Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Hisato Maruoka; Ryosuke Kamei; Shunsuke Mizutani; Qingrui Liu; Shigeo Okabe

doi:10.3791/64111

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

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

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08:26 min

August 23, 2022

DOI: 10.3791/64111-v

Hisato Maruoka¹, Ryosuke Kamei¹, Shunsuke Mizutani¹, Qingrui Liu¹, Shigeo Okabe¹

¹Department of Cellular Neurobiology, Graduate School of Medicine and Faculty of Medicine,The University of Tokyo

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Overview

This study outlines a protocol that combines adeno-associated virus (AAV) injection with cranial window implantation to enable the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. The method allows researchers to investigate the surveillance behavior of microglia and their interactions with neurons while minimizing motion artifacts during imaging.

Key Study Components

Area of Science

Neuroscience
Neuroimaging
Neurobiology

Background

Microglia play an essential role in brain health and disease.
Real-time imaging of microglial dynamics is crucial for understanding their functions.
AAV is commonly used for delivering genetic material in neuroscience applications.
Head fixation in awake mice reduces motion artifacts during imaging.

Purpose of Study

To develop a reliable method for imaging microglia and neuron interaction.
To enhance understanding of microglial dynamics and neuronal activity under physiological conditions.
To minimize data contamination from motion artifacts during imaging.

Methods Used

The protocol involves AAV injection and cranial window implantation in the primary visual cortex of awake mice.
The biological model consists of transgenic mice expressing fluorescent proteins.
Key steps include precise stereotaxic coordinates for injection and surgical procedures for cranial window placement.
Imaging was conducted using two-photon microscopy at a frame rate of 30 Hz.
Calcium traces from neurons and microglia were analyzed in response to visual stimuli.

Main Results

Fast dynamics were observed in microglial processes, which changed morphology within 10 seconds.
Simultaneous imaging revealed neuronal activity and microglial dynamics in response to visual stimuli.
The protocol validated the effectiveness of AAV and cranial windows for high-quality imaging.

Conclusions

This method enables real-time observation of microglia and neuronal interactions, providing insights into brain dynamics.
The study contributes valuable tools for understanding the roles of microglia in neuronal mechanisms.
Future applications may include investigating the effects of various interventions on microglial and neuronal activity.

Frequently Asked Questions

What are the advantages of the combined AAV injection and cranial window implantation?

This approach minimizes motion artifacts during imaging, allowing for clear visualization of both microglial dynamics and neuronal activity in awake mice.

How is the biological model of transgenic mice used in this study?

Transgenic mice expressing fluorescent proteins enable the real-time imaging of microglial and neuronal activity under physiological conditions.

What types of data are obtained from this imaging technique?

The imaging technique captures calcium traces from neurons and microglia, providing insights into their dynamics and interactions in response to stimuli.

Can this method be adapted for other brain regions or disorders?

Yes, this protocol can be adapted to target different brain regions or to investigate various neurological disorders by altering the injection site or the AAV serotype used.

What are the key limitations of this method?

Technical challenges may arise during surgery and AAV injection, particularly for novice researchers. Patience and practice are necessary to achieve consistent results.

How does the setup prevent light contamination during imaging?

Black aluminum foil is used to cover the objective lens, reducing light contamination from external sources such as LCD monitors used for visual stimuli.

在这里，我们描述了一种将腺相关病毒注射与颅窗植入相结合的方案，用于同时对清醒小鼠的小胶质细胞动力学和神经元活动进行成像。

我们的协议能够同时对清醒小鼠的小胶质细胞动力学和神经元活动进行成像。它可以广泛用于研究小胶质细胞的监视行为或与神经元的相互作用。这种方法的优点是，由于头部固定牢固，运动伪影不容易污染成像数据。

此外，以高帧率成像后的恢复使我们能够消除数据中的运动伪影。在这种方法中，AAV注射和颅窗植入手术在技术上要求很高。一开始失败是很常见的，所以请不要沮丧，多练习。

为了制备注射装置，将连接到26号汉密尔顿注射器的玻璃移液器通过管子放在立体定位仪器的移液器支架上。然后，将移液器支架从垂直轴向前倾斜 60 度。用液体石蜡填充玻璃移液器、注射器和连接管，并将注射器放在微量注射器上。

对麻醉小鼠进行镇痛并连接辅助耳杆。然后，将动物固定在立体定位仪器上，其背侧朝上。从手术部位去除毛发并对手术区域进行消毒后，沿中线在头皮上做一个两厘米长的切口，确保右侧初级视觉皮层上方的颅骨良好暴露。

使用镊子去除暴露的颅骨上的骨膜。在中线外侧 3 毫米和λ线前方 5 毫升的立体定位坐标上钻颅骨，以创建一个直径约为 0.5 毫米的小孔。然后，将一块大约两厘米乘两厘米的透明薄膜放在暴露的小鼠头骨上。

使用移液器将一微升AAV溶液液滴排出薄膜上。推进注射器。并将玻璃移液器尖端放入薄膜上的AAV溶液液滴中，轻轻拉动注射器以吸出AAV溶液。

接下来，将玻璃移液管通过颅骨中形成的孔从大脑表面插入 500 微米的深度。然后，使用微量注射器以每小时2微升的注射体积流速注入0.5微升AAV溶液。抽出针头并用生理盐水冲洗脑表面。

通过钻孔沿着头骨上的标记创建一个圆形凹槽。清洁碎屑以确保头骨的可见性，并涂抹盐水以防止钻孔过程中加热。用镊子轻轻按压中央颅骨。

如果垂直移动且阻力很小，则钻孔深度就足够了。当凹槽达到足够的深度时，将镊子的尖端插入中央颅骨碎片的底部。轻轻提起并取下它以露出大脑表面。

使用27号针，刺破并撕裂暴露的大脑表面边缘的硬脑膜。将镊子的尖端插入硬脑膜边缘的孔中，然后将其剥离以露出大脑表面。在盐水中逐个分散止血纤维后，将止血纤维沿着存在横断面硬脑膜的孔的边缘放置。

将颅窗放在暴露的脑表面，然后用即时胶将其粘附在头骨上，同时轻轻按压窗户。之后，在整个暴露的头骨上涂抹即时胶水。然后，小心地将头板连接到头骨上，将颅窗定位在头板方孔的中心。

一旦胶水充分硬化，将牙科水泥涂在暴露的头骨上，以加强头部和头刀片之间的连接。要安装定制的着色装置和用于视觉刺激的LCD监视器，请首先将镜头定位为聚焦在大脑表面，然后将该镜头位置设置为原始Z位置。保持XY坐标恒定并抬高物镜。

从物镜上取下鼠标和立体定位仪器。使用硅胶在顶板顶部安装遮阳装置，确保顶板和遮阳装置之间的空间密封良好。用蒸馏水填充遮阳装置。

然后，将鼠标与物镜下方的立体定位框架固定。小心地重置大脑表面的焦平面，检查物镜的深度。用黑色铝箔盖住物镜，以避免液晶显示器的光污染。

将 10 英寸 LCD 显示器设置在鼠标眼前 12.5 厘米处，以呈现视觉刺激。配置EGFP和R-CaMP的荧光提交收集滤光片，并将激发波长配置为1， 000纳米。采集空间分辨率为每像素 0.25 微米的图像。

找到可以同时对R-CaMP阳性神经元和EGFP阳性小胶质细胞进行成像的区域。在第 2、3 层中。以 30 赫兹的帧速率采集图像。

在图像采集的同时，以6个方向从0到150度，以30度的步长，在12个方向上向鼠标呈现漂移光栅视觉刺激。图像采集后，将鼠标从显微镜载物台上移开。从鼠标上拆下着色装置和立体定位仪器，然后将鼠标放回其主笼。

使用该协议，在八周龄转基因小鼠的初级视觉皮层中进行AAV注射和颅窗植入，然后在第2层，第3层对基于R-CaMP的神经活动和小胶质细胞动力学进行两次光子成像。将光栅视觉刺激呈现给小鼠，并使用钙迹线分析脊椎中的视觉反应。小胶质细胞过程显示出快速的动力学并在10秒内改变了它们的形态。

在12周龄转基因小鼠初级视觉皮层的第2层，第3层中使用双光子成像，在神经元中观察到R-CaMP，此处在洋红色中观察到。在小胶质细胞中观察到EGFP，呈绿色。还观察到EGFP和R-CaMP的个体信号。

成功的AAV注射对于该方法至关重要。AAV注射失败的主要原因是时钟玻璃移液器和组织损伤。小胶质细胞的监视行为和SNAP的小胶质细胞相互作用已被确定为在各种致病机制中普遍存在，如阿尔茨海默病。

我们的方法有利于专注于该领域的研究。

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