这项工作是由美国心脏协会（博士后奖学金AYS）和国立卫生研究院（MH085499，EB003832，并OD006831 DK）的支持。我们感谢贝思·弗里德曼和巴勃罗·布林德手稿上的评论。

1。准备手术， 我 <ol><li>清洁及手术Maxizyme超声波清洗机的牛奶混合物中超声分散的手术工具。高压灭菌之前，每个实验的外科手术工具。 </li><li>确保所有必要的试剂和耗材可用。表2中提供的试剂和耗材清单。试剂和耗材，在接触到暴露组织应该是无菌的，在可能的情况下。 </li><li>诱导麻醉。表1描述了适合生存研究的典型麻醉药。为缺乏脚趾掐反射的检查，以确保手术的麻醉平面。鼠标的最佳年龄是3至6周龄。年轻小鼠的头骨更柔软，更困难的要薄。年纪较大的老鼠有较厚的头骨，在细化过程中，会流血。 </li><li>固定在一个立体的第1帧的动物。表2中提供手术设备清单。 </li><li>维持身体温度TURE在37°C使用一个反馈调节直肠探头和热垫。 </li><li>眼药膏的眼睛，以保持水分。 </li><li>一个小的电动剃须刀刮胡子的头皮。 </li><li>优碘，70％（V / V）异丙醇擦拭清洁头皮。 </li><li>如果需要，检查心脏和呼吸率正常使用脉搏血氧饱和度的范围内。鼠标，这些数字应该围绕10，分别为2赫兹。 </li><li>暖取无菌过滤的人工脑脊髓液（学联）37℃（125毫米氯化钠，葡萄糖10毫米，10毫米肝素钠， 氯化钙 3.1毫米，1.3毫米氯化镁2，pH值7.4）（从Sigma的所有化学品） 2。 </li></ol> 2。安装一个头架<ol><li>一双镊子和手术剪，删除整个背头骨表面的头皮。修剪皮肤的头骨和后两侧颞肌边缘横向erior到颈部肌肉（ 图1A）。 </li><li>使用手术刀叶片去除颅骨表面薄薄的骨膜。 </li><li>清理用潮湿的棉花头骨放倒撒施，干燥的空气流从灰尘可以颅骨表面。然后应用到整个头骨表面的氰基丙烯酸酯胶的薄层。让胶水彻底干燥。这层胶需要适当的坚持在后续步骤中的牙科水泥。氰基丙烯酸酯胶，不需要进行消毒。 </li><li>附加一个金属连接器的颅骨表面，离所需的窗口面积。对于麻醉的准备，一个小螺母可固定的颅骨上，民建联的氰基丙烯酸酯胶，以后可以成像设置（ 图1B，1L和1N）使用螺栓拧。螺母背面用胶带密封，以确保胶不进入线程，在附件的头骨。螺母和磁带应消毒<html>到手术前灭菌。 </li><li>对于清醒的成像准备，有两个附着点（ 图1M及1N）附加一个更加刚性的自定义连接器。对侧半球皮质与一个半毫米钻孔毛刺钻两个洞的头骨，然后引入两个＃000自攻螺钉。这些螺丝将有助于锚头安装到头骨。适用于只有一个螺丝的全面转向，以避免底层皮质施加压力。在小脑的小民建联的氰基丙烯酸酯胶，然后，将自定义的金属横栏。允许的氰基丙烯酸酯胶彻底晾干。这种金属连接器，大大降低了自由度，并简化了在相同的成像纵向研究领域的搬迁。宽的横栏提供了充足的空间电极安置和触须的刺激。生成自定义的头部安装及附件设备的详细计划，可在网上（SICS / links.html“&gt; http://physics.ucsd.edu/neurophysics/links.html）。螺钉和横栏应消毒灭菌手术前。 </li><li>覆盖整个颅骨表面，不包括窗口的位置，用牙科水泥层，（ 图1C和1D）。确保所有暴露的皮肤边缘，被水泥覆盖。牙科水泥组件不需要进行消毒。 </li></ol> 3。抛光的生成和增强减薄的头骨（端口）“窗口” <ol><li>确保钻孔毛刺是雪亮的，并避免重复使用它们。使用低速牙钻，一个半毫米毛刺地区体感皮层2毫米薄2毫米。湿冷却，疏干;润湿与学联的头骨，然后一个温柔的空气流气体除尘器干燥颅骨表面之间的替代。这就需要通过松的头骨，这可能会流血层变薄，但可以治理工程ED冲洗学联（ 图1E）。头骨开始弯曲轻微的压力下钻时〜50微米，应该通过湿骨（ 图1F）可见软脑膜血管。在此厚度，骨内的白色小点，将成为几秒钟后立即蘸干颅骨表面可见。 </li><li>在这一点上，瘦骨，甚至进一步。使用钻速度较慢，即每分钟1000转，刮胡子颅骨表面只有轻微的触摸。使用小的控制动作，而像一支钢笔钻，只适用于在横向方向的力量。骨应在其最终厚度（ 图1G）...... 10至15微米。当足够薄，骨是骨的小白斑将不再可见时蘸干颅骨表面。 </li><li>擦亮窗口区域与氧化锡粉。附加一个预制的钻头已在硅水族馆密封胶和蘸画，留下一个锥形鞭（ 插图，图1H）。硅鞭应在手术前准备至少一天，有70％的异丙醇，使用前消毒。学联（ 图1H）下降的窗口上放置一小捏粉。鼓动长达十几分钟的泥浆在窗户上轻轻抚摸头骨表面移动鞭尖。彻底冲掉锡氧化物粉末，从使用学联的窗口和干骨彻底温柔的空气流。在前面的步骤进行钻探留下的不规则表面和壁骨片应拆除抛光后（ 图1H和1I）。锡氧化物粉末，不需要进行消毒。 </li><li>切不见方的小块。 0盖玻片略高于窗口的大小要小得多。使用文士轻轻地使用直边盖滑得分分离的水平和垂直线。然后将其放置在培养皿和KN盖玻片玉珠对桌子边缘的菜分开的玻璃碎片。然后可以在整个培养皿灭菌，确保无菌。 </li><li>将适当大小的玻璃盖附近的干的窗口。应用使用一个破碎的木制棉棒撒施尖端的窗口小民建联氰基丙烯酸酯胶，并迅速推向胶水预切一块玻璃盖之上。避免产生气泡的玻璃盖下面。对头骨表面的玻璃盖轻轻一推，并保持几秒钟（ 图1J和1O）。允许胶彻底晾干后超过15分钟。可以去掉多余的氰基丙烯酸酯胶，干燥后用手术刀刀片从表面上的玻璃盖。用牙科水泥密封玻璃盖的边缘形成一个略有提高，以及举办水浸渍镜头（ 图1K和1O）。 </li></ol> 4。复苏<ol><li>放置在一个温暖的笼子手术后的动物。监察动物定期，直到它完全从麻醉中恢复。 </li><li>如果超过一天的准备工作是为了生存，提供镇痛丁丙诺啡（0.03微克每克灭鼠）。我们通常形象的初始植入后动物至少一天。 </li></ol> 5。成像的制备<ol><li>稳定的动物成像的光学线路板，用头支持（ 图1L及1M）的框架。限制装置，可从从Qioptiq或ThorLabs商业可用光学机械部件。我们的独立板可运送手术和双光子成像与动物和所有生理监测设备组装为一个单位，3套房。 </li><li>如果所需的血流成像，注入0.05毫升的5％（W / V），荧光葡聚糖染料溶解在无菌生理盐水中，通过尾静脉或眶下静脉标记血清（ 图2A和2C至2H </strong&gt;）4-7。这必须在全身麻醉下完成。眶下静脉注射可能会更容易，对于初学者来说，葡聚糖解决方案是更粘稠。尾静脉难以找到深色小鼠，静脉趋于崩溃失败后的注射。成像血管与绿色发光，用异硫氰酸荧光素葡聚糖。为红光发射，使用德州红葡聚糖。超高分子量右旋糖酐，染料将继续留在流通了好几个小时。另外，与内源性荧光标记的动物，可以直接在适当的双光子激发波长成像。右旋糖酐液可以冻结等分，以供将来使用。 </li><li>潮湿的棉布轻轻擦拭窗口表面倾斜涂药。 </li><li>麻醉准备长期成像，体积3μL注入无菌乳酸林格氏液腹腔每克每2个小时，以维持体液和能源需求。/ LI&gt; <li>当成像在清醒状态的动物，只在几个小时的时间限制头枕，以减轻压力水平。返回动物的食物和水成像会话之间的家庭笼。 </li></ol> 6。代表结果一个成功的窗口将允许成像深度可达250微米以下脑膜表面几个月。使用该方法已在体内毛细血管血流量4，8，小胶质细胞活化8，9，和树突状结构的研究，在皮层薄壁8。举一个例子，我们使用双光子成像显示麻醉Thy1系黄色荧光蛋白（YFP的）鼠标皮质血管后，血清标记是由得克萨斯红葡聚糖（ 图2A）静脉注射。硬脑膜血管往往略高于可见硬脑膜皮质表面（ 图2C，箭头 ）。大脑膜动脉和静脉李皮质表面E（图2D）。从这个表面网络和潜水到皮质，他们ramify成一个密集的毛细血管床，饲料皮层组织（ 图2E 2H）穿透血管分支。深表达YFP的皮层神经元，这鼠标线信号内源性的树突状乔木，同时在第二个通道10（ 图2B，2H），可成像。骨的第二谐波信号被收集在第三个通道，可以用来衡量厚度减薄后的图像栈收集头骨（ 图2A 2C）。 皮质血管动力学正在深刻地影响麻醉药11。在第二个例子中，我们展示了双光子显微镜收集从习清醒鼠标自发vasoactivity视频。突出管腔直径的血管收缩振荡与脑膜动脉，但不与邻近小静脉。这减少乌拉坦麻醉4基底范围vasoactivity的。量化自发和诱发血流的变化，我们使用改编线扫描技术，捕捉血管直径和红细胞速度，个别船只。定量血流成像，利用双光子显微镜的详细资源，3，12。 <img alt="图1" src="/files/ftp_upload/3742/3742fig1.jpg" /> 图1。一个端口“窗口中的程序。 （A至K）连续过程中的步骤生成一个端口“窗口形象。看到详细的说明文字。 β=前囟门和λ=拉姆达。 （L）的系统，确保在麻醉准备成像头螺栓和螺母。 （男）自加工清醒准备横栏头安装。在这个例子中，连接器也被植入重复脑电录音。 （N）的示意图头部安装各部件的位置和背视图。作为替代使用在面板研究两个横栏面板大号螺母是＃000自攻螺钉与横栏安装与稳定清醒成像准备增加补充。 （O）的示意图显示的端口“窗口中的横截面。 <img alt="图2" src="/files/ftp_upload/3742/3742fig2.jpg" /> 图2。双光子成像的血管和神经元在小鼠皮层的结构 。所有图像采集在Thy1系YFP的鼠标在窗口植入后10天内，通过一个端口“窗口。组织超过150微米的最大投影在冠状血管（A）和树突（二）稀释头骨方向。通过收集450 nm发射900 nm激发二次谐波荧光检测骨（蓝色） <sup&gt; 8。标记的血管（红色），静脉注射70 kDa的得克萨斯州红葡聚糖6。神经元的树突领域（绿色）是内源性的Thy1系YFP的转基因小鼠线。超过50微米的组织，在水平方向不同深度低于PIA（CH）的最大预测。数据是从面板中显示相同的图像栈A和B.硬脑膜血管可能略高于皮质表面可见（ 在C箭头 ）。 缩略语学联=人工脑脊髓液 PORTS =抛光和钢筋变薄颅骨窗口 YFP的=黄色荧光蛋白 确保所述的程序批准由当地机构动物护理和使用委员会。

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+cerebral+blood+flow+using+laser+speckle.">Dynamic imaging of cerebral blood flow using laser speckle.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 21, 195-201 (2001).</li><li>Villringer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+perfusion+of+the+rat+brain+cortex:+An+in+vivo+confocal+microscopy+study.">Capillary perfusion of the rat brain cortex: An in vivo confocal microscopy study.</a> Circulation Research. 75, 55-62 (1994).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248, 73-76 (1990).</li><li>Srinivasan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+for+the+quantitative+study+of+cerebrovascular+physiology.">Optical coherence tomography for the quantitative study of cerebrovascular physiology.</a> Journal of Cerebral Blood Flow &#38; Metabolism. 31, 1339-1345 (2011).</li><li>Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+imaging+and+characterization+of+the+microvasculature.">Photoacoustic imaging and characterization of the microvasculature.</a> Journal of Biomedical Optics. 15, 011101-011101 (2010).</li><li>Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory animal anesthesia. , (1987).</li></ol>

没有透露。

通过一个端口“窗口中的双光子成像需要通过传输薄骨和硬脑膜，衰减激光和光学像差增加了更大的深度8。然而，尽管这个缺点，成像深度可达250微米以下脑膜表面可以达到900 nm激发。大成像深度可能在原则上有可能与较长的激发波长13。这种方法的主要优点是皮质炎症的情况下，可能存在短暂的涉及全开颅14，15的方法。一个精心准备的端口窗口应显示血管生成或炎症没...

<table border="1"><tbody><tr><td> 代理人 </td><td> 交货路线 </td><td> 剂量为鼠标 </td><td> 时间 </td><td> 笔记 </td><td> 源 </td><td> 参考价 </td></tr><tr><td> 巴比妥（戊巴比妥） </td><td>知识产权</td><td> 90微克/克</td><td> 15-60分钟</td><td>狭窄的安全保证金。工作到适当的麻醉剂量缓慢。所需补编诱导剂量的10％。 </td><td> 036093;巴特勒施恩</td><td> 7 </td></tr><tr><td> 氯胺酮（Ketaset）混二甲苯胺噻嗪（Anased） </td><td>知识产权</td><td> 60微克/克（金） 10微克/ G（X）（混合在同一注射器） </td><td> 20-30分钟</td><td>甲苯噻嗪联合注入作为肌肉松弛剂和止痛药。氯胺酮诱导剂量的50％只补充需要。 </td><td> （金）010177（X）033198;巴特勒施恩</td><td> 7 </td></tr><tr><td> 异氟醚（Isothesia） </td><td>吸入</td><td> 4％，平均诱导肺泡浓度（MAC）; 1-2％的维修MAC </td><td> 4-6小时。 </td><td>提供70％氧气和30％的一氧化二氮的混合物。长时间麻醉导致的缓慢复苏。 </td><td> 029403;巴特勒施恩</td><td> 26 </td></tr></tbody></table> 表1。推荐为生存研究麻醉剂。 <table border="1"><tbody><tr><td> 项目 </td><td> 公司 </td><td><stroNG&gt;目录＃/型号</td></tr><tr><td>优碘</td><td>巴特勒施恩</td><td> 6906950 </td></tr><tr><td>丁丙诺啡（Buprenex） </td><td>巴特勒施恩</td><td> 031919 </td></tr><tr><td> 2 MDA分子量右旋糖酐，异硫氰酸荧光素</td><td>西格玛</td><td> FD2000S </td></tr><tr><td>异丙醇</td><td>费舍尔</td><td> AC42383-0010 </td></tr><tr><td>乳酸林格氏液的解决方案</td><td>巴特勒施恩</td><td> 009846; </td></tr><tr><td>利多卡因溶液，2％（V / V） </td><td>巴特勒施恩</td><td> 002468 </td></tr><tr><td>盐水</td><td>巴特勒施恩</td><td> 009861 </td></tr><tr><td>手术牛奶</td><td>巴特勒施恩</td><td&gt; 014325 </td></tr><tr><td>德州红，70 kDa的分子量右旋糖酐</td><td> Invitrogen公司</td><td> D1864 </td></tr><tr><td> maxizyme </td><td>巴特勒施恩</td><td> 035646 </td></tr><tr><td> 一次性 </td></tr><tr><td>硬质合金旋转锉，1/2毫米针尖大小</td><td>罚款科学工具</td><td> 19007-05 </td></tr><tr><td>棉田尖端喷头</td><td> Fisher Scientific则</td><td> 23-400-100 </td></tr><tr><td>覆盖玻璃，没有。 0厚度</td><td>托马斯科学</td><td> 6661B40 </td></tr><tr><td>氰基丙烯酸酯胶</td><td> ND工业</td><td> 31428 H04308 </td></tr><tr><td>煤气除尘器</td><td>新蛋</td><td> N82E16848043429 </td></tr><tr><td>握水泥粉</td><td>登士柏</td><td> 675571 </td></tr><tr><td>握水泥溶剂</td><td>登士柏</td><td> 675572 </td></tr><tr><td>胰岛素注射器，0.3 mL体积29.5针头</td><td>巴特勒施恩</td><td> 018384 </td></tr><tr><td>螺母和螺栓，以确保头</td><td> digikey </td><td>螺母，H723-ND;螺栓，R2-56X1/4-ND </td></tr><tr><td>眼科软膏</td><td>巴特勒施恩</td><td> 039886 </td></tr><tr><td>手术刀片</td><td> Fisher Scientific则</td><td> 12-460-448 </td></tr><tr><td>螺钉，自攻＃000 </td><td>姬·莫里斯公司</td><td> FF000CE125 </td></tr><tr><td>硅水族馆密封胶</td><td> perfecto制造</td><td> 31001 </td></tr><tr><td>锡氧化物粉末</td><td>妈妈的矿物</td><td>殷拓，TINOX </td></tr><tr><td> 设备 </td></tr><tr><td>玻璃划线</td><td> Fisher Scientific则</td><td> 08-675 </td></tr><tr><td>解剖显微镜</td><td>卡尔·蔡司</td><td> OPMI-1的FC </td></tr><tr><td>电动钻</td><td> foredom或Osada </td><td> K.1020（Foredom）或EXL-M40的（Osada） </td></tr><tr><td>电剃刀</td><td>华尔</td><td>系列8900 </td></tr><tr><td>钳，杜蒙没有。 55 </td><td>罚款科学工具</td><td> 11255-20 </td></tr><tr><td>反馈调节热垫</td><td>金控</td><td> 40-90-8（直肠热敏电阻40-90-5D-02，H吃垫，40-90-2-07） </td></tr><tr><td>异氟醚蒸发器</td><td>美达</td><td> IsoTec4 </td></tr><tr><td>脉搏血氧仪</td><td>斯塔尔生命科学</td><td> MouseOx </td></tr><tr><td>螺丝刀，微型</td><td>阁楼韦德</td><td> 26B09.01 </td></tr><tr><td>立体框架</td><td> kopf仪器</td><td>型号900（鼠标麻醉面罩和非破裂耳酒吧） </td></tr><tr><td>手术剪，钝端</td><td>罚款科学工具</td><td> 14078-10 </td></tr><tr><td>超声波清洗机</td><td> Fisher Scientific则</td><td> 15-335-30 </td></tr></tbody></table> 表2。特定的试剂，耗材和设备的名单。

a polished and reinforced thinned-skull window for long-term imaging of the mouse brain

在体内的皮质功能成像需要光纤接入无脑颅内环境的破坏。我们提出了一个方法，在小鼠颅骨，跨越直径几毫米个月的稳定形成抛光和钢筋颅骨变薄（端口）窗口。手举行演习，以实现光学清晰度头骨10至15微米的厚度变薄，然后覆盖氰基丙烯酸酯胶和玻璃盖：1）提供刚性，2）抑制骨的再生和3）减少光散射从骨表面上的违规行为。由于颅骨没有违反，任何可能会影响正在研究过程中的炎症，会大大减少。可以实现利用双光子激光扫描显微镜成像深度可达250微米以下的皮质表面。这个窗口非常适合研究脑血流和细胞的功能，在麻醉和清醒的准备。它进一步提供运portunity操纵细胞的活性，利用光遗传学或破坏由照射循环光敏剂的目标船只的血流量。

Watch this Scientific Journal Video about A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain at JoVE.com

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

我们提出了一个方法，形成一个横跨毫米，是没有大脑的炎症个月稳定在小鼠颅骨的影像窗口。这种方法非常适合于使用双光子显微镜的血液流通，细胞动力学，细胞/血管结构的纵向研究。

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

a-polished-reinforced-thinned-skull-window-for-long-term-imaging

Research

JoVE Journal

Neuroscience

26.7K 次观看.  University of California, San Diego. 我们提出了一个方法，形成一个横跨毫米，是没有大脑的炎症个月稳定在小鼠颅骨的影像窗口。这种方法非常适合于使用双光子显微镜的血液流通，细胞动力学，细胞/血管结构的纵向研究。

视频: A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

灭绝培训在彭兰霞窗口无法还原的恐惧

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

稀疏的头骨皮层的窗口技术<em在体内</em光学相干断层成像

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...