小鼠上丘的钙成像
Summary
该方案详细介绍了在清醒小鼠的上丘（SC）中对钙反应进行成像的过程，包括用双光子显微镜成像单神经元活动，同时在野生型小鼠中保持皮层完整，以及在部分皮层突变小鼠中用宽场显微镜对整个SC进行成像。
Abstract
上丘（SC）是所有脊椎动物进化上保守的中脑结构，是大脑皮层出现之前最复杂的视觉中心。它接收来自 ~30 种视网膜神经节细胞 （RGC） 的直接输入，每种细胞编码特定的视觉特征。SC 是否只是继承视网膜特征，或者 SC 中是否发生了额外的和潜在的 从头 处理，仍然难以捉摸。为了揭示SC中视觉信息的神经编码，我们在这里提供了一个详细的协议，以在清醒小鼠中使用两种互补方法光学记录视觉反应。一种方法使用双光子显微镜以单细胞分辨率对钙活性进行成像，而不消融覆盖的皮层，而另一种方法使用宽视场显微镜对皮层基本未发育的突变小鼠的整个SC进行成像。该协议详细介绍了这两种方法，包括动物制备、病毒注射、头板植入、栓子植入、数据采集和数据分析。代表性结果表明，双光子钙成像揭示了单细胞分辨率下的视觉诱发神经元反应，而宽视野钙成像揭示了整个SC的神经活动。通过结合这两种方法，人们可以揭示SC中不同尺度的神经编码，并且这种组合也可以应用于其他大脑区域。
Introduction
上丘（SC）是所有脊椎动物的重要视觉中枢。在哺乳动物中，它接收来自视网膜和视觉皮层的直接输入1。虽然光学记录已广泛应用于皮层 2,3,4,5，但其在 SC 中的应用受到不良光学访问的阻碍 6,7,8,9,10,11,12,13,14,15,16,17 ，18,19.该协议的目标是提供有关用于光学记录SC中神经活动的两种互补方法的详细信息。
SC 位于皮层和横窦下方，这限制了对胶状神经元的光学接触。克服这一限制的一种方法是吸出覆盖的皮层并暴露前外侧 SC 7,9,10,13,14,19。然而，由于 SC 接收皮层输入，这样的操作可能会影响 SC 神经元对视觉刺激的反应。为了克服这一限制，我们在这里详细介绍了一种替代方案，即用硅塞对后内侧 SC 的浅层进行成像，同时保持皮层完整 8,11。具体来说，为了实现单细胞分辨率，我们应用双光子显微镜对野生型小鼠后内侧SC的钙反应进行成像。此外，为了实现广泛的覆盖范围，我们应用宽视野显微镜对后皮层尚未发育的突变小鼠的整个SC进行成像20。
该协议中描述的两种方法是相互补充的。不消融皮层的双光子钙成像适用于以单细胞分辨率记录具有完整皮层输入的神经活动。宽视场钙成像适用于记录整个SC的神经活动，同时牺牲空间分辨率。
Protocol
所有实验程序均按照动物福利指南进行，并得到北京中国脑科学研究院IACUC的批准。 注意：该协议的时间表如下：1）制作吸盘;2）注射病毒;3）植入头板;4）3周后，植入塞子;5） 在跑步机上恢复和适应 ~3 天后，进行双光子/宽视场成像。 1.吸盘的准备（图1A） 将一滴磷酸盐缓冲盐水（PBS，1x）放入丙烯…
Representative Results
图 1A、B 分别显示了如何制作吸盘和塞子。图 2 显示了如何成功植入插头。植入塞子后，后内侧SC暴露在外，如图2D所示。图 3 显示了使用双光子显微镜成像的野生型小鼠示例的 SC 神经元的钙反应。在显微镜下很容易捕捉到的三棱柱可用于定位成像部位（图3B）。视觉反应?…
Discussion
协议中的关键步骤
最关键的一步是步骤 5.2 和 5.3 中的开颅手术。首先，λ后方 0.5 毫米处的骨头很厚，里面有血管，在钻孔过程中会导致出血。应准备足够的凝胶泡沫来止血。其次，切除横窦上方的骨头时，很有可能发生血管出血。对于故障排除，另一种方法是将椭圆形内的骨头变薄并一块一块地移除。另一个技巧是在提升前浸泡骨头，使硬脑膜更容易从骨头上分离并防止出血。</…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金（32271060）的支持。Y.-t.L.设计了研究，进行了实验，分析了数据，并撰写了手稿。Z.L.和R.W.进行了实验。
Materials
| 16x objective | Nikon | ||
| 50-mm lens | Computar | M5018-MP2 | |
| 5-mm coverslip | Warner instruments | CS-5R | |
| bandpass filter | Chroma Technology | HQ575/250 m-2p | |
| butyl cyanoacrylate | Vetbond, World Precision Instruments | ||
| camera for monitoring pupil | FLIR | BFS-U3-04S2M-CS | |
| camera for widefield imaging | Basler | acA2000-165µm | |
| corona treater | Electro-Technic Products | BD-20AC | |
| dichroic | Chroma Technology | T600/200dcrb | |
| galvanometers | Cambridge Technology | ||
| glass bead sterilizer | RWD | RS1502 | |
| microdrill | RWD | 78001 | |
| micromanipulator | Sutter Instruments | QUAD | |
| photomultiplier tube | Hamamatsu | R3896 | |
| rotory encoder | USdigital | MA3-A10-125-N | |
| self-curing dental adhesive resin cement | SuperBond C&B, Sun Medical Co, Ltd. Moriyama, Japan | ||
| thermostatic heating pad | RWD | 69020 | |
| Ti:Sapphire laser | Spectra-Physics | Mai Tai HP DeepSee | |
| translucent silicone adhesive | Kwik-Sil, World Precision Instruments | ||
| treadmill | Xinglin Biology | ||
| Virus Strains | |||
| rAAV2/9-hsyn-Gcamp6m | Vector Core at Chinese Institute for Brain Research, Beijing | ||
| Animals | |||
| C57BL/6J wild type | Laboratory Animal Resource Center at Chinese Institute for Brain Research, Beijing | ||
| Emx1-Cre | The Jackson Laboratory | 5628 | |
| Pals1flox/wt | Christopher A. Walsh Lab | ||
| Software | |||
| ImageJ | NIH Image | ||
| Labview | National Instruments | ||
| MATLAB | Mathworks |
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Citar este artigo
Li, Z., Wu, R., Li, Y. Calcium Imaging in Mouse Superior Colliculus. J. Vis. Exp. (194), e65181, doi:10.3791/65181 (2023).