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。

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）
<ol><li>将一滴磷酸盐缓冲盐水（PBS，1x）放入丙烯酸培养皿中，并用21G扁平针触摸它，以通过毛细管填充尖端。</li>
	<li>用半透明的硅胶粘合剂覆盖针尖，并放置约~10分钟。</li>
	<li>用剪刀将硅胶粘合剂剪成直径为 ~2 毫米的圆盘。</li>
</ol>2.插头的准备（图1B）
<ol><li>准备一个 0.75 毫米厚的塑料垫片，并在中心切出一个 1 厘米 x 1 厘米的正方形。准备两个亚克力块。用酒精清洁垫片和亚克力块，然后用镜头纸擦拭。</li>
	<li>将垫片放在一个亚克力块上，将硅胶粘合剂沉积到中心以填充~90%的开口（避免气泡），添加另一个亚克力块和~1 kg力，然后等待20分钟。</li>
	<li>在手术显微镜下，用手术刀将硅胶粘合剂切割成 1 毫米高 x 1.5 毫米宽的三棱柱，上面印有三角形。</li>
	<li>用塑料胶带清除三棱镜上的所有灰尘，并将其放在直径为 5 毫米、厚度为 0.15 毫米的玻璃盖玻片上。</li>
	<li>将带有三棱柱的盖玻片放在电晕处理机下，打开电晕处理器，并将其靠近盖玻片，直到出现闪电。将它保持在该位置几分钟，直到它们相互连接。 注意： 对于其他电晕处理机，此步骤可能有所不同。</li>
	<li>将塞子放入培养皿中，并将其置于70°C的培养箱中过夜。</li>
</ol>3.动物的制备和病毒构建体的注射
<ol><li>在6周龄（成像时9周）使用C57BL / 6J（野生型）和Emx1-Cre：Pals1flox / wt （部分皮层）20 只小鼠。</li>
	<li>对用于外科手术的所有材料和器械进行消毒。</li>
	<li>用5%异氟烷麻醉小鼠，然后在手术过程中以1L / min的流速将异氟烷维持在1%-2%。通过缺乏脚趾捏合反射来确认麻醉水平。</li>
	<li>将鼠标头部固定在带有耳杆的立体定位框架上。在整个手术过程中，将眼药膏涂在小鼠眼睛上以防止干燥，并用恒温加热垫将其体温保持在37°C。</li>
	<li>剃掉毛皮并用碘伏和75%乙醇对皮肤表面进行消毒。注射利多卡因（5 mg/kg，皮下注射 [SQ]）、替利定（2 mg/kg，SQ）和美洛昔康（2 mg/kg，SQ）。 注意：绝育、镇痛和局部麻醉的手术程序可能因机构而异。</li>
	<li>用手术剪刀去除SC上的皮肤，露出颅骨。用棉签清洁头骨。</li>
	<li>调整立体定位框架，直到颅骨表面平坦。在λ的侧面和0.42毫米的侧面钻一个孔，直到硬脑膜暴露出来。</li>
	<li>用斜面玻璃微量移液管（尖端斜面至25°，直径为40-50μm）在距λ1mm和1.6mm的深度注射表达GCaMP6m（rAAV2 / 9-hsyn-GCaMP6m;1×1013GC / mL溶解在1x PBS中的腺相关病毒（AAV）。</li>
	<li>在每个深度，以 50 nL/min 的速度进样 100 nL，每次进样后等待 5 分钟。注射后，慢慢抽出注射器。</li>
</ol>4.头板的植入
<ol><li>清洁颅骨上方的肌肉和组织，并用刀片刮擦颅骨。如果发生出血，请用凝胶泡沫将其止住并清洁血液。用自固化牙科粘合剂树脂水泥将头板（图1C）连接到颅骨上。具体来说，将 3/4 勺聚合物、三滴单体和一滴催化剂混合在冰上的陶瓷碗中。将混合物涂抹在头板和头骨上。将它们连接在一起并等待 5 分钟让粘合剂凝固。</li>
	<li>注射抗生素（头孢噻呋钠，5 mg/kg，SQ）以预防感染。将鼠标从立体定位框架中取出，并将其放在加热垫上进行恢复。从麻醉中恢复后将其放回家笼。为了控制疼痛，在手术后向小鼠提供含布洛芬的饮用水（0.5 mL / 50 mL）3天。</li>
</ol>5.插入插头（图2）
<ol><li>在病毒注射后 3 周植入塞子。麻醉鼠标并将其固定在立体定位框架上，如步骤 3.2-3.5 中所述。准备浸泡在盐水中的凝胶泡沫以阻止潜在的出血。</li>
	<li>用微钻钻在λ后方0.5mm处钻出3 mm x 2 mm的椭圆形。将椭圆形的边界变薄，直到它破裂。在椭圆形记录窗口前将头骨变薄，使盖玻片靠近SC。</li>
	<li>使用细镊子取下骨瓣。在横窦后方的硬脑膜上做一个切口，取出硬脑膜。如有必要，用 3 mL 注射器向大脑中添加人工脑脊液 （ACSF） 以防止干燥。用凝胶泡沫和棉签擦干头骨和记录窗口。</li>
	<li>将一滴有机硅粘合剂沉积到记录窗口中。使用吸盘负压固定塞子。使用电动显微操纵器移动吸盘并将塞子放入硅胶粘合剂中，直到盖玻片接触颅骨。然后，将插头向前移动 ~1 毫米以推开横窦并露出 SC。此步骤应在有机硅粘合剂变粘之前 1-2 分钟内完成。</li>
	<li>清洁顶板，在塞子边界周围涂上氰基丙烯酸丁酯和树脂水泥，将其固定在顶板上。 注意：提供术后护理，如步骤 4.2 所述。</li>
</ol>6. 双光子成像（图3）
<ol><li>将动物头部固定在带有头板的旋转跑步机上。将跑步机放在双光子显微镜下，并将高度调整到合适的位置。在物镜和头板之间放置一个黑色铝制锥体，以防止显示器的光污染，从而获得视觉刺激。使鼠标习惯~15分钟。</li>
	<li>使用16倍放大倍率、0.8数值孔径（NA）和3毫米工作距离（WD）物镜在显微镜上进行双光子成像。使用由两个振镜扫描仪控制的具有锁模技术的钛蓝宝石激光器在 920 nm 处激发 GCaMP6m。在 20-80 mW 之间调整样品平面的激光功率。</li>
	<li>以 4.8 Hz 的频率扫描 600 μm x 600 μm 的视野，空间分辨率为 2.4 μ m/像素，并在深度达 350 μ m 的范围内对神经活动进行成像。 注意： 采样率和空间分辨率适用于振镜扫描仪，应针对共振扫描仪进行调整。</li>
	<li>用二向色镜收集发射的荧光，并在通过带通滤光片后使用光电倍增管（PMT）进行检测。</li>
	<li>使用旋转编码器记录鼠标在跑步机上的运动。使用带有 50 毫米镜头的相机记录其瞳孔大小和位置。通过记录刺激计算机发送的触发器，将这些记录和图像采集与视觉刺激同步。</li>
</ol>7. 双光子成像测量的钙反应分析
<ol><li>使用 SIMA21 或 NoRMCorre22 纠正成像过程中的大脑运动。</li>
	<li>使用细胞魔棒工具 （ImageJ） 手动绘制感兴趣区域 （ROI），并在 MATLAB 中用椭圆拟合。去除神经细胞污染后提取每个ROI的荧光痕迹： <img alt="Equation 1" src="/files/ftp_upload/65181/65181eq01.jpg" style="margin: auto;"> F true 和 F raw分别是 ROI 的校正和原始荧光振幅，F neuropil 是周围 neuropil 的荧光振幅，r 是离焦的 neuropil污染因子（我们的设置为 0.7）。r 是通过测量血管上的信号来估计的，其中 Ftrue = 0，如前所述23,24。</li>
	<li>通过从以每帧为中心的 15 秒窗口中减去第八个百分位值来消除缓慢的基线波动25.</li>
	<li>将神经元的视觉反应定义为刺激期间其 ROI 中荧光振幅的相对变化： <img alt="Equation 2" src="/files/ftp_upload/65181/65181eq02.jpg" style="margin: auto;"> F峰 是视觉刺激期间荧光迹线的峰值振幅，F基线是刺激前0.5秒内的平均振幅。</li>
</ol>8. 宽视场成像和数据分析（图4）
<ol><li>使用串联透镜4 构建宽视场宏镜（图4A）。串联镜头宏量镜是通过 适配器连接的两个 相机镜头（50 mm 和 105 mm），放大倍率为 ~2 倍。</li>
	<li>如步骤3.2-3.5所述制备部分皮层突变小鼠。在SC每侧中心200μm的深度注射100nL表达GCaMP6m的AAV。</li>
	<li>~3 周后，在 SC 上植入直径为 5 mm 的盖玻片，以 SC 为中心进行 4 mm x 3 mm 的椭圆形开颅手术，并稀释其周围的骨头。然后，将盖玻片直接压在SC上，并用树脂水泥固定。</li>
	<li>使用带有激发滤光片（469 nm ± 35 nm）的蓝光发光二极管 （LED） 激发 GCaMP6m。在以 10 Hz 通过发射滤光片（525 nm ± 39 nm）后，使用互补金属氧化物半导体 （CMOS） 相机采集图像。该相机覆盖面积为 5.36 mm x 2.85 mm，空间分辨率为 2.63 μm/像素。</li>
	<li>使用归一化盒式滤波器对每个采集的图像进行卷积，并将其下采样到原始大小的 1/4。对于图像中的每个像素，定义响应，如步骤 7.4 中所述。 注意：提供术后护理，如步骤 4.2 所述。</li>
</ol>

上丘的双光子和宽场钙成像

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作者没有什么可透露的。

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。</...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-mm lens</td>
			<td>Computar</td>
			<td>M5018-MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mm coverslip</td>
			<td>Warner instruments</td>
			<td>CS-5R</td>
			<td></td>
		</tr>
		<tr>
			<td>bandpass filter</td>
			<td>Chroma Technology</td>
			<td>HQ575/250 m-2p</td>
			<td></td>
		</tr>
		<tr>
			<td>butyl cyanoacrylate</td>
			<td>Vetbond, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera for monitoring pupil</td>
			<td>FLIR</td>
			<td>BFS-U3-04S2M-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>camera for widefield imaging</td>
			<td>Basler</td>
			<td>acA2000-165&#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>corona treater</td>
			<td>Electro-Technic Products</td>
			<td>BD-20AC</td>
			<td></td>
		</tr>
		<tr>
			<td>dichroic</td>
			<td>Chroma Technology</td>
			<td>T600/200dcrb&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>galvanometers</td>
			<td>Cambridge Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bead sterilizer</td>
			<td>RWD</td>
			<td>RS1502</td>
			<td></td>
		</tr>
		<tr>
			<td>microdrill</td>
			<td>RWD</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>QUAD</td>
			<td></td>
		</tr>
		<tr>
			<td>photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>R3896</td>
			<td></td>
		</tr>
		<tr>
			<td>rotory encoder</td>
			<td>USdigital</td>
			<td>MA3-A10-125-N</td>
			<td></td>
		</tr>
		<tr>
			<td>self-curing dental adhesive resin cement&#160;</td>
			<td>SuperBond C&#38;B, Sun Medical Co, Ltd. Moriyama, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>thermostatic heating pad&#160;</td>
			<td>RWD</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser</td>
			<td>Spectra-Physics</td>
			<td>Mai Tai HP DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>translucent silicone adhesive&#160;</td>
			<td>Kwik-Sil, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>treadmill</td>
			<td>Xinglin Biology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Virus Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV2/9-hsyn-Gcamp6m</td>
			<td>Vector Core at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J wild type</td>
			<td>Laboratory Animal Resource Center at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emx1-Cre</td>
			<td>The Jackson Laboratory&#160;</td>
			<td>5628</td>
			<td></td>
		</tr>
		<tr>
			<td>Pals1flox/wt</td>
			<td>Christopher A. Walsh Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH Image</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium imaging in mouse superior colliculus

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。

The superior colliculus (SC) is an important visual center in all vertebrates. In mammals, it receives direct inputs from the retina and the visual cortex1. While optical recording has been widely applied to the cortex2,3,4,5, its application in the SC is hindered by poor optical accesss6,7,8,9,10,11,12,13,14,15,16,17,18,19. The goal of this protocol is to provide details about two complementary methods for optical recording of the neural activity in the SC.
The SC is located beneath the cortex and transverse sinus, which limits optical access to the collicular neurons. One approach to overcome this limitation is to aspirate the overlaying cortex and expose the anterior-lateral SC7,9,10,13,14,19. However, because the SC receives cortical inputs, such an operation might affect how the SC neurons respond to visual stimuli. To overcome this limitation, we detail here an alternative protocol to image the superficial layer of the posterior-medial SC with a silicon plug, while leaving the cortex intact8,11. Specifically, to achieve single-cell resolution, we applied two-photon microscopy to image calcium responses in the posterior-medial SC of wild-type mice. In addition, to achieve broad coverage, we applied wide-field microscopy to image the entire SC of a mutant mouse whose posterior cortex has not developed20.
The two methods described in this protocol are complementary to each other. The two-photon calcium imaging without ablating the cortex is appropriate for recording neural activity at&#160;single-cell resolution with intact cortical inputs. The wide-field calcium imaging is appropriate for recording neural activity in the entire SC while sacrificing spatial resolution.

作者聚焦：揭示上丘的神经编码和视觉处理机制 

小鼠上丘的钙成像

My research focuses on neural coding and mechanisms for visual processing. We are trying to answer how various information is encoded in the brain, especially in the superior colliculus under the underlying neuro-mechanisms. To understand visual processing, scientists combine various approaches, including neuronal morphology, antegrade and retrograde tracing, IL sequencing, neuro modeling, machine learning, is understood well.The challenge is to remove the skull over the SSA, cutting and left in the bone in one larger piece, is likely to fail because the bone is attached to the dura. By combining two photo on a wide field calcium image, our recent work revealed the function architecture of motion direction in the mouse SC at both single cell resolution and a global scale. We found that neurons with similar preference form patches up to 500 micrometer under global to the upward on the nasal motion.With our protocol, we are addressing two research gaps. First, researchers can perform long-term cast imaging in mouse SC at a single cell resolution without breaking the cortex. Secondly, researchers can record the neuroactivity across the entire SC using widefield microscoping.Our protocol provides a measure to image the posterior medial SC at single cell resolution with a intact cortex in mice. Also, we use a biocompatible plug to expose the SC, which reduces infection for chronic imaging. Our results provide a way to study the neural coding of visual information across a large visual field.Combined with optogenetics, one can study half inputs from different brain regions modulating the neuroactivity in SC.

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?...

Watch this Scientific Journal Video about Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus at JoVE.com

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。

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

我的研究重点是神经编码和视觉处理机制。我们正试图回答各种信息是如何在大脑中编码的，特别是在潜在神经机制下的上丘中。为了理解视觉处理，科学家们结合了各种方法，包括神经元形态学、顺行和逆行追踪、IL测序、神经建模、机器学习，都很好理解。挑战在于移除SSA上方的头骨，切开并留在骨头中，因为骨头附着在硬脑膜上，因此可能会失败。通过在宽视场钙图像上结合两张照片，我们最近的工作揭示了小鼠SC在单细胞分辨率和全局尺度上运动方向的功能结构。我们发现，具有相似偏好的神经元在鼻腔运动中形成高达500微米的斑块。通过我们的协议，我们正在解决两个研究差距。首先，研究人员可以在不破坏皮层的情况下以单细胞分辨率对小鼠SC进行长期铸造成像。其次，研究人员可以使用宽场显微镜记录整个SC的神经活动。我们的方案提供了一种以单细胞分辨率对小鼠皮层完整的后内侧SC进行成像的措施。此外，我们使用生物相容性塞子暴露皮状细胞瘤，从而减少慢性成像的感染。我们的研究结果提供了一种在大视野中研究视觉信息的神经编码的方法。结合光遗传学，人们可以研究来自不同大脑区域调节 SC 神经活动的一半输入。

calcium-imaging-in-mouse-superior-colliculus

Research

JoVE Journal

Neuroscience

Calcium Imaging in Mouse Superior Colliculus

1.8K 次观看.  Chinese Institute for Brain Research. 我的研究重点是神经编码和视觉处理机制。我们正试图回答各种信息是如何在大脑中编码的，特别是在潜在神经机制下的上丘中。为了理解视觉处理，科学家们结合了各种方法，包括神经元形态学、顺行和逆行追踪、IL测序、神经建模、机器学习，都很好理解。挑战在于移除SSA上方的头骨，切开并留在骨头中，因为骨头附着在硬脑膜上，因此可能会失败。通过在宽视场?...

视频: 作者聚焦：揭示上丘的神经编码和视觉处理机制 

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.

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.

科研

神经科学

Preparation of a Suction Cup and Plugs for Calcium Imaging in Mouse

Begin the process of creating a suction cup by depositing a drop of PBS in an acrylic dish. Use a 21 gauge flat needle to fill the tip of the needle with PBS by capillarity. Cover the tip of the needle with a translucent silicone adhesive, and set it for approximately 10 minutes.Cut the silicone adhesive with scissors to a disc with a two millimeter diameter. To prepare the plugs, take a 0.75 millimeter thick plastic shim stock and cut out a one by one centimeter square in the center. Clean the shim stock and two acrylic blocks with alcohol, and wipe them with lens paper.Place the shim stock on one acrylic block, and deposit silicone adhesive into the center to fill 90%of the opening. Add another acrylic block. Apply approximately one kilogram of force, and wait 20 minutes.Under a surgical microscope, cut the silicone adhesive with a scalpel into one by 1.5 millimeter triangular prisms. Remove any dust from the triangular prisms with sticky plastic tape, and place them on a glass cover slip. Place the cover slip with the triangular prisms under a corona treater and turn it on.Bring the corona treater closer to the cover slip until lightning appears, and hold it in that position for a few minutes until the prisms and cover slip are attached. After placing the plugs in a Petri dish, incubate at 70 degrees Celsius overnight.

用于小鼠钙成像的吸盘和吸盘的制备

Viral Construct (AAV) Injection in Mouse Superior Colliculus for Calcium Imaging

Begin by securing the anesthetized mouse onto a stereotaxic frame with ear bars. Apply ophthalmic ointment on the mouse's eyes to prevent drying, and maintain the body temperature of the mouse at 37 degrees Celsius with a thermostatic heating pad. After shaving the fur and sterilizing the surgical site, inject lidocaine, tilidine, and meloxicam.Once the skin over the SC is removed, clean the skull with a cotton swab. Adjust the stereotaxic frame until the skull surface is flat. Using a drill, create a whole 0.5 millimeter lateral, and 0.42 millimeter anterior to the lambda, until the dura is exposed.Inject an adeno-associated virus, or AAV, expressing GCaMP6m, at depths of one millimeter and 1.6 millimeters from the Lambda using a beveled glass micro-pipette. After the injection, slowly withdraw the injector and proceed with the implantation of the head plate.

在小鼠上丘细胞中注射病毒构建体 （AAV） 用于钙成像

Implantation of the Headplate in Mouse for Calcium Imaging 

After injecting the viral construct into the mouse brain, clean the muscles and tissues over the skull, and scratch the skull with a blade. Prepare the dental adhesive resin cement by mixing 3/4 spoonful of polymer, three drops of monomer, and one drop of catalyst in a ceramic bowl on ice. Apply the mixture to the head plate and the skull.Attach them and wait for the adhesive to set for five minutes. Finally, inject antibiotics to prevent infections. After removing the mouse from the stereotaxic frame, place it on a heating pad for recovery, and return it to the home cage after recovering from anesthesia.

在小鼠体内植入头板进行钙成像 

Implanting the Plug in the Mouse for Calcium Imaging

Three weeks after the viral infection, anesthetize and fix the mouse with a head plate on a stereotaxic frame. Soak a gel foam in saline to stop potential bleeding. Use a micro-drill to create a three by two millimeter oval, centered at 0.5 millimeter posterior to the lambda.Thin the boundary of the oval until it cracks, then thin the skull before the oval recording window to close the cover slip to the SC.Using fine forceps, take off the bone flaps. Make a cut down the dura, posterior to the transverse sinus and remove the piece of dura. Dry the skull and the recording window with gel foam and cotton swabs.Then deposit a drop of silicone adhesive into the recording window. Use the suction cup to hold the plug with negative pressure, and use a motorized micro-manipulator to move the suction cup and lower the plug into the silicone adhesive until the cover slip touches the skull. Once the head plate is cleaned, apply butyl cyanoacrylate and resin cement around the boundary of the plug to fix it to the head plate and proceed with imaging.

将塞子植入鼠标进行钙成像

Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons

After implanting the head plate and plug, secure the animal on a rotating treadmill with its head plate. Place the treadmill beneath the two-photon microscope and adjust the height to an appropriate position. Place a black aluminum cone between the objective and the head plate to prevent light contamination from the monitor for visual stimulation.To perform two-photon imaging, adjust the laser power at the sample plane between 20 and 80 milliwatts. Scan a 600-by-600 micrometer field of view at 4.8 hertz with a spatial resolution of 2.4 micrometers per pixel and image neural activity up to 350 micrometers in depth. Using a rotary encoder, record the mouse's locomotion on the treadmill.Use a camera with a 50 millimeter lens to record its pupil size and position. Lastly, synchronize these recordings, and image acquisitions to visual stimulation by recording triggers sent by the stimulation computer. The calcium responses of SC neurons from a wild type mouse and the standard deviation projection of the fluorescence across images are presented.

小鼠上丘神经元的双光子钙成像

Wide-Field Calcium Imaging of Mouse Superior Colliculus Neurons

To begin, inject 100 nanoliters of AAV expressing GCaMP6m at a depth of 200 micrometers at the center of each side of the SC to the mutant mouse whose posterior cortex has failed to develop. After approximately three weeks, implant a five millimeter diameter cover slip over the SC.After fixing the cover slip, acquire images using a CMOA camera after passing an emission filter at 10 hertz. The calcium responses of SC neurons from a partial cortex mutant mouse were imaged using wide-field microscopy.The acquired image was down sampled to one quarter of the original size, and visually evoked calcium responses are shown.