这项工作得到了上海市基础研究试点项目 - 复旦大学 21TQ1400100 （22TQ019）、上海市科技重大专项、临港实验室（批准号。LG-QS-202203-09） 和中国国家自然科学基金 （32371036）。

所有动物程序均由复旦大学机构动物护理与使用委员会 （202109004S） 批准。本研究中使用的所有动物均为 6 个月大的 C57BL/6J;使用了两性。小鼠从上午 8 点到晚上 8 点保持 12 小时的光照周期。我们在 DG 中使用以下坐标进行病毒注射：A/P：-2.2 mm，M/L：1.5 mm，D/V：距脑表面 1.7 mm。
1. 病毒注射到齿状回
<ol><li>穿戴防护设备，包括一次性无菌手套和防护服。</li>
	<li>准备所需的手术器械、两管 5 mL 生理盐水（0.9% NaCl [无菌] 或人工脑脊液）和两根 25 G 鲁尔锁钝针。 注意：手术前必须对所有手术器械进行彻底消毒。我们在高温 （121-134 °C） 和压力 （20 psi） 下高压灭菌对手术器械进行消毒。此外，我们还用含酒精的消毒剂喷洒了表面。在所有外科手术中，我们使用 0.9% 无菌生理盐水溶液。在手术过程中，用加热的玻璃珠对器械进行间歇性消毒。</li>
	<li>准备 10% 的漂白剂水溶液，对可能与病毒接触的任何用品进行消毒。</li>
	<li>如有必要，使用无菌盐水稀释AAV-CaMKIIα-GCaMP6f病毒，并将其储存在4°C冰箱或冰上。病毒的目标最终滴度为 1 × 1012 GC/mL。 注意：我们建议避免病毒的重复冻融循环，以保持病毒的完整性。为此，我们通常将制造商提供的病毒分装成小体积（例如，每管 2 μL），并将它们保存在 -80 °C 冰箱中。病毒应在解冻当天使用，不宜在 4 °C 下长期保存。</li>
	<li>使用微量移液器拉拔器将微量移液器拉至细尖，用手术剪刀剪掉一小部分尖端，然后用矿物油填充试管。确保移液器可以正常吸取液体，然后将其安装在注射器上。 注意：我们使用了一步拉法，将加热器设置为最大功率的 ~60%。此步骤可以使用其他拉拔器。总体目标是使尖端直径为 ~50-100 μm;直径太小会影响液体的流动;太厚可能会对小鼠的脑组织造成损害。</li>
	<li>打开所有仪器，包括小鼠麻醉机、保温机（加热垫）和真空泵。此外，在手术前测量小鼠的体重。 注意：在此过程中，我们目视监测了小鼠的呼吸，并偶尔检查了体温以确保它们处于良好状态。</li>
	<li>将鼠标放入含有 2.5% 异氟醚和 1 L 氧气/分钟的气室中。监测小鼠的呼吸状况以测量麻醉状态。 注意：呼吸频率应约为 1 次呼吸/秒，以保持鼠标健康。</li>
	<li>将鼠标从毒气室中取出，并将其放在立体定位装置上。</li>
	<li>在开始下一次手术之前，确保鼠标已充分麻醉。通过捏住双后脚的脚垫来测试踏板撤退反射。如果鼠标对脚垫捏有反应，请在继续之前进行额外的麻醉并重新测试反射。</li>
	<li>用面罩盖住小鼠鼻子，并将异氟醚流速设置为 1.5%。用耳杆牢固地固定鼠标头（图 2A）。 注意： 小心不要将耳栏固定在鼠标上太紧，因为这很容易影响鼠标的呼吸，在某些情况下会导致死亡。</li>
	<li>用手术剪刀修剪小鼠的头顶，并使用脱毛膏去除任何残留的毛发，直到皮肤完全暴露。将脱毛霜涂抹在小鼠的头皮上，静置约 5 分钟，然后用纱布擦去霜。</li>
	<li>在小鼠的眼睛上涂抹眼药膏。</li>
	<li>使用棉签用碘伏消毒剂和 75% 乙醇对无毛区域进行消毒。</li>
	<li>使用手术剪刀（或手术刀刀片）沿头皮中线连续切开，并切掉部分头皮以露出颅骨表面（图2B）。用生理盐水冲洗颅骨表面;然后，使用真空泵去除筋膜和过量的盐水。多次重复此过程，直到伤口周围区域停止流血。用棉签擦拭颅骨表面，使其保持干燥。 注意： 还有其他方法可以去除筋膜，例如用棉球或棉签将其擦掉。</li>
	<li>当前囟和 lambda 可见时，使用定位针调整颅骨表面。进行多次调整，直到整个头部水平（Z 轴方向的整个误差小于 0.05 毫米）。</li>
	<li>将针尖从前囟移动到适当的目标位置。对于目标坐标，使用前囟作为参考点。标记四个点以定义病毒注射部位的周长： A/P： -2.0 mm， M/L： -1.5 mm;A/P： -2.5 毫米， M/L： -1.5 毫米;A/P -2.25 毫米，M/L： -1.0 毫米;和 A/P -2.25 毫米，M/L：-2.0 毫米，带可擦除标记。 注意：该协议在同一手术中执行病毒注射和 GRIN 晶状体植入。在该协议中，所有手术均在右半球进行，但可以想象可以使用任何一个半球。在上述四个点定义的注射区域内，在不同位置以三个单独的 80 nL 剂量注射病毒。尽管这三个地点的选择是任意的，但我们建议将它们分散开来，以便更好地传播病毒。这将增加受感染细胞的数量。</li>
	<li>用微钻在该区域进行开颅手术，并将这四个点设置为边界。当脑组织可见时停止钻孔（图 2C）。 注意： 尝试慢慢钻孔头骨。钻头可能会因用力过大而损伤脑组织。缓慢钻孔还可以防止大脑过热。</li>
	<li>使用超细镊子去除骨碎屑和硬脑膜，并不断用无菌盐水冲洗该区域。 注意：在此步骤中发生少量出血是正常的，因为一些毛细血管会破裂;只需继续用生理盐水冲洗，直到不再流血。</li>
	<li>使用注射器的控制面板弹出一些矿物油，为随后加载病毒产生所需的空间。以 100 nL/s 的速度向微量移液器中注入至少 240 nL 的稀释病毒。 注意：应使用控制面板排出管中的气泡，以确保微量移液器分配适量的液体。如果多次尝试后液体体积仍然不准确，请考虑更换微量移液器并重新开始。我们建议吸出比实际注射体积更多的病毒;这种方法可以防止无意中将矿物油注射到小鼠大脑中。</li>
	<li>将微量移液器移动到开颅区域上方;然后，慢慢地将其向下移动到脑组织中，到达 D/V 的目标 Z 轴：大脑表面以下 1.70 毫米。 注意：许多参考图集使用头骨表面作为 D/V 0。根据我们的经验，使用来自大脑表面的坐标可以产生更可靠的 DG 定位。</li>
	<li>使用控制面板将注射器设置为以 1 nL/s 的流速注射 80 nL 病毒（图 2D）。点击 INJECT 控制面板上的按钮注入病毒。随后，在开颅手术中选择另外两个位置并重复上一个操作。 注意：每次注射需要 ~2 分钟。完成注射后，再等待 8-10 分钟，然后再移动到另一个位置。如果在取出过程中出现出血，请立即用生理盐水清洗。</li>
	<li>慢慢地将微量移液器从大脑中取出（图 2E）。</li>
</ol>2. GRIN 晶状体植入齿状回
注意：完成 240 nL 病毒注射后立即执行此步骤。
<ol><li>将直径为 1 mm 的 GRIN 镜片在 75% 乙醇中消毒 20 分钟;植入前用生理盐水正确冲洗。</li>
	<li>使用微钻头扩大开颅手术，将 A/P 方向的开口增加到大约 1 毫米。用盐水清除碎屑。</li>
	<li>用支架将 GRIN 镜头夹住到位，并确保露出足够的长度。 注意：在后续步骤中使用 UV 树脂将支架固定在颅骨上时，很容易将支架粘在 GRIN 镜片上。但是，如果镜头的曝光长度足够，则可以避免这种情况。</li>
	<li>使用支架将 GRIN 晶状体移动到开颅区域上方。当 GRIN 镜头的底部接触脑组织表面时，将 Z 轴位置设置为零。然后，取下 GRIN 镜头支架并将其放在一边。</li>
	<li>将 25 G 鲁尔锁钝针连接到真空泵抽吸管上，并将压力调节至 0.04 MPa。 注意：如果是第一次执行此步骤，则可以适当降低压力以缓慢吸出脑组织。</li>
	<li>通过将钝针的尖端紧贴脑组织上方旋转来吸取脑组织，然后轻轻按压脑组织以促进抽吸。在抽吸过程中不断用生理盐水冲洗（储存在 4 °C），以保持大脑的清晰视野。仔细观察组织特征以监测抽吸深度：皮质组织为淡粉色，下面的胼胝体显示为白色纤维组织，海马 CA1 层为深红色和灰色。当组织表面变得光滑并且大面积的 CA1 暴露时，停止抽吸（图 3）。 注意：此步骤对于整个手术的成功至关重要，并且通常是最困难的部分。我们提供了一份常见问题列表，以帮助读者对此步骤进行故障排除（表 1）。此外，我们建议用冷盐水不断冲洗伤口，因为这可以在一定程度上减少出血。<ol><li>如果针头堵塞;将堵塞的针头连接到注射器上并冲洗掉堵塞物。如果这不起作用，请尝试更换针。</li>
		<li>如果出血持续，它会阻塞脑组织的视野。如果发生这种情况，请等待血液凝结，然后用生理盐水冲洗。</li>
	</ol></li>
	<li>将刀柄移动到钻孔上方。当 GRIN 镜头接触脑组织表面时，将 Z 轴设置为 0。如果 GRIN 透镜超过孔的直径，请使用微钻扩展孔。然后，重复此过程。</li>
	<li>将 GRIN 镜头插入 D/V 处的孔中：-1.32 mm。让支架静置 5-10 分钟。抬起支架后，用生理盐水冲洗孔。重复此步骤 3 次（图 4A）。 注意：在此步骤中脑组织出血是正常的。反复用生理盐水冲洗后，孔中不应再有血。这一步对成像质量影响很大，如果血液没有清理干净，可能会导致成像视野变黑。</li>
	<li>使用生理盐水彻底清洁头骨，然后让头骨干燥，然后再涂抹 UV 树脂。</li>
	<li>用针头将 UV 树脂涂抹在 GRIN 镜片周围。用紫外线照射该区域 15 秒，并确保 UV 树脂变成固体（图 4B）。 注意：一次涂抹一点 UV 树脂，然后重复此操作几次，直到覆盖 GRIN 镜片周围的区域。使用紫外线照明时，请注意不要将 GRIN 镜头固定在支架上。</li>
	<li>小心地从 GRIN 镜头上取下支架。用 UV 树脂覆盖整个裸露的头骨，并将头板放在头骨上的适当位置。用紫外线照射整个颅骨和头板 15 秒（图 4C）。 注意：头板是一个组件，用于在连接底板时将鼠标的头部牢固地固定到位（有关设计，请参阅 补充文件 1 ）。尽可能牢固地固定头板;头板脱落将导致整个手术失败。根据我们的经验，彻底涂抹 UV 树脂应该能够提供牢固的附着。如果需要更坚固的连接，可以考虑安装颅骨螺钉。</li>
	<li>用 3D 打印的保护盖盖住曝光的 GRIN 镜头（有关设计，请参阅 补充文件 2 ）。使用 M1.6 螺钉将盖子固定到头板上（图 4D）。</li>
	<li>手术后腹膜内注射地塞米松（溶于 0.9% 生理盐水，2 mg/kg）以防止术后炎症。此外，皮下注射卡洛芬（溶于 0.9% 盐水，5 mg/kg）以减轻术后疼痛。</li>
	<li>将小鼠放回笼子中，并将它们转移到热毯中以促进其恢复。然后，在小鼠可以自由活动后将其转移到实验动物舍。 注意：为避免因打架而损坏底板，接受过手术的小鼠可以单独饲养或与最少数量的同居者一起饲养。我们建议手术后笼子里的小鼠不超过三只。</li>
	<li>使用 10% 漂白剂溶液对工作台面进行消毒。取下一次性个人防护装备 （PPE） 并将其丢弃在生物危害袋中。</li>
	<li>手术后 3 天每天监测小鼠。记录小鼠的体重并观察它们的伤口愈合状态和运动活动。每天腹膜内注射地塞米松和卡洛芬溶液（与步骤 2.13 中的剂量相同），持续 3 天。必要时提供湿润的食物或治疗，以促进恢复。</li>
</ol>3. 检查成像场的质量
注意：小鼠在手术后 2-3 周内恢复，然后进行第一次 体内 钙成像。此步骤的目的是检查手术质量和鼠标的恢复情况。如果在成像视野中可以观察到单细胞活性并且小鼠状况良好，请将底板固定在小鼠头骨上。如果成像质量或健康状况不佳，应通过颈椎脱位对小鼠实施安乐死。
<ol><li>使用 USB 3.0 电缆将微型望远镜数据收集盒连接到计算机。然后通过 To Scope 连接器 （SMA） 将同轴电缆连接到盒子。</li>
	<li>打开微型显微镜控制软件。在软件中，单击 Select Config File |使用 Configuration Files V4 plus 查看实时图像。</li>
	<li>使用定制的支架通过夹紧头板将鼠标固定在滚轮上。用微型瞄准镜支架（由 3D 打印制成;有关设计，请参阅 补充文件 3 ）握住微型瞄准镜，并使用立体定位仪器将微型瞄准镜移动到鼠标头部上方（图 4E）。</li>
	<li>用螺丝刀取下盖子。用 75% 乙醇轻轻擦拭 GRIN 镜片的表面。将微型望远镜放置在曝光的 GRIN 镜头的正上方，并使瞄准镜的底面与镜头表面平行。通过将微型望远镜慢慢向下朝向 GRIN 镜头来找到最佳焦平面。 注意：将焦平面设置为 0，然后调整微型望远镜的位置;这为调整焦点提供了更多空间。选择具有最大单元格数清晰可视化的焦平面。</li>
	<li>在微型显微镜控制软件中，将 LED 灯功率调整到最佳水平。 注：要观察单细胞活性，成像视野既不能过度曝光，也不应太暗。 LED 强度 通常在 10% 以内。</li>
	<li>如果血管血流可见，并且细胞众多且均匀分布在整个成像视野中，请继续进行实验的下一部分。 注意：在此步骤中，能够观察单细胞活性非常重要。这可能会影响数据分析的结果。通常，区域活动（荧光波动斑点）很难解释。这可能表明目标神经元不在 GRIN 镜头的焦点上。如果成像区域显示为黑色、细胞表现出区域活性或细胞数量不足，则不会在后续实验中使用鼠标。</li>
	<li>断开电缆与微型望远镜的连接。</li>
</ol>4. 将 miniscope 底板连接到鼠标头骨上
<ol><li>将底板安装到微型望远镜上，并重新调整微型望远镜的位置，以获得成像质量良好的视野。</li>
	<li>在搅拌碗中混合义齿基托材料。 注意：重要的是混合物既不能太自由流动，也不能太硬。过多的流动性往往会覆盖 GRIN 镜头的表面，从而导致成像场的损失。如果太坚固，义齿基托材料将无法紧密覆盖基板和头板之间的间隙。</li>
	<li>小心地在微型内窥镜底板周围应用第一层义齿基材时，注意不要改变微型内窥镜的位置。等待第一层水泥硬化，然后再从底板到头板上涂抹第二层水泥。所有义齿基材硬化后，松开固定螺钉并将微型内窥镜从底板上拆下。 注意：使用义齿基材时，请注意软件中的成像区域，并在粘接剂凝固之前进行任何必要的调整。</li>
	<li>轻轻地从微型望远镜上取下固定臂。</li>
	<li>将底板盖放入底板以保护暴露的 GRIN 镜头并拧紧固定螺钉（图 4F）。</li>
	<li>将鼠标放回其 Home Cage。在进行行为实验之前，给鼠标几天的时间来恢复。</li>
</ol>5. 数据采集
注意： 体内 钙成像可以与任何行为测试同时进行。在这个协议中，我们以线性轨道为例。这条线性轨道长 1.5 m，两端都有水，这是对老鼠的奖励。小鼠可以佩戴微型显微镜（微型显微镜）并沿着轨道来回奔跑 20 分钟。在此期间，小鼠通常能够运行 ~60 次试验。
<ol><li>使用 USB 3.0 电缆将微型望远镜数据收集盒连接到计算机。然后通过 To Scope （SMA） 连接器将同轴电缆连接到盒子。</li>
	<li>打开微型显微镜控制软件。在 miniscope control Software 中，单击 Select Config File |使用 Configuration Files V4 plus 查看实时图像。</li>
	<li>使用 USB 3.0 电缆将工业相机连接到计算机。</li>
	<li>开启相机控制软件。在软件中，单击 Open Equipment，然后选择文件以导入数据收集参数。选择视频格式作为 未压缩的视频 in AVI 容器.点击 Start 开始 按钮查看实时图像。 注意：要减小行为录制视频的文件大小，视野应排除轨道之外的任何不必要区域。</li>
	<li>使用定制的支架通过夹紧头板将鼠标固定在滚轮上。</li>
	<li>用螺丝刀松开固定螺钉，取下底板盖，然后用 75% 乙醇清洁 GRIN 镜头表面。</li>
	<li>将微型瞄准镜安装到其底座上。将微型瞄准镜固定在鼠标头上的底座上。</li>
	<li>通过滑动焦平面按钮找到最佳焦平面。</li>
	<li>轻轻地将鼠标从滑轮移动到线性轨道。</li>
	<li>单击并按住 Record 按钮开始使用微型望远镜进行录制。接下来，点击 开始收集 按钮开始相机录制。录制 20 分钟。</li>
	<li>分别保存 体内 钙成像数据、行为数据和 Arduino 数据。使用日期、会话编号和鼠标编号命名这些文件。 注意：使用微型望远镜的研究往往对单个实验动物具有长期的数据收集期。清楚地命名文件有助于数据处理程序。</li>
	<li>将微型望远镜从其底座上拆下。</li>
	<li>关闭相机控制软件、微型望远镜控制软件和计算机。</li>
	<li>断开同轴电缆与 To Scope （SMA） 的连接，以及断开 USB 3.0 电缆与摄像机的连接。</li>
	<li>拧下底座中的固定螺钉，将底板盖放回原处，然后拧紧螺钉。</li>
	<li>将鼠标放回其 Home Cage。</li>
</ol>6. 数据处理
<ol><li>在 MATLAB 中加载记录的钙成像数据。 注意：我们认识到数据分析应根据实验设计进行定制。在这里，我们提供了一个预处理管道，可将原始钙成像视频转换为来自单个细胞的活动轨迹。我们相信此过程相对通用且对用户有用。本节中描述的所有脚本都可以在 补充文件 4 中找到。</li>
	<li>将成像数据从 AVI 转换为 HDF5 （.h5） 格式。 注意：微型望远镜的原始输出会生成经过压缩的 AVI 文件。未压缩的文件通常为数十 GB。HDF5 文件格式允许虚拟和部分访问，可以轻松可视化和操作以进行后续分析。</li>
	<li>应用非刚体运动校正 （NoRMCorre）19。</li>
	<li>对运动校正的影片进行下采样，以防计算机在后续步骤中受到 RAM 限制。 注意：miniscope 收集的原始数据大小为 600 x 600。通过使用空间下采样，我们可以将视频大小减小到 200 x 200。这种方法显著降低了数据存储要求，并允许更快的数据处理。</li>
	<li>按照在线存储库20 （https://github.com/schnitzer-lab/EXTRACT-public） 中 EXTRACT 代码的说明，对下采样数据应用 EXTRACT 以识别单细胞信号。</li>
</ol>

小鼠齿状回细胞神经元活动的实时成像 

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作者声明没有竞争性的经济利益。

在这里，我们描述了在小鼠 DG 中进行 体内 钙成像的程序。我们相信该协议对于旨在研究各种认知过程中 DG 功能的研究人员很有用，尤其是在对遗传鉴定的亚群感兴趣的情况下。在这里，我们解释了我们的方案的优势，强调了手术中的一些关键点，并讨论了这种方法的局限性。
我们已经从现有文献中测试了 DG 成像的各种程序，并优化了本协议中详述的实验程序。在我...

以前的研究发现，海马体是处理和检索记忆所必需的大脑结构 1,2。自 1950 年代以来，啮齿动物海马体的神经回路一直是研究记忆形成、存储和检索的重点3。海马内的解剖结构包括齿状回 （DG）、CA1、CA2、CA3、CA4 和亚眼4 的亚区。这些亚区之间存在复杂的双向连接，其中 DG、CA1 和 CA3 形成一个突出的三突触环，由颗粒细胞和锥体细胞组成5。该回路从内嗅皮层 （EC） 接收其主要输入，一直是研究突触可塑性的经典模型。先前关于海马功能的体内研究主要集中在 CA1 6,7 上，因为它更容易接近。虽然 CA1 神经元在记忆形成、巩固和检索中起着重要作用，特别是在空间记忆的原位细胞中，但海马体的其他亚区域也至关重要 8,9。特别是，最近的研究强调了 DG 在记忆形成中的作用。据报道，DG 中的位置细胞比 CA1 中的细胞更稳定10，并且它们的活性反映了特定环境的信息11。此外，DG 颗粒细胞的活性依赖性标记可以被重新激活以诱导记忆相关行为12。因此，为了更深入地理解 DG 中的信息编码，在动物执行记忆依赖任务时研究 DG 子区域的活动至关重要。
DG 活性的先前研究主要使用 体内 电生理学13。然而，这种技术也有一些缺点：首先，在电记录中，可能很难直接识别产生信号的各种类型的细胞。记录的信号来自抑制性和兴奋性细胞。因此，需要进一步的数据处理方法来分离这两种细胞类型。此外，很难将其他细胞类型信息（例如投影特异性亚组或活性依赖性标记）与电记录相结合。此外，由于 DG 的解剖形态，记录电极通常以正交方向植入，这极大地限制了可以记录的神经元数量。因此，电记录很难实现对同一动物 DG 结构中数百个单个神经元的监测14。
在 DG 中记录神经元活动的补充技术是使用 体内钙成像 15。钙离子是生物体细胞信号传导过程的基础，在许多生理功能中起着至关重要的作用，尤其是在哺乳动物神经系统中。当神经元活跃时，细胞内钙浓度迅速增加，反映了神经元活动和信号传递的动态性质。因此，记录神经元细胞内钙水平的实时变化为了解神经编码机制提供了重要的见解。
钙成像技术利用专门的荧光染料或基因工程钙指示剂 （GECI） 通过检测荧光强度的变化来监测神经元中的钙离子浓度，然后通过显微成像捕获这些变化16。通常，采用钙指示基因的 GCaMP 家族，包括绿色荧光蛋白 （GFP）、钙调蛋白和 M13 多肽序列。GCaMP 与钙离子结合时可发出绿色荧光17，从而可通过成像记录绿色荧光的波动18。此外，为了获得目标大脑区域的清晰图像，通常在感兴趣区域上方植入梯度折射率透镜（GRIN 透镜）。GRIN 镜头能够对无法直接从表面进入的大脑深部区域进行成像。
该技术相对容易与其他遗传工具结合使用来标记不同的细胞类型。此外，由于成像平面与 DG 中细胞的方向平行，因此每次成功的手术都可以访问数百个神经元进行成像。在这项工作中，我们提出了一个完整而详细的手术方案，用于小鼠齿状回的 体内 钙成像（图 1）。该程序涉及两个主要操作。第一种是将 AAV-CaMKIIα-GCaMP6f 病毒注射到 DG 中。第二种操作是在病毒注射部位上方植入 GRIN 晶状体。这两个程序在同一次会议上进行。从这些手术中恢复后，下一步是使用小型显微镜（微型显微镜）检查成像质量。如果成像区域有数百个活性细胞，则后续程序是使用牙科水泥将微型望远镜底板附着在小鼠头骨上;然后，鼠标可用于成像实验。我们还提出了一个基于 MATLAB 的预处理管道，用于简化对收集的钙数据的分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 &#956;L universal pipette tips</td>
			<td>Transcat Pipettes</td>
			<td>1030-260-000-9</td>
			<td>For removing the blood and saline</td>
		</tr>
		<tr>
			<td>25 G luer lock blunt needle (Prebent dispensing tips)</td>
			<td>iSmile</td>
			<td>20-0105</td>
			<td>For removing the brain tissue</td>
		</tr>
		<tr>
			<td>3D printed protective cap</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To protect the GRIN Lens</td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Shanghai Hushi Laboratory Equipment Co.,Ltd</td>
			<td>bwsj-230219105303</td>
			<td>For disinfection and cleaning the GRIN lens surface</td>
		</tr>
		<tr>
			<td>AAV2/9-CaMKII&#945;-GCaMP6f virus</td>
			<td>Brain Case</td>
			<td>BC-0083</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe</td>
			<td>cc 2018 version 22.1</td>
			<td>To draw figures</td>
		</tr>
		<tr>
			<td>Anesthesia air pump</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-30</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Camera control software</td>
			<td>Daheng Imaging</td>
			<td>Galaxy Windows SDK_CN (V2)</td>
			<td>For recording the behavioral data</td>
		</tr>
		<tr>
			<td>Cannula/Ceramic Ferrule Holders (GRIN lens holder)</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>68214</td>
			<td>To hold the GRIN lens</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>MedChemExpress</td>
			<td>53716-49-7</td>
			<td>To reduce postoperative pain of the mouse&#160;</td>
		</tr>
		<tr>
			<td>Coax Cable</td>
			<td>Open ephys</td>
			<td>CW8251</td>
			<td>To connect the miniscope and the miniscope DAQ box</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus Life Science&#160;</td>
			<td>FV3000</td>
			<td>For observing the brain slices</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Nanchang Xiangyi Medical Devices Co.,Ltd</td>
			<td>20202140438</td>
			<td>For disinfection</td>
		</tr>
		<tr>
			<td>Customized headplate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For holding the mouse on the running wheel</td>
		</tr>
		<tr>
			<td>Customized headplate holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To hold the headplate of the mouse</td>
		</tr>
		<tr>
			<td>Denture base matierlals (self-curing)</td>
			<td>New Centry Dental</td>
			<td>430205</td>
			<td>For attaching the miniscope</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td>ASIN : B001DUUPQ0</td>
			<td>For removing the hair of the mouse</td>
		</tr>
		<tr>
			<td>Desktop digital stereotaxic in strument, SGL M</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>68803</td>
			<td>For viral injection and GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Huachu Co., Ltd.</td>
			<td>N/A</td>
			<td>To prevent postoperative inflammation of the mouse</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>RWD Life Science Co., Ltd</td>
			<td>MZ62-WX</td>
			<td>For observing the conditions during surgeries</td>
		</tr>
		<tr>
			<td>Gas filter canister, large, packge of 6</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-31-6</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>GRIN lens</td>
			<td>GoFoton</td>
			<td>CLHS100GFT003</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>GRIN lens</td>
			<td>InFocus Grin Corp</td>
			<td>SIH-100-043-550-0D0-NC</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Induction chamber-mouse (15 cm x 10 cm x 10 cm)</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>V100</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Industrial camera</td>
			<td>Daheng Imaging</td>
			<td>MER-231-41U3M-L, VS-0618H1</td>
			<td>For acquiring the behavioral data</td>
		</tr>
		<tr>
			<td>Iodophor disinfectant</td>
			<td>Qingdao Hainuo Innovi Disinfection Technology Co.,Ltd</td>
			<td>8861F6DFC92A</td>
			<td>For disinfection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-22-10</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Liquid sample collection tube (Glass Capillaries micropipette for Nanoject III)</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2021b</td>
			<td>For analyzing the data</td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>78001</td>
			<td>For craniotomy</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige International USA</td>
			<td>PC-100</td>
			<td>For pulling the liquid sample collection tube</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Miniscope DAQ Software</td>
			<td>Github (Aharoni-Lab/Miniscope-DAQ-QT-Software)</td>
			<td>N/A</td>
			<td>For recording the calcium imaging data</td>
		</tr>
		<tr>
			<td>Miniscope Data Acquisition (DAQ) Box (V3.3)</td>
			<td>Open ephys</td>
			<td>V3.3</td>
			<td>To acquire the calcium imaging data</td>
		</tr>
		<tr>
			<td>Miniscope V4</td>
			<td>Open ephys</td>
			<td>V4</td>
			<td>For in vivo calcium imaging</td>
		</tr>
		<tr>
			<td>Miniscope V4 base plate (Variant 2)</td>
			<td>Open ephys</td>
			<td>Variant 2</td>
			<td>For holding the miniscope</td>
		</tr>
		<tr>
			<td>nanoject III Programmable Nanoliter Injector</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-207</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Cisen Pharmaceutical Co.,Ltd.</td>
			<td>H37022025</td>
			<td>To keep the eyes moist</td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>LabServ</td>
			<td>309101009</td>
			<td>For dilue the virus</td>
		</tr>
		<tr>
			<td>Personal Computer (ThinkPad)</td>
			<td>Lenovo</td>
			<td>20W0-005UCD</td>
			<td>To record the calcium imaging data and behavioral data</td>
		</tr>
		<tr>
			<td>Running wheel</td>
			<td>Shanghai Edai Pet Products Co.,Ltd</td>
			<td>NA-H115</td>
			<td>For holding the mouse when affixing the base plate</td>
		</tr>
		<tr>
			<td>Screwdriver (M1.6 screws)</td>
			<td>Greenery (Yantai Greenery Tools Co.,Ltd)</td>
			<td>60902</td>
			<td>To unscrew the M1.6 screws</td>
		</tr>
		<tr>
			<td>Screwdriver (set screws)</td>
			<td>Greenery (Yantai Greenery Tools Co.,Ltd)</td>
			<td>S2</td>
			<td>For unscrew the set screws</td>
		</tr>
		<tr>
			<td>Set screw</td>
			<td>TBD</td>
			<td>2-56 cone point set screw</td>
			<td>For fasten the miniscope to its base plate</td>
		</tr>
		<tr>
			<td>Small animal anesthesia machine</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R500</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>Jiangsu Great Wall Medical Equipment Co., LTD</td>
			<td>20163140236</td>
			<td>For rinse the blood</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>S14016-13</td>
			<td>For cutting off the hair and scalp</td>
		</tr>
		<tr>
			<td>ThermoStar temperature controller,69025 pad incl.</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>69027</td>
			<td>To maintain the animal&#39;s body temperature</td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>F11020-11</td>
			<td>For removing the bone debris and dura</td>
		</tr>
		<tr>
			<td>USB 3.0 cable</td>
			<td>Open ephys</td>
			<td>N/A</td>
			<td>To connect the miniscope DAQ box and the computer</td>
		</tr>
		<tr>
			<td>UV light</td>
			<td>Jinshida</td>
			<td>66105854002</td>
			<td>To fix the GRIN lens on the skull</td>
		</tr>
		<tr>
			<td>UV resin (light cure adhesive)</td>
			<td>Loctite</td>
			<td>32268</td>
			<td>To fix the GRIN lens on the skull</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Kylin-Bell</td>
			<td>GL-802B</td>
			<td>To remove the blood, saline and the brain tissue</td>
		</tr>
	</tbody>
</table>

in vivo calcium imaging of granule cells in the dentate gyrus of hippocampus in mice 

学习和记忆研究通常需要实时方法， 体内 钙成像为研究清醒动物在行为任务期间的神经元活动提供了可能性。由于海马体与情景记忆和空间记忆密切相关，因此它已成为该领域研究中必不可少的大脑区域。在最近的研究中，通过使用微型显微镜记录小鼠海马 CA1 区域的神经活动，同时执行包括开场和线性跟踪在内的行为任务，研究了记忆蛋白细胞和位置细胞。虽然齿状回是海马体的另一个重要区域，但由于其更大的深度和成像的难度，它很少被用于 体内 成像研究。在该协议中，我们详细介绍了钙成像过程，包括如何注射病毒、植入 GRIN（梯度指数）透镜以及连接用于对海马齿状回成像的底板。我们进一步描述了如何使用 MATLAB 预处理钙成像数据。此外，对其他需要成像的大脑深部区域的研究可能受益于这种方法。

图 1 显示了实验程序的示意图，包括病毒注射、GRIN 晶状体植入、底板固定、通过微型镜 进行体内 钙成像和数据处理。一般来说，整个过程需要 1 个月。 图 2 显示了病毒注射的示例程序，包括在颅骨上钻孔的位置和 GRIN 晶状体植入前脑组织的状况。
图 3 显示了去除脑组织的过程，这对该手术的成功至...

作者聚焦：用微型显微镜研究齿状回状颗粒细胞的神经活动

海马体的齿状回在学习和记忆中执行重要而独特的功能。该协议描述了一组稳健而有效的程序，用于对自由移动的小鼠齿状回中的颗粒细胞 进行体内 钙成像。

小鼠海马齿状回管颗粒细胞的体内钙成像 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

in-vivo-calcium-imaging-granule-cells-dentate-gyrus-hippocampus

Research

JoVE Journal

Neuroscience

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice 

906 次观看.  Fudan University. 好吧，在这个协议中，我们使用微型显微镜来研究海马齿状回中的神经活动。我们旨在研究不同疾病条件下的神经元编码和空间表示。我相信研究体内齿状回的关键挑战在于获得高质量的钙成像数据，病毒表达是否令人满意，梯度是否植入适当的位置。像这样的细节，整个手术对于研究的成功至关重要。在传统的钙成像手术中，病毒注射和 GRIN ...

视频: 作者聚焦：用微型显微镜研究齿状回状颗粒细胞的神经活动

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

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

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

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

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

在基于生物发光钙指标GFP的水母体内脑功能成像方法

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

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

在体内 小鼠劣质橄榄中的钙成像

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

活体转基因小鼠脑围产层钙和血液学成像

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.