brain tissue harvesting, cryosectioning, and imaging post stereotaxic surgery in mice

Optimizing Stereotaxic Microinjection Accuracy in Mice

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.

Author Spotlight: Accelerating Cognitive Impairment Research in Mice Through Stereotaxic Injection and Cost-Effective Dyes

Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

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.

Brain Tissue Harvesting, Cryosectioning, and Imaging Post Stereotaxic Surgery in Mice

brain-tissue-harvesting-cryosectioning-imaging-post-stereotaxic

Research

JoVE Journal

Neuroscience

240 次观看.  Shanghai Jiaotong University, School of Medicine. 首先，从立体定位框架中释放溴酚蓝染料注射的小鼠。麻醉后，用胶带将鼠标固定在泡沫板上。在右心耳做一个 1 到 2 毫米的切口。将输液针插入左心室。注入 20 毫升预冷盐水，然后注入 20 毫升冰镇的 4% 多聚甲醛溶液。现在沿着从颈部到鼻子的中线切开一个切口，去除皮肤并露出头骨。使用镊子或剪刀清除颅骨上的残留肌肉。将细剪刀的一根刀片的尖端放在枕骨...

视频: 小鼠立体定向手术后的脑组织采集、冷冻切片和成像

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 ...