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 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.
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'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.
All animal experiments were conducted in compliance with the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The present study was approved by the Animal Care and Use Committee of Renji Hospital, Shanghai Jiaotong University School of Medicine. Eight-week-old C57BL/6J male mice were used for the present study. The animals were commercially obtained (see Table of Materials) and housed in standard cages (22 °C ± 2 °C, 12 h/12 h light/dark cycle, food, and water ad libitum).
1. Selection of target brain regions
NOTE: This is a sterile procedure. Ensure all surgical instruments are sterilized. Spray the gloves with alcohol prior to contact with surgical instruments. Avoid touching the tip of the instruments or the surgical area with gloves. Ensure the mouse is adequately anesthetized before starting the procedure by checking for the loss of righting reflexes and no response to cutaneous stimulation. Before the drilling and injection, closely monitor the respiratory rate (60-220 breaths per min, average 100-150 breaths per min) and the breathing amplitude (should be consistent and not excessively shallow or deep). Any significant changes in the respiratory rate or amplitude should be noted.
2. Preparing dye solution and brain microinjection
3. Brain tissue harvest and cryosectioning
4. Imaging
This study successfully identified the injection site within 30 min using the demonstrated method. Initially, an SDS-PAGE sample loading solution containing bromophenol blue was injected into the LDTgV in the C57/BL mice. Figure 1A shows a schematic representation of the dye solution injection. The distribution of the blue dye in the LDTgV is illustrated in Figure 1B.
Bromophenol blue was also injected into the mPFC prelimbic cortex, mPFC infralimbic cortex, and basomedial amygdala (BMA) to assess the universality of this protocol. As shown in Figure 1C–E and Figure 2A, the blue dye was distributed in the mPFC prelimbic cortex, mPFC infralimbic cortex, and BMA.
Furthermore, an adeno-associated virus carrying a fluorescent mCherry protein was injected into the LDTgV (the coordinates were verified using the dye solution). The expression of the virus took four weeks. The distribution of mCherry+ neurons in the LDTgV is depicted in Figure 2B.
Figure 1: Injection of dye solution. (A) A schematic diagram of the dye solution injection. (B) Distribution of blue dye in LDTgV. (C) Distribution of blue dye in mPFC prelimbic cortex. (D) Distribution of blue dye in mPFC infralimbic cortex. (E) Distribution of blue dye in BMA. Please click here to view a larger version of this figure.
Figure 2: Blue dye distribution and mCherry distribution viewed under the microscope. (A) Distribution of blue dye in mPFC prelimbic cortex (10x magnification, scale bar = 500 µm). (B) Representative image of mCherry distribution in LDTgV region acquired by fluorescent microscopy (10x magnification, scale bar = 500 µm). Please click here to view a larger version of this figure.
This article has described a stable strategy to verify the accuracy of stereotaxic brain injections5,6more quickly and simply before viral tracing, but the irreplaceable aspect of reporter gene expression in the brain region is crucial for brain region labeling. The blue dye we used allowed for the immediate visualization of the injection site.
Several critical steps in this protocol contribute to improving the accuracy of the injection site. Firstly, ensuring the left-right levelness of the brain in the stereotaxic instrument from step 1.5; this step requires operators to be familiar with skull anatomy, ensuring that the ear bars are placed in the right position. The scales on the ear bars and the stereotaxic frame also serve as references. Secondly, finding the exact zero point, the bregma, in step 1.6 and step 2.3. Occasionally, a significant discrepancy in the coordinates found in steps 1.6 and 2.3 is due to a considerable difference in the zero-point positioning. This can result in the microinjector needle being unable to penetrate the cranial window. In such cases, it is necessary to reposition the zero point and if needed, a new cranial window needs to be created. Despite this, it is still essential to reestablish the zero point and find the coordinates anew in step 2.3; one cannot directly insert the micro-syringe from the previously made cranial window.
Compared with cutting a non-frozen brain in a mouse brain-slice mold, this strategy provides a more accurate result because the solid specimen block avoids the blade squeezing the brain and causing the dye to be displaced from the injection points. Moreover, brain sections cut by a brain slice mold are relatively thicker and not feasible to localize small cerebral nuclei. However, compared with the standard brain tissue cryosectioning process11,12,13,14,15, we shortened the processing duration by at least 72 h by skipping the dehydration steps, which resulted in the brain sections being more fragile and more difficult to pick up on a glass slide, representing a major weakness of this protocol.
While making the cranial windows for dye injection, attaching the dental drill tightly to the stereotaxic arm, rather than holding it by hand, makes it easier to control the drilling depth using the fine-adjustment knob, thereby reducing the possibility of damaging the brain tissue.
In conclusion, this study has provided a promising method to preliminarily verify the exact stereotaxic coordinates before viral tracing, making the subsequent neuron labeling and tracing process more reliable.
The authors have nothing to disclose.
National Natural Science Foundation of China (grant NO. 82101249 to XY Sun), Postdoctoral Research Foundation of China (grant NO. 2022M722125 to XY Sun). Shanghai Sailing Program (grant NO. 21YF1425100 to SH Chen). Special Project for Clinical Research of Shanghai Municipal Health Commission (grant NO. 202340088 to J Zhou). National Natural Science Foundation of China (grant NO. 82101262 to X Zhang, grant NO. 82101287 to SH Chen).
1.0 µL, Neuros Syringe, Model 7001 KH, 32 G, Point Style 3 | Hamilton | 65458-01 | |
200 μL pipette tips | biosharp | BS-200-T | |
20 mL syringe | Kindly group | ||
3%H2O2 solution | Lircon Company | ||
6-well plate | Shengyou Biotech | 20006 | |
Anerdian | Likang High-tech | 31001002 | |
Anti roll plate | Leica | 14047742497 | |
BD insulin syringe | Becton,Dickinson and Company | 328421 | |
Bend toothed dissecting forceps | Jinzhong | JD1050 | |
Cellsens dimension software | Olympus | ||
Cotton swab | Fisher Scientific | 23-400-122 | |
Dapi-Fluoromount-G | Southernbiotech | 0100-20 | |
Drill | Longxiang | ||
Fine brushes | HWAHONG | ||
Fine scissors | Jinzhong | y00030 | |
Fluorescent microscopy | Olympus | BX63 | |
freezing microtome | Leica | CM1950 | |
Hemostatic forceps straight with tooth | Jinzhong | J31010 | |
Infusion needle 0.7 mm | Kindly group | ||
Lidocaine hydrochloride injection | Harvest Pharmaceutical Company | 71230803 | |
Magnifying glass | M&G Chenguang Stationery | ||
Male C57/BL mice | The Shanghai Institute of Planned Parenthood Research–BK Laboratory | ||
Mice coronal brain slice mold | RWD Life Science | 68713 | |
Microcentrifuge tube | biosharp | BS-02-P | |
Microtome blades | Leica | 819 | |
Ophthalmic ointment | Cisen Pharmaceutical Company | G23HDM9M4S5 | |
paraformaldehyde | Biosharp | BL539A | |
Peristaltic pumps | Harvard Apparatus | 70-4507 | |
Phosphate buffered saline | Servicebio | G4202 | |
Piette 2-200 μL | thermofisher | 4642080 | |
SDS-PAGE sample loading containing bromophenol blue | Beyotime | P0015A | |
Shaving blades | BFYING | 91560618 | |
Slides | Citotest Scientific | 188105 | |
Stereotaxic apparatus | RWD Life Science | 68807 | |
Straight toothed dissecting forceps | Jinzhong | JD1060 | |
Syringe Holder | RWD Life Science | 68206 | |
Tissue scissors | Jinzhong | J21040 | |
Tissue-Tek O.C.T compound | Sakura | 4583 | |
Tribromoethanol | Aibei Biotechnology | M2910 |