存在可用于在体内映射谷氨酸的其他方法。使用基因编码谷氨酸荧光记者（iGluSnFR）的ð双光子成像，研究人员能够通过在体外和体内 7，8，9的神经元和星形胶质细胞可视化的谷氨酸释放。值得注意的是，这允许从更大的视场记录和不破坏大脑的内在联系。尽管这些新的光学技术允许谷氨酸动力学和感官诱发反应和神经元活性的测量可视化，它们缺乏量化离散脑区域外空间的谷氨酸的量的能力。 另一种方法是酶联微电极阵列（MEA），其可以选择性地测量细胞外神经递质水平，如谷氨酸盐，通过使用自参考记录方案。该MEA技术已用于研究创伤性脑细胞外谷氨酸的变化损伤10，11，12，老化13，14，应力15，16，癫痫17，18，阿尔茨海默氏病19，20，和病毒模拟物21的注射和代表了在微透析中固有的空间和时间限制的改进。而微透析限制测量突触22，23附近的能力，具有的MEA具有高的空间分辨率，其允许细胞外谷氨酸溢出的选择性措施附近突触24,25。第二，微透析的低时间分辨率（1 - 20分钟）限制了调查的能力谷氨酸释放和清除的快速动力学在毫秒发生的第二范围26。因为谷氨酸的释放或间隙差异补品的措施，休息谷氨酸水平可能并不明显，这可能是必要的谷氨酸释放和间隙直接测量。 MEA的允许这样的措施，由于其高的时间分辨率（2赫兹）和检测的下限（&lt;1微米）。第三，允许的MEA用于特定脑区域内神经递质分区域变型中，如大鼠或小鼠海马的检查。例如，使用的MEA我们可以分别针对齿状脑回（DG），海马，它们经由trisynaptic电路27连接的大角ammonis 3（CA3）以及大角ammonis 1（CA1），以检查细胞外谷氨酸的分区域的差异。造成通过植入28 &lt; - （4毫米的长度1）和损坏，因为微透析探针的大小的/ SUP&gt;，29，分区域的差异是难以解决。此外，光学系统仅允许通过刺激的外部刺激，例如晶须刺激或光的闪烁，这不允许分区域的刺激7。比其他方法的多边环境协定的最后一个好处是研究在体内这些次区域没有打乱他们的外在和内在联系的能力。 在这里，我们描述了如何在组合的记录系统（ 例如 ，FAST16mkIII）用的MEA，其由基于陶瓷的多点微电极，可以有差别地涂覆在记录位点以允许干扰试剂要被检测和从分析物信号中除去。我们还证明这些阵列可用于麻醉RTG的CA1海马亚区的DG，CA3内体内谷氨酸调节基于电流分析法研究，和（TauP301L）4510只小鼠中，通常使用的米阿尔茨海默病的乌斯模型。此外，我们通过用利鲁唑小鼠提供MEA系统对谷氨酸释放和清除的快速动力学的敏感性的确认， 在体外所示的药物以减少谷氨酸释放和增加谷氨酸盐摄取30，31，32，33，并展示在 TauP301L小鼠模型中的体内这些相应的改变。

<table><tbody><tr><td>FAST-16mkIII-8 channel</td><td>Quanteon</td><td>16mkIII</td><td></td></tr><tr><td>Microelectrode arrays</td><td>CenMet</td><td>W4 or 8-TRK</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A-3059</td><td>10 g (expires after 1 month)</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G-6257</td><td>100 mL (expires after 6 months)</td></tr><tr><td>Glutamate Oxidase</td><td>US Biological or Sigma Aldrich</td><td>G4001-01 or 100646</td><td>50 UI (expires after 6 months)</td></tr><tr><td>Hamilton Syringes</td><td>Hamilton</td><td>#80383</td><td>2 syringes</td></tr><tr><td>Methanol</td><td>BDH</td><td>UN1230</td><td>4 L</td></tr><tr><td>m-Phenylenediamine dihydrochloride (mPD)</td><td>ACROS Organics</td><td>1330560250</td><td>25 g</td></tr><tr><td>Reference Electrodes (RE-5B)</td><td>BAS</td><td>MF-2079</td><td>3 electrodes</td></tr><tr><td>Battery-powered Magnetic stir plate</td><td>Cole-parmer</td><td>EW-04804-01</td><td>Can purchase from different supplier</td></tr><tr><td>Glutamate</td><td>Sigma-Aldrich</td><td>G-1626</td><td>100 g</td></tr><tr><td>Ascorbic Acid</td><td>TCI</td><td>50-81-7</td><td>500 g</td></tr><tr><td>Dopamine Hydrochloride</td><td>Alfa Aesar</td><td>62-31-7</td><td>5 g</td></tr><tr><td>Perchloric acid</td><td>VWR</td><td>UN2920</td><td>500 mL</td></tr><tr><td>Postassium chloride</td><td>VWR</td><td>7447-40-7</td><td>1 kg</td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>7647-40-7</td><td>1 kg</td></tr><tr><td>Calcium Chloride</td><td>MP</td><td>153502</td><td>100 g</td></tr><tr><td>Sodium Hydroxide</td><td>BDH</td><td>1310732</td><td>500 g</td></tr><tr><td>Glass pressure ejection pipettes</td><td>CenMet</td><td></td><td></td></tr><tr><td>Sticky wax</td><td>Kerrlab</td><td>625</td><td>Can purchase from different supplier</td></tr><tr><td>Microsyringe</td><td>World Precision Instruments</td><td>MF28G-5</td><td></td></tr><tr><td>Modeling clay</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Picospritzer III</td><td>Parker</td><td></td><td></td></tr><tr><td>Silver wire</td><td>AM systems</td><td>#786500</td><td></td></tr><tr><td>Hydrochloric acid</td><td>BDH</td><td>7647010</td><td>2.5 L</td></tr><tr><td>Platinum wire</td><td>AM Systems</td><td>778000</td><td></td></tr><tr><td>Solder gun</td><td>Lowes or Home Depot</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Multimeter</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>PhysioSuite</td><td>Kent Scientific</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>SomnoSuite</td><td>Kent Scientific</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Stereotaxic device</td><td>Stoelting</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Digital Lab Standard</td><td>Stoelting</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Meiji EMZ microscope</td><td>Meiji</td><td>EMZ-5</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>Micro</td><td></td></tr><tr><td>Metricide</td><td>Metrex</td><td>102800</td><td></td></tr><tr><td>Scalpel</td><td>VWR</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Surgery scissors</td><td>VWR</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Sterile cotton swabs</td><td>Puritan</td><td>25806</td><td>Can purchase from different supplier</td></tr><tr><td>Eye ointment</td><td>Puralube Vet Ointment</td><td></td><td>Obtain from the vet</td></tr><tr><td>Iodine swabs</td><td>VWR</td><td>S48050</td><td>Can purchase from different supplier</td></tr><tr><td>Alcohol swabs</td><td>Local drug store</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Sterile surgery drape</td><td>Dynarex</td><td>4410</td><td>Can purchase from different supplier</td></tr><tr><td>Sterile saline</td><td>Teknova</td><td>S5815</td><td>Can make own soltuion using filters</td></tr><tr><td>Hydrogen Peroxide (3%)</td><td>Local drug store</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Heating Pad</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr></tbody></table>

using enzyme-based biosensors to measure tonic and phasic glutamate in alzheimer's mouse models

神经递质的破坏往往是中枢神经系统（CNS）疾病的一个重要组成部分，发挥着病理阿尔茨海默氏症，帕金森氏症，抑郁症和焦虑潜在的作用。传统上，微透析一直是最常见的（赞美）技术检测发生在这些疾病中神经递质的变化。但由于微透析具有测量跨组织的大面积慢1-20分钟变化的能力，它具有侵袭性的缺点，可能会破坏大脑和慢速采样能力范围内的内在联系。一个相对较新的技术中，微电极阵列（MEA），具有用于所发生的测量的离散的大脑区域中的特定神经递质的变化，使得用于空间和时间上精确的方法的许多优点。此外，使用的MEA是微创，允许体内的神经递质的改变的测量。在我们的实验室，我们哈去过在神经递质谷氨酸，与阿尔茨海默氏病的病理变化特别感兴趣。这样，这里所描述的方法已经被用于评估在阿尔茨海默病的转基因小鼠模型中的潜在谷氨酸海马中断。简言之，该方法涉及使用涂布用酶对感兴趣，并使用自参照位点以减去背景噪声和干扰神经递质非常有选择性的多站点微电极。电镀和校准之后，可以MEA用微量被构造和使用立体定位设备下降到感兴趣的大脑区域。在此，所描述的方法涉及麻醉RTG（TauP301L）4510只小鼠和使用立体定位装置精确地定位子区域海马（DG，CA1，CA3和）。

虽然这种技术可用于测量谷氨酸的改变在许多类型的动物模型，如创伤性脑损伤，衰老，压力和癫痫的信号，在这里我们展示了MEA技术如何可以用于检查转基因小鼠模型的谷氨酸改变的人tau蛋白病19,20。 RTG的（TauP301L）4510小鼠表达与额颞叶痴呆和帕金森综合征与染色体17相关的tau蛋白突变P301L，和通常用来研究与阿尔茨海默氏病?...

Watch this Scientific Journal Video about Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models at JoVE.com

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

在这里，我们描述了用于测量补药和采用酶联微电极阵列（MEA） 在体内阶段性细胞外谷氨酸的变化的空间和时间上精确的方法的设置，软件导航，和数据分析。

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

using-enzyme-based-biosensors-to-measure-tonic-phasic-glutamate

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.2K 次观看.  West Virginia University. 在这里，我们描述了用于测量补药和采用酶联微电极阵列（MEA） 在体内阶段性细胞外谷氨酸的变化的空间和时间上精确的方法的设置，软件导航，和数据分析。 

视频: Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

一个串行收集脑脊液从池鼠标麦格纳技术

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

科研

生物学

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

雌性叙利亚仓鼠自然奖赏行为中细胞外神经递质的测量体内变化

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

神经科学

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

医学

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

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 ...

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

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