作者要感谢霍华德休斯医学研究所Janelia研究所的遗传编码神经元指标和效应因子（GENIE）项目的V. Jayaraman，DS Kim，LL Looger和K. Svoboda慷慨捐赠AAV1-GCaMP6f宾夕法尼亚大学矢量核心。他们还要感谢斯坦福大学的奥尔森和神经科学显微镜核心，由NIH NS069375授予的共焦显微镜服务支持。

涉及动物受试者的程序已得到加利福尼亚州美国航空航天局艾姆斯研究中心的LifeSource生物医学服务机构动物保护和使用委员会（IACUC）的批准。 术前准备<ol><li>在热珠灭菌器中灭菌外科手术中使用的工具，并用70％乙醇擦拭手术区域。打开放置在立体定位台上的加热垫，并将其保持在37°C。 </li><li>使用异氟醚麻醉动物（5％用于诱导，1-2％用于维持，0.6-0.8L / min O 2 ）。检查没有脚趾反射以评估麻醉深度。 </li><li>将动物安装在装有耳朵和牙齿条的立体定位框架中。 </li><li>将眼科软膏涂在动物的眼睛上，用一张深色纸盖住它们，以防止它们干燥和强烈的手术灯。 </li><li>用酮洛芬皮下注射动物（2.5mg / kg）或卡洛芬（2.5mg / kg） </li></ol>病毒注射手术<ol><li>修剪和剃刮眼睛和耳朵之间的头皮，并用3个70％乙醇和betadine的替代拭子消毒皮肤。 </li><li>通过在头皮中切开头部，从眼睛之间开始并用无菌外科手术刀片延伸1.5厘米的腰带，露出头骨。打开皮肤暴露头骨，并使用棉签和手术刀去除所需注射部位周围的骨膜。用无菌PBS冲洗颅骨。用棉签清洁头发并擦亮。 </li><li>调整头骨，并用标记，标记病毒注射的立体定位坐标。在高速微型钻（使用约7,000-10,000转/分钟）上使用0.5毫米毛刺，在颅骨上创造一个小洞。在钻孔时施加轻微的压力，并间歇地清洁骨灰，并用无菌PBS润湿该区域，以防止脑组织过热，直到脑表面恢复疼痛难忍。保持脑部潮湿，钻孔。 </li><li>在微量注射器中使用26 G针头接种病毒（ 如 AAV1.CaMKII.GCaMP6f.WPRE.SV40），然后用35 G针进行注射。将装有病毒的微量注射器连接到立体定位装置的操纵臂上。 </li><li>将注射器靠近注射部位孔并调整针的角度，使其与脑表面成90度角。降低针头直到触碰皮肤并穿透硬脑膜。开始以10μm/ s的增量降低针，直至达到所需的深度（z）。使用立体定位臂固定针位置。 </li><li> 设置微量注射泵以25 nL / min注入250 nL病毒。 <ol><li>临界步骤:由于待注射的病毒体积取决于滴度和稀释度，事先进行稀释实验，以确定成像细胞的最佳体积和浓度标准在实验中。 </li></ol></li><li>如果看到某些病毒从注射部位渗出，请暂停注射，等待脑组织吸收病毒滴。注射总体积之后等待5-7分钟，然后取出针头。染色体如绿色绿也可以添加到病毒溶液中，以帮助控制在病毒被渗出脑部表面的情况下的注射速率。 </li><li>为了在皮质中标记多层，必要时使用多次注射。首先从最腹部的部位开始，注射后等待5-7分钟，然后将针头拉至下一个最背部注射点（ 例如注射-1.0毫米AP，1.5毫米±ML和400和600微米DV）。在最后一次注射后10分钟，然后拔出针头并将其从立体定位装置中取出。 </li><li>从双筒注射器中混合少量体内生物相容的透明弹性体粘合剂（例如Kwik-Sil），并用它覆盖颅骨中的孔。将氰基丙烯酸酯粘合剂涂在弹性体粘合剂层的顶部并使其固化。 </li><li>缝合头皮，并允许动物在温暖的恢复笼中从麻醉中恢复，直到它走动。皮下注射酮洛芬（2.5 mg / kg）或卡洛芬（2.5 mg / kg），然后将动物送回家中。单独住院手术对象保护手术部位，并在24小时后重复使用。 </li><li>取出微量注射器后，用蒸馏水冲洗26 G和35 G针头7-10次，待储存前进行清洗。 </li></ol>棱镜探头植入手术<ol><li>病毒注射1-2周后，准备进行棱镜探针植入手术。在70％乙醇中消毒棱镜探头，并用透镜纸清洁。将棱镜探头插入镜头固定器工具，然后用螺丝刀拧紧六角螺丝。将显微镜置于基座（的位置）磁铁将其固定到位）。 </li><li>根据术前准备部分所述准备动物。 </li><li>修剪和剃刮眼睛和耳朵之间的动物头部，并用70％乙醇和betadine的替代拭子消毒皮肤。 </li><li>通过用一把无菌剪刀切开皮肤暴露头骨，并移除皮瓣和下面的骨膜。用棉签擦干头骨。确保充分去除周围的肌肉组织，以创建一个干净，干燥，宽的骨基础，准备以下步骤。 </li><li>植入颅骨螺钉在对侧半球，使植入物稳定和安全。如果选择植入用于清醒头部固定的头条来准备动物以进行第5节中的实验成像会话，这些也可能是有用的。 </li><li> 对头骨进行调整，并用标记标记AP和ML坐标以进行镜头插入。在微型钻机上使用0.5毫米毛刺打开圆形开颅手术，确保在这种情况下，开颅直径大于棱镜直径， 即 1.0mm。在间歇性地暂停冲洗，用无菌PBS冲洗颅骨，并用棉签将其吸出。清除所产生的骨灰尘。 <ol><li>临界步骤:放置开颅手术，使得当棱镜插入皮层时，其平坦的边缘（成像表面）面向病毒注射部位，半径在150-200μm之内。 </li></ol></li><li>在头骨完全变薄之前就停止钻孔。血管应通过变薄的骨骼可见。用精细的45°镊子轻轻取出骨头。 </li><li> 用＃5镊子去除硬脑膜。 <ol><li>关键步骤:一旦脑组织暴露，始终保持组织湿润。放置一个棉签浸在无菌盐水上开颅手术。这也将保持对组织的压力。 </li></ol></li><li>减轻脑组织压力环形插入棱镜探头，提前插入。将直边切割刀连接到立体定位装置的电极支架臂上，并将其安装在立体定位装置上，角度使得刀片垂直于颅骨的曲率（在这种情况下为10°角），并且在平行于病毒注射柱的平面。 </li><li>在这种情况下，小心地将刀沿其前内侧位于开颅上方，并且病毒注射部位的侧面约为200μm，刀刃朝后（ 图1 ）。当刀尖接触皮肤并逐渐降低（以10μm/ s为增量）将Z轴切除到棱镜探针插入的深度。然后将刀移动到后方1 mm处，以创建棱镜前缘的路径。暂停和控制在使用预先无菌生理盐水浸泡的美沙佛明的切片进行切口时可能发生的任何出血。 </li><li&gt;一旦刀在这个位置，用无菌盐水冲洗现场，等到任何出血消退。然后用立体定位臂显微操纵器以10微米/秒的速度慢慢收缩刀，并将一块置于无菌盐水中的海藻海绵浸在切口上。 </li><li>将镜头支架（带棱镜探头和显微镜）以与上一步相同的角度与立体定位机械手臂相连。对准棱镜，使得棱镜的平坦侧面在切口上方并且平行于病毒注射柱。该步骤可能需要对立体定位臂位置进行一些精细校正。通过保持靠近头骨的方式调整对准以获得更快的效果。 </li><li>一旦棱镜处于正确的角度，从脑部开始，以10μm的增量将其逐渐降低至大脑中1.1 mm的最终z。脑组织会在棱镜周围扩张，任何产生的压力都在视觉上在飞机上，不会影响视野。将显微镜插入计算机，并通过USB3端口安装采集软件，并通过打开LED显示现场荧光。 </li><li>使用25G针将非常薄的弹性体粘合剂保护层覆盖开颅手术周围棱镜周围的任何暴露组织。 </li><li>弹性体粘合剂固化后（通常为〜3-5分钟），使用25G针头涂抹一些氰基丙烯酸酯粘合剂，将棱镜透镜的玻璃贴在弹性体粘合剂层上的相邻颅骨上，以防透镜移动开颅手术包括棱镜探针袖口的边缘以获得更好的粘附力。不要在植入的棱镜探头的顶面上看到任何粘合剂。一旦氰基丙烯酸酯粘合剂固化，拧下镜头支架，并小心地取出显微镜。然后缓慢缩回立体定位机械手臂，使棱镜探针牢固植入。 </li><li> 涂一层牙科丙烯酸或氰基丙烯酸酯粘合剂在植入物周围覆盖所有暴露的颅骨表面，直到但不接触周围的收缩的肌肉组织。用这种颅盖覆盖大面积的头骨将有助于基板附件。植入物周围的皮肤应自行修复颅盖周围。 <ol><li>关键步骤:不要让粘合剂接触任何周围的皮肤或肌肉组织，并且不要吞没颅盖中的任何皮肤。这样做会刺激皮肤，并可能导致植入物过度刮伤和潜在的损伤。 </li></ol></li><li>可选:如果希望使用清醒的头枕固定装置在实验成像过程中将显微镜连接到动物的基板上，而不是简单地麻醉或擦伤动物，则将头盖植入头盖，选择的固定设置（在本协议中未示出）。 </li><li>从硅中混合催化剂和碱e粘合剂注射器，并将一滴弹性体放入棱镜探针袖口内以覆盖探头镜头顶部，以防止任何损坏和灰尘沉降。 </li><li>从立体定位框架中取出动物，并允许在温室中从麻醉中恢复。皮下注射酮洛芬（2.5mg / kg）或卡洛芬（2.5mg / kg），一旦移动，将动物返回到干净的家中。单独安置所有受试者保护植入物并在24小时后重复使用。 </li></ol> 4.微型显微镜安装基板附件<ol><li>在植入棱镜探针后一周至10天，通过植入式棱镜探头检查组织中的病毒表达，如果制剂显示细胞活性，则将底板附着在颅骨上。在实时成像期间，显微镜将停靠在底板上。 </li><li>按照术前手术中列出的准备动物基板附件的步骤进行。 </li><li>在植入的棱镜探针镜头顶部的表面上取下硅胶胶盖。检查镜头探头表面，并用镜头纸和70％乙醇轻轻清除任何碎屑，以确保成像表面清洁。 </li><li>将显微镜插入其DAQ盒，并将其通过USB3端口连接到PC。 </li><li>打开计算机上的采集软件，并通过USB3端口连接显微镜。使用采集软件检查神经活动，以及测量和记录本主题中将来录制的视野设置。 </li><li>将底板安装在显微镜上，并固定底板固定螺丝，将底板固定就位，并将显微镜固定在显微镜主体上的立体定位显微操纵臂上的显微镜夹具上。将夹具连接到可以安装在立体定位显微操纵臂上的Newport杆。 </li><li>使用s将显微镜放置在棱镜探头镜头的上方立体定位微型制导臂。通过从动物阶段的侧面和背面观察棱镜，目视检查方向。显微镜物镜和棱镜探头镜头的光轴必须对齐。 </li><li>通过软件打开显微镜LED。通过在采集软件中聚焦在植入式棱镜探针镜头的顶面来评估显微镜对准的质量。当正确对准时，棱镜探头镜头顶面的边缘应该是锋利的。 </li><li>使用立体定位机械手臂调整显微镜在植入式棱镜探头上方的物理距离，以获得组织内所需的焦平面。显微镜物镜和植入的GRIN透镜之间的光学优化距离为〜500μm。 </li><li>一旦捕获所需的成像平面，保存参考荧光图像。 关键点:从这一点上，不要调整显微镜的位置，因为这会改变位置在组织中的成像平面上。 注意:在下一步中涂抹粘合剂以永久固定基板相对于颅盖的位置。粘合剂在第二天或两天会经历一些体积收缩，这可能改变组织中的焦平面。通过测量粘合剂混合物的收缩量和离体距离，先预先说明这一点，然后在进行粘合剂涂布步骤之前将显微镜+底板的最终Z位置支撑该量。 </li><li> 使用牙科丙烯酸或氰基丙烯酸酯将基板永久地附着在覆盖动物头骨的丙烯酸盖上，与丙烯酸或粘合剂桥接间隙。逐步应用牙科丙烯酸/氰基丙烯酸酯和多级固化可以最小化上述收缩对显微镜图像平面最终位置的影响。 <ol><li>关键步骤:应用牙科时要小心丙烯酸/氰基丙烯酸酯，以防止任何材料接触显微镜的物镜，定位螺丝或显微镜主体，这将阻碍仪器的适当操作。 </li><li>关键步骤:使用粘合剂时，请勿推动显微镜。显微镜或底板上的压力可能导致显微镜物镜相对于棱镜探针镜头的移动，这可能导致组织中的未对准或焦平面的变化，这将需要及时的重新调整。 </li></ol></li><li>验证牙科丙烯酸/氰基丙烯酸酯通过用一对镊子或注射器尖端敲击丙烯酸酯已经固化和硬化。使用采集软件获取最终参考荧光图像。 </li><li>从夹具上取下显微镜并从显微镜上收回夹具。如果使用氰基丙烯酸酯或其他透明粘合剂，用黑色指甲油或一层黑色牙科水泥覆盖以防止mbient光泄漏到头盖，这可能会污染在实验期间获得的未来图像。 </li><li>此时，如果需要，请取出显微镜。为了将显微镜与基板分离，请通过逆时针方向旋转固定螺丝约1/2圈来松开底板固定螺丝。夹住显微镜主体，另一只手支撑底板和丙烯酸盖，然后将显微镜向上拉。将其更换在其存储容器中。 </li><li>用底板盖保护植入的棱镜探头。这将防止任何灰尘颗粒沉淀在透镜表面上，这在安装底板后可能会很麻烦。 </li><li>将基板盖板安装在底板上，顺时针方向将定位螺丝推入约1/2圈，或直到固定螺丝与底板盖齐平。不要过度紧张</li><li>从麻醉中取出动物，并在温暖的恢复室中进行监测，直到不动。回来了e动物到它的家笼。用植入的基板单独安置所有动物，以保护植入物。 </li></ol> 5.在自由移动的鼠标中成像多个皮质层<ol><li>通过清洁和消毒，并用10％漂白溶液擦拭，准备行为装置（ 如 Phenotyper，Noldus）。 </li><li>将显微镜插入其DAQ盒子，并将其连接到计算机并启动采集软件。 </li><li>检查采集计算机上是否有足够的文件存储空间，为钙成像电影腾出空间。直接从软件保存到本地硬盘，而不是写入外部硬盘驱动器，以适应显微镜和计算机之间的高数据传输速率，并防止录制过程中的数据丢失。 </li><li>在感应室中用异氟烷麻醉麻醉（5％氧气）以附着显微镜。或者，轻轻擦伤动物或使用清醒的头部固定设置如果已知麻醉剂干扰选择的行为范式，则使用头条。 </li><li>逆时针转动底座固定螺丝，取出底板盖，并将底板盖取出。 </li><li> 将显微镜放在动物的底板上。借助于底板上的磁铁，显微镜应该卡入到位。推进底板固定螺丝，直到感觉到轻微的阻力。 <ol><li>关键步骤:不要过度拧紧底板固定螺丝，以免损坏显微镜外壳。 </li></ol></li><li> 通过在软件中获取荧光快照检查组织中的成像平面，如果需要，通过松开显微镜转塔固定螺丝来调整组织中的焦平面，旋转显微镜转台调整精细对焦，然后重新拧紧转盘外壳固定螺丝。 <ol><li>关键步骤:切勿在没有松开固定螺丝的情况下强制转动转盘，而且不要拧紧转塔固定螺丝。 </li></ol></li><li>如果进行纵向研究，返回到物理炮塔位置以捕获相同的视野。在硬件中，请注意使用相同显微镜拍摄的每只动物的炮塔转弯数量或炮塔的物理位置，以便快速恢复到相同的视野。 </li><li> 将携带显微镜的动物释放到其家中的笼子或行为室适应适应，并等待穿戴麻醉（如果适用）。 <ol><li>关键步骤:使用虚拟显微镜训练动物携带显微镜的重量几次，直到确保戴上显微镜不会干扰其正常行为，才能开始实验。定期处理和训练清醒约束将防止动物过度的压力。 </li></ol></li><li>选择要用于收集数据的采集设置。这包括我捕获数据的速率（ 例如 20 fps，增益为1，LED功率为50％）。选择设置时检查图像直方图以确保良好的信噪比。 注意:1 mm棱镜探头的荧光采集数值孔径为0.35，而直径为1mm的探针则为0.5。 </li><li>启动行为软件并对其进行编程，以在所需的成像记录周期（ 例如 ，4X 5分钟ON 2分钟OFF）触发显微镜。将Noldus IO盒上的TTL端口通过RJ45至BNC电缆连接到DAQ盒上的TRIG端口。 </li><li>如果不在那里，将动物放在行为领域，并开始实验。 </li><li>获取所需数据后，在感应室中用异氟烷（5％氧气）重新麻醉动物，或轻轻地清醒动物。 </li><li>松开底板固定螺丝，轻轻拉起显微镜，将显微镜从底板上取下。更换底板盖板，轻轻拉紧底板te固定螺丝。 </li><li>将动物返回家庭笼子直到下一个记录会话。使用参考荧光图像作为后续成像会话的指南，以返回到相同的视野。 </li></ol> 6.评估大规模Ca 2+成像数据<ol><li>为了从数据中提取视场内的细胞位置和Ca 2+动力学，可以使用不同的数据分析平台。马赛克是一种专门用于处理大型Ca 2+成像电影的数据分析平台，用于本研究。 </li><li>纠正缺陷像素，并在预处理步骤中插入原始动画中的任何单个丢弃帧。将空间中的图像从完整的1,440 x 1,080像素视野中分解为720 x 540像素，以减少数据占用。 </li><li>为了校正相对于显微镜图像传感器的大脑中的运动伪像，使用严格的基于ImageJ的图像记录电影触发算法（TurboReg）。 </li><li>为了识别单个神经元，将图像重新表达为荧光的相对变化ΔF/ F 0 = FF 0 / F 0 ，其中F 0是通过对整个电影进行平均而获得的平均图像。 </li><li>使用建立的细胞分选算法识别与各个细胞相对应的空间滤波器。在这里，我们使用主成分和单个成分分析21来识别个体神经元。 注意:当轨迹中的事件的峰值幅度与我们的数据集中的迹线基线超过8个标准偏差时，指定了一个事件，并且输出了细胞位置和Ca 2+动力学数据进行进一步分析。 </li></ol>

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Rev. 63 (2), 291-315 (2011).</li><li>Berdyyeva, T., 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=Zolpidem+reduces+hippocampal+neuronal+activity+in+freely+behaving+mice:+a+large+scale+calcium+imaging+study+with+miniaturized+fluorescence+microscope.">Zolpidem reduces hippocampal neuronal activity in freely behaving mice: a large scale calcium imaging study with miniaturized fluorescence microscope.</a> PloS One. 9 (11), e112068 (2014).</li></ol>

作者已阅读期刊的政策，并具有以下竞争利益:SG，SO和VC是Inscopix的有偿雇员。

在清醒行为期间了解神经回路活动是有效剖析健康和疾病中脑功能所需的神经科学调查的重要水平。皮质是醒目行为背景下研究的一个特别重要的领域，因为它在许多重要的感官，认知和执行功能28,29中起重要作用。 皮质柱被认为是皮层中的基本功能单位，已知基于其在柱内的物理位置，皮质细胞的群体水平活性不同。例如，躯体感觉皮层中2/3层的兴奋性神经元主要对其他新皮质区域进行调节，并调制其他皮质网络30 ，而较深层的细胞则主要投入皮质下区域，如丘脑31 。记录一百的活动随着时间的推移，预先指定的皮质细胞随着时间的推移在自由行为的受试者中可以大大提高我们对皮质信息流的理解，允许通过实时行为信息和任务相关的时间信息通过皮质柱进行更精细的功能解剖，秤。 通过使用有效和流线型的小型化显微镜平台，在自由行为的受试者（或需要的头部固定的受试者）中进行大规模的Ca 2+成像，可以收集这一级别的神经回路数据。与遗传编码的钙指示剂一起使用以实现细胞型特异性靶向，并且通过长期植入的棱镜探针提供的多层视野成像，该方案在许多可能的应用中探讨了一种情况:观察小鼠体感皮质加工中的层流差异物理上与一个新物体接合（ 图5 ）。这是这种细胞型特异性体内方法的第一个程序说明， 用于在清醒，自由行动的动物中研究多个皮层，并扩大了可用于了解活跃脑中层状结构的实验方法。 期望通过棱镜探针在这种技术中实现的透视视野可以适用于其它脑结构，当保留直接背向感兴趣区域的组织时;例如，可以在不破坏海马功能的情况下实现CA3成像。 用于成像Ca 2+活性的基于Prism探针的方法需要物理插入并将微棱镜永久性植入皮质，这等同于创建透镜探针被插入的皮质损伤。这可能导致局部神经电路的破坏，包括顶端树突和过程的切断。 Ť他的手术也会导致该区域胶质细胞的初始激活，尽管预计这种胶质细胞定位于距离棱镜面约15​​0微米的组织，并在大脑已经愈合22后消退。在规划实验时，考虑这种技术是否会影响动物的正常电路解剖和/或行为是非常重要的。应始终进行行为控制组，以确保基线行为无显着变化，从而产生混杂的实验结果。 使用这种小型化，移动的Ca 2+成像技术与神经药理学操作，各种认知，社会，运动或内在的行为模式，并将其与其他生理指标结合，可以深化和丰富研究，重点是了解神经回路在行为和信号中的功能作用处理32 。抑制或激活通过药物调节的某些途径可影响相关行为，可以使用该技术轻松研究33 。通过修改钙指示剂的靶向来分化成不同的细胞类型是另一个强大而有用的应用，并且实现了许多实验工具的创造性组合来解决各种神经回路问题。

皮质是许多复杂的心理和行为功能的重要参与者，从注意力，感觉知觉和自上而下的认知控制1，2，3到激励，回报和成瘾途径4,5 。了解作为其功能的计算过程是推动对许多精神和行为障碍的更好的临床理解的重要目标。 许多目前的精神疾病理论主要围绕皮层神经回路功能障碍或失调的原因可能是认知和行为异常的基础，这些认知和行为异常是诸如精神分裂症6 ，自闭症7或强迫症8等症状的标志。因此，从公司获得种群水平的神经活动数据在同时行为信息的适当背景下的理论电路是非常重要的，并且理想地可以针对特定细胞类型进行更精细的神经电路解剖。 微型显微镜结合植入式梯度折射率（GRIN）微透镜使得能够在自由移动的条件下从多种可能的大脑区域9,10,11,12,13包括皮层14,15,16进入神经元组合。使用与遗传编码的钙指示剂结合的移动显微镜系统允许在许多脑区域9中在数天至数周内包含数百个神经元的相同细胞群体的一致成像，并且可以使用病毒载体或转基因技术遗传地靶向特定细胞类型。 由于已知皮质支持不同的功能并且连接到不同的脑区域，这取决于皮质层17,18,19 中细胞的位置，我们有兴趣在清醒行为主体中获得同时多层神经活动。在这里，我们演示如何使用与植入式棱镜探针配对的小型化荧光显微镜20 ，在几天内自由行为的小鼠中成像数百个荧光标记的神经元，这提供了皮层的多层视图（ 图1 ）。 这里使用的棱镜探头由两个独立的GRIN透镜组成:棱镜和圆柱形中继透镜（ 图1 ）。来自显微镜的光激发荧光标记在沿着棱镜探针的成像面位于探针的棱镜部分的斜边上的单元之后。来自细胞的发射光也从棱镜的斜边反射出来，通过显微镜的目的被收集，并在显微镜中到达传感器。本程序中使用的棱镜探头适用于标准立体定位设备。 微型荧光显微镜20在用Ca 2+敏感遗传编码的荧光指示剂特异性标记细胞之后，检测单细胞分辨率的神经元群体中的动作电位诱发Ca 2+瞬变。在该方案中，我们注入编码在病毒载体（AAV1.CaMKII.GCaMP6f.WPRE.SV40）中的Ca 2+指示剂，植入棱镜探针，安装显微镜，然后获得多天的体感（S1后肢）神经活动数据从动物暴露d在自由探索期间的新物体表面（ 图2 ）。

<table><tbody><tr><td>Neurostar Motorized&lt;br/&gt; Ultra Precise Small Animal Stereotaxic Instrument</td><td>Kopf</td><td>Model 963SD</td><td>Surgery</td></tr><tr><td>Stereoscope</td><td>Labomed</td><td>Prima DNT</td><td>Surgery and Imaging</td></tr><tr><td>Mini Rectal Thermistor Probe (.062&quot;/1.6 mm diameter) - 1/4&quot; Jack</td><td>FHC</td><td>40-90-5D-02</td><td>Surgery</td></tr><tr><td>Heating Pad 5 X 12.5 cm&amp;nbsp;</td><td>FHC</td><td>40-90-2-07</td><td>Surgery</td></tr><tr><td>DC Temperature Controller</td><td>FHC</td><td>40-90-8D</td><td>Surgery</td></tr><tr><td>Microsyringe Pump</td><td>World Precision Instruments</td><td>UMP3 model; serial 155788 F110</td><td>Surgery</td></tr><tr><td>NanoFil 10 &amp;mu;L Syringe</td><td>World Precision Instruments</td><td>NANOFIL</td><td>Surgery</td></tr><tr><td>35 G Beveled Tip Nanofil NDL 2PK</td><td>World Precision Instruments</td><td>NF35BV-2</td><td>Surgery</td></tr><tr><td>Omnidrill35, 115 - 230 V</td><td>World Precision Instruments</td><td>503598</td><td>Surgery</td></tr><tr><td>Burrs for Micro Drill</td><td>Fine Science Tools</td><td>19007-05</td><td>Surgery</td></tr><tr><td>nVista</td><td>Inscopix</td><td>100-001048</td><td>Imaging</td></tr><tr><td>AAV1.CaMKII.GCaMP6f.WPRE.SV40</td><td>Penn Vector Core</td><td>AV-1-PV3435</td><td>Surgery</td></tr><tr><td>Ketoprofen</td><td>Victor Medical</td><td>5487</td><td>Surgery</td></tr><tr><td>Carprofen</td><td>Victor Medical</td><td>1699008&amp;nbsp;</td><td>Surgery</td></tr><tr><td>Isoflurane</td><td>Victor Medical</td><td>1001054</td><td>Surgery</td></tr><tr><td>Gelfoam (Patterson Veterinary Supply Inc Gelfoam Sponge 12 cm x 7 mm)</td><td>Pfizer (Fisher Scientific)</td><td>NC9841478</td><td>Surgery</td></tr><tr><td>Dumont #5/45 forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>Surgery</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-30</td><td>Surgery</td></tr><tr><td>Dissecting knives</td><td>Fine Science Tools</td><td>10055-12</td><td>Surgery</td></tr><tr><td>ProView Implant Kit</td><td>Inscopix</td><td>100-000756</td><td>Surgery and Imaging</td></tr><tr><td>ProView Prism Probe 1.0 mm-Dia. ~4.3 mm Length</td><td>Inscopix</td><td>100-000592</td><td>Surgery and Imaging</td></tr><tr><td>Kwik-Sil adhesive pack of 2</td><td>World Precision Instruments</td><td>KWIK-SIL</td><td>Surgery</td></tr><tr><td>Kwik-Cast Sealant</td><td>World Precision Instruments</td><td>KWIK-CAST</td><td>Surgery and Imaging</td></tr><tr><td>Miniature Optical Mounting Post</td><td>Newport</td><td>M-TSP-3</td><td>Imaging</td></tr><tr><td>Microscope Baseplate</td><td>Inscopix</td><td>BPL-2</td><td>Imaging</td></tr><tr><td>Microscope Baseplate Cover</td><td>Inscopix</td><td>BPC-2</td><td>Imaging</td></tr></tbody></table>

multi-layer cortical ca2+ imaging in freely moving mice with prism probes and miniaturized fluorescence microscopy

体内电路和细胞水平功能成像是了解大脑在行动中的关键工具。双光子显微镜对小鼠皮层神经元的高分辨率成像提供了皮质结构，功能和可塑性的独特见解。然而，这些研究仅限于头部固定动物，大大降低了可用于研究的行为复杂性。在本文中，我们描述了在自由行为的小鼠中进行多个皮质层的细胞分辨率的慢性荧光显微镜的程序。我们使用与植入式棱镜探针配对的集成小型荧光显微镜，同时将数百个神经元的钙动力学视觉化并记录在多层体感皮质中，因为鼠在几天内就进行了一项新颖的对象探索任务。这种技术可以适应不同动物物种的其他脑区域，用于其他行为paradigms。

这里概述的方案描述了一种有效和有效的方法，使用棱镜探针，在数百个皮质神经元自由行为的小鼠中进行纵向多层Ca 2+成像（ 图1 ）。以前的多层皮层成像方法主要限于头部固定动物22,23,24,25,26,27。为了在自由行为的上下文中获得这种级别的数据，使用微型显微镜平台进行行为灵活性;使用遗传编码的钙指示剂（GCaMP6f）靶向特定细胞群（皮质中的CAMKII +细胞）;并选择棱镜探针提供慢性多层视野。 图2 ，步骤1）（ 图2 ，步骤2）。作为一个安全的临时码头，用于在成像期间定位显微镜的基板然后安装在动物头部（ 图2 ，步骤3）），使得能够在清醒行为实验中跨多个细胞层的皮层活动的可视化设置（ 图2 ，步骤4）。 为了确保所需的细胞群体被靶向， 图3中显示了来自代表性小鼠的死后冠状脑部分，其中棱镜探针管和视野相对于GCaMP6f实验室标记体细胞感觉皮层的2/3和5层的ele神经元。 在系统唤醒行为成像期间，当小鼠暴露于三个不同的环境时，记录体感皮层神经元的活动 - 开放领域（第1天），对象熟悉（第2-4天）和新对象（第5天） 图4 ）。在第1天，鼠标被放置在没有任何物体的行为竞技场。在第2-4天，将鼠标放在与相同的两个不同物体（凝胶垫和木块）的竞技场中。在第5天，其中一个对象被一个新的对象所取代。动物每天在5天内成像20分钟。 在使用Ca 2+图像数据分析软件进行细胞提取后，将对应于细胞位置的空间滤光片覆盖在显微镜记录数据的平均荧光强度投影上（ 图5） 。白色虚线分隔2/3和5个细胞。来自每个层的5个细胞的相应的Ca 2+痕迹显示了两种不同行为环境中的细胞的发射模式 - 对象熟悉和新对象曝光。当小鼠暴露于新物体当天时，层2/3细胞与第5层细胞相比更有活性。光栅图显示了在第1,4和5天所有成像细胞的阈值焙烧活性。 <img alt="图1" src="/files/ftp_upload/55579/55579fig1.jpg" /> 图1: 在自由移动的小鼠中多个皮质层的体内 Ca 2+成像。 （ A ）棱镜探头的规格和成像平面的描绘。斜边内侧的反射涂层允许从棱镜探头的插入平面成像90°。镜头cuff与透镜架集成，其简化了植入过程，并允许在植入期间潜在地观察周围组织荧光（ B ）（i）。相对于病毒注射部位放置棱镜探头开颅刀和刀切口的图示，以及（ii）相对于刀切口和病毒注射部位的棱镜探针平面侧的位置说明。 （ C ） 体内 Ca 2+成像装置的示意图，其通过植入小鼠皮层的棱镜探针在整个视野内显示出小面积的光路。 （ D ）棱镜探头安装时的示例视场。微型显微镜连接到透镜架上，其保持棱镜探头，允许在棱镜探头安装期间检查病毒表达。 （ E ）显微镜与棱镜探头的一体化，用于多层皮质成像的GCaMP6f标记的S1细胞。 F示例视图在基板安装过程中。在基板安装时，原始图像中有一些细胞可见清晰的血管图案。在采集软件窗口中打开DF / F时，更多的单元格清晰可见。 <a href="http://ecsource.jove.com/files/ftp_upload/55579/55579fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55579/55579fig2.jpg" /> 图2:显示棱镜探针植入和显微镜安装工作流事件的时间线的示意图。周数在X轴上以及沿着Y轴的程序的工作流步骤表示。 （ A ）沿着相同的背腹轴的病毒注射（AAV1.CaMKII.GCaMP6f.WPRE.SV40）的图示，以标记多层小鼠体感皮质。 （ B ）病毒注射后2周，棱镜问题e植入在与病毒注射部位平行的轴上。 （ C ）棱镜探针植入后约一周，用显微镜检查动物的表达，如果细胞群可见，则将底板安装在头上。 （ D ）随后，在相关行为任务（UW-Madison生物化学MediaLab许可之后修改了小鼠剪贴画），该动物随时准备进行慢性成像。 <a href="http://ecsource.jove.com/files/ftp_upload/55579/55579fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55579/55579fig3.jpg" /> 图3:棱镜探针位置和GCaMP表达的尸检组织学验证。 （ A ）来自代表性小鼠脑的冠状切片，显示棱镜探针管及其成像面朝向GCaMP6f表达细胞（AAV1.CaMKII.GCaMP6f在第2/3和5层的神经元中表达）。 （ B ）DAPI染色后的冠状脑部分相同。比例尺= 250μm（ C ）放大在体感皮层中表达GCaMP6f细胞的视图。刻度棒=100μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55579/55579fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55579/55579fig4.jpg" /> 图4:使用视频软件视频跟踪习惯中的鼠标活动，熟悉和新对象曝光测试。 （ A ）在第1天，动物被放置在没有任何物体的行为竞技场（露天场）。 （ B ）在第2-4天，将相同的两个不同的物体（凝胶垫和木块）放置在竞技场中（Object Familiarization）。 （ C ）在第5天，其中一个物体被一个新物体（带有砂纸的木块）（新物体曝光）所取代。 <a href="http://ecsource.jove.com/files/ftp_upload/55579/55579fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" src="/files/ftp_upload/55579/55579fig5.jpg" /> 图5:与显微镜成像的代表性小鼠的体感皮层的浅层和深层的钙动力学。 （ A ）神经元空间滤波器（绿色斑点）的合并图像和通过棱镜探头视野的显微镜记录的平均荧光强度投影。由白色虚线表示的超颗粒层和颗粒层之间的边界。刻度棒=100μm。 （ B ）来自五个表面和深层细胞的钙痕迹（填充蓝色和红色c主要和独立成分分析后荧光标准偏差的单位。水平比例尺50秒和垂直比例尺10 SD（ C ）显示在开放场，对象熟悉和新对象探索中的表面（层2/3）和深层（层5）的细胞的光栅图。比例尺= 100秒。 <a href="http://ecsource.jove.com/files/ftp_upload/55579/55579fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy at JoVE.com

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

在这里，我们提出了一种在自由移动的小鼠中通过多个皮质层进行细胞分辨率的大规模Ca 2+成像的程序。可以使用与植入式棱镜探头耦合的微型头戴式显微镜同时观察数百个活性细胞。

多层皮质Ca<sup&gt; 2+</sup&gt;使用棱镜探针和小型荧光显微镜成像自由移动的小鼠

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

multi-layer-cortical-ca2-imaging-freely-moving-mice-with-prism-probes

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Behavior

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

30.9K 次观看.  Inscopix Inc.. 在这里，我们提出了一种在自由移动的小鼠中通过多个皮质层进行细胞分辨率的大规模Ca 2+成像的程序。可以使用与植入式棱镜探头耦合的微型头戴式显微镜同时观察数百个活性细胞。 

视频: Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

在小鼠导航虚拟现实环境的双光子钙成像

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

科研

行为学

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

生物学

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 ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

在使用微深皮质层的活体成像

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

神经科学

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

体内 使用大颅窗的小鼠宽场和双光子钙成像

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

上丘的双光子和宽场钙成像

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

作者聚焦:揭示上丘的神经编码和视觉处理机制 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

小鼠上丘的钙成像

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

多层皮质Ca

摘要

探索更多视频

此视频中的章节

在小鼠导航虚拟现实环境的双光子钙成像

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

在体内2光子层钙成像对小鼠的2 / 3

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

在使用微深皮质层的活体成像

小鼠大脑皮层细胞组织的大尺度三维成像

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

体内使用大颅窗的小鼠宽场和双光子钙成像

小鼠上丘的钙成像

多层皮质Ca

摘要

探索更多视频

此视频中的章节

在小鼠导航虚拟现实环境的双光子钙成像

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

在体内2光子层钙成像对小鼠的2 / 3

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

在使用微深皮质层的活体成像

小鼠大脑皮层细胞组织的大尺度三维成像

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

体内 使用大颅窗的小鼠宽场和双光子钙成像

小鼠上丘的钙成像

体内使用大颅窗的小鼠宽场和双光子钙成像