targeted brain region microinjection of bromophenol dye in mice

优化小鼠立体定向显微注射的准确性

Nearly all modern neuromodulation tools, including in vivo calcium recording, optogenetic, and chemogenetic tools, require the use of stereotaxic coordinates to target the brain area of interest1,2,3, forming the foundation of neural manipulation. Stereotaxic coordinates for mouse brain regions are defined in relation to bregma and lambda, the bony landmarks on the cranium, forming the so-called skull-derived stereotaxic coordinate system. Either bregma or lambda can serve as the zero point of the three-dimensional coordinates. The three axes are anteroposterior (AP), mediolateral (ML), and dorsoventral (DV), representing the y, x, and z axes on the digital display of stereotaxic instruments. For well-known brain regions, the stereotaxic coordinate parameters of a specific area can be obtained from mouse brain atlases4 (e.g., Paxinos and Franklin&#39;s mouse brain in stereotaxic coordinates) and/or the published literature5,6. However, further validation is necessary due to variations in stereotaxic equipment and the age/populations of mice used by different researchers.
Structure is the basis of function. Neural circuits form the foundation for many brain functions, such as cognitive activities, emotion, memory, sensory, and motor functions1. Labeling the structure and manipulating the activity of neural circuits are vital for understanding the function of a specific neural circuit. Over the past decades, neural tracers have evolved through many generations; early research adopted wheat germ agglutinin (WGA) and phaseolus vulgaris agglutinin (PHA) as anterograde tracers, and fluorogold (FG), cholera toxin subunit B (CTB), carbocyanine as retrograde tracers. However, unlike viral tracers, these traditional neural tracers cannot integrate exogenous genes into the host, nor do they have cell type selectivity. Nowadays, the viral strategy has become an important proposition during basic neuroscience research. For different research purposes, various viral tools can be selected7,8. There are non-transsynaptic viruses, trans-multisynaptic viruses (retrograde and anterograde), and trans-monosynaptic viruses (retrograde and anterograde). Each category contains several types with respective characteristics.
The process of viral administration and expression is highly time- and resource-intensive, often taking weeks or even longer. Among various viral vectors, adeno-associated virus has been identified as a promising means for gene delivery, providing a wide window ranging from 3 to 8 weeks post-injection for the experimental procedure7,8. As AAV evolves, analysis can be performed 2-3 weeks after administration9,10. Other neural circuit tracers, such as pseudorabies virus (PRV) and rabies virus (RV), also require a tracing period of at least 2-7 days11,12,13,14,15. Thus, a preliminary verification of the injection site before observing fluorescence signals is both time- and cost-effective.
To facilitate a simple and rapid verification of stereotaxic injections, in this study, a dye is administered before viral vectors, and cryosectioning enables researchers to observe the injection site and track it within 30 min post-injection.

作者聚焦：通过立体定向注射和经济高效的染料加速小鼠认知障碍研究

通过冷冻切片初步验证立体定向注射坐标

Our research primarily focus on investigating the neural circuits mechanism underlying cognitive impairment in mice. And we frequently employ brain stereotaxic techniques in these studies. Our objective is to optimize and accelerate the verification process for stereotaxic injection sites using dyes, ensuring that it becomes a simple and rapid procedure.Various strategies have become crucial in neuroscience research through stereotaxic injection of virus. This is followed by observation of fluorescent markers in tissue slice to confirm the injection sites, a process that takes several weeks to complete. Our protocol offers advantages over other techniques by saving at least three days of dehydration time and using a cost-effective dye for quicker identification of injection sites.It may save weeks compared with no preliminary verification.

小鼠靶向脑区显微注射溴酚染料

首先将无菌手术器械放在工作台上。为了将麻醉的小鼠固定在立体定位框架中，将上切牙与鼻夹混合，并用两个耳杆稳定头部。用眼剪刀在头皮上做一个0.5厘米的切口，露出头盖骨。用棉签蘸 3% 过氧化氢，擦洗暴露的颅骨以去除骨膜。让颅骨干燥。将钻头固定在立体定位仪器上并调整立体定位臂的旋钮。在放大镜的帮助下，将钻尖精确地定位在前臂上。使用 LCD 数字显示器记录前囟处的 Z 轴值。现在，将钻头放置在 lambda 处并记录 Z 轴值。调整鼻夹以最小化两个 Z 值之间的差异。将钻头放置在 X 坐标减 5.2 处，Y 坐标减 0.8 处，以测量背腹方向的 Z 轴值。在坐标减去 5.2（X 值）减去 0.8（Y）（值）处重新定位钻孔，然后再次测量 Z 轴值。将耳杆调整到指定的刻度。将钻头定位到前门，并在 LCD 数字显示屏上将三个坐标设置为零。使用参考坐标，将钻头放置在感兴趣区域上方。启动钻头，使用垂直臂逐渐降低钻头，直到钻头穿透颅骨。用棉签清除碎屑和血液。将 25 微升含有溴酚蓝的 SDS-PAGE 上样缓冲液和 50 微升双蒸水加入 200 微升管中。用盐水湿润的化妆棉盖住颅窗。将 0.3 微升染料加载到微量注射器中。将注射器和电动立体定位微注射器连接到立体定位臂。将注射器尖端放在前囟处，并在 LCD 数字显示屏上将坐标设置为零。根据参考坐标，将针头调整到感兴趣的区域。将注射器速度设置为每分钟 0.1 微升，然后启动电动微注射器，将 0.3 微升染料输送到目标区域。10 分钟后，慢慢抽出注射器。

targeted-brain-region-microinjection-of-bromophenol-dye-in-mice

Research

JoVE Journal

Neuroscience

Targeted Brain Region Microinjection of Bromophenol Dye in Mice

183 次观看.  Shanghai Jiaotong University, School of Medicine. 首先将无菌手术器械放在工作台上。为了将麻醉的小鼠固定在立体定位框架中，将上切牙与鼻夹混合，并用两个耳杆稳定头部。用眼剪刀在头皮上做一个0.5厘米的切口，露出头盖骨。用棉签蘸 3% 过氧化氢，擦洗暴露的颅骨以去除骨膜。让颅骨干燥。将钻头固定在立体定位仪器上并调整立体定位臂的旋钮。在放大镜的帮助下，将钻尖精确地定位在前臂上。使用 LCD 数...

视频: 小鼠靶向脑区显微注射溴酚染料

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their choice of stereotaxic injection parameters on mouse brain atlases or published materials that employed various populations/ages of mice and different stereotaxic equipment, necessitating further validation of the stereotaxic coordinate parameters. The efficacy of calcium imaging, chemogenetic, and optogenetic manipulations relies on the precise expression of reporter genes within the region of interest, often requiring several weeks of effort. Thus, it is a time-consuming task if the coordinates of the target brain region are not verified in advance. Using an appropriate dye instead of a virus and implementing cryosectioning, researchers can observe the injection site immediately following dye administration. This facilitates timely adjustments to coordinate parameters in cases where discrepancies exist between the actual injection site and the theoretical position. Such adjustments significantly enhance the accuracy of viral expression within the target region in subsequent experiments.

The present protocol describes a practical strategy to expedite the verification step of stereotaxic injection coordinates before conducting viral tracing using dyes and frozen sections.

Stereotaxic injection of a specific brain region constitutes a fundamental experimental technique in basic neuroscience. Researchers commonly base their ...