Preliminary Validation of Stereotaxic Injection Coordinates via Cryosectioning

Summary

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.

Abstract

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.

Introduction

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 interest^1,2,3, 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 literature^5,6. 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 selected^7,8. 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 procedure^7,8. As AAV evolves, analysis can be performed 2-3 weeks after administration^9,10. Other neural circuit tracers, such as pseudorabies virus (PRV) and rabies virus (RV), also require a tracing period of at least 2-7 days^{11,12,13,14,15}. 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.

Protocol

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.

1. Anesthetize the mouse with an intraperitoneal injection of 1.25% tribromoethanol (250 mg/kg) using a 29 G needle. Shave the hair on the skull once the righting reflex disappears. Secure the mouse in the stereotaxic frame (see Table of Materials) by fixing the upper incisors with a nose clamp and stabilizing the head using two ear bars.
2. Apply ophthalmic ointment to prevent eye dryness. Disinfect the scalp by swabbing it three times with Anerdian (disinfectant solution; see Table of Materials). Inject 0.2 mL of 1% lidocaine subcutaneously using a 29 G insulin needle to provide local analgesia.
3. Make a 0.5 cm incision using eye scissors to expose the cranium. Utilize a cotton swab dipped with 3% H₂O₂ to scrub the exposed skull to remove the periosteum. Allow the cranium to dry.
4. Affix the drill to the stereotaxic instrument. Adjust the stereotaxic arms' knobs to position the drill tip precisely at the bregma with the help of a magnifier and record the z-axis value using the LCD digital display.
   1. Adjust the knobs and position the drill tip at the lambda and record the value of the z-axis. Adjust the nose clamp and ear bars to make two z-values closer, and repeat the steps above until the difference between the two z-values is less than 0.1 mm.
      NOTE: The x, y, and z axes on the LCD digital display correspond to mediolateral (ML), anteroposterior (AP), and dorsoventral (DV), respectively. The bregma and lambda can be considered to be on the same horizontal plane once the difference between the two z-values is less than 0.1 mm.
5. Ensure the left-right levelness of the brain. Adjust the ear bars attached to the stereotaxic frame with the same scale. Take the laterodorsal tegmental nucleus, ventral part (LDTgV), for example; its stereotaxic coordinates are -5.2 for x, +0.8 for y, -4.0 for z, according to Paxinos and Franklin's mouse brain atlas¹⁶.
   1. Adjust the ear bar to make the drill at (-5.2, +0.8) to measure the z value in the dorsal-ventral direction and move the drill to (-5.2, -0.8) to measure the z value.
      NOTE: The left-right is considered to be at the equal level when the difference between the two z-values is less than 0.2 mm. The ear bars need to be inserted into the right place because the external auditory meatus is symmetric. If the difference between the two z-values is more than 0.5 mm, the ear bar may be inserted into the wrong place.
6. Position the drill to the bregma again and set three coordinates on the LCD digital display to zero. Position the drill above the region of interest using reference coordinates. Start the drill.
   1. Gradually lower the drill using the vertical arm until it penetrates the cranium. Clear away debris and blood using cotton swabs.
      NOTE: Be careful not to damage brain tissue.

2. Preparing dye solution and brain microinjection

1. Take 25 µL of SDS-PAGE loading buffer containing bromophenol blue (see Table of Materials) into a 200 µL microcentrifuge tube. Add 50 µL of ddH₂O to the tube to prepare the dye solution.
2. Cover the cranial window with a saline-moistened cotton pad. Ensure microsyringe patency by aspirating and expelling saline repeatedly. Load 0.3 µL of dye into the micro syringe. Attach the syringe and the motorized stereotaxic microinjector to the stereotaxic arm.
3. Position the syringe tip at the bregma and set the coordinates to zero on the LCD digital display. Adjust the needle into the region of interest according to reference coordinates. Set the injection speed to 0.1 µL/min and start the motorized microinjector, delivering 0.3 µL of blue dye into the target region. Leave the syringe in position for at least 10 min before slowly withdrawing the injector.
   NOTE: Different color dyes can be used in the same mice to distinguish closely located regions. Test other dyes for suitable concentrations based on their physical properties. It's recommended to use the bromophenol blue formula and thoroughly rinse the syringe before and after use to prevent blockages. Adjust injection volumes based on dye diffusion.

3. Brain tissue harvest and cryosectioning

1. Release the mouse from the stereotaxic frame. Anesthetize the mouse with an intraperitoneal injection of 50 mg/kg pentobarbital sodium using a 29 G needle. Secure the mouse on a foam board with tape.
2. Open both sides of the thorax to induce rapid suffocation. Make a 1-2 mm incision in the right atrial appendage. Insert an infusion needle into the left ventricle. Infuse 20 mL of pre-cooled saline and 20 mL of iced 4% paraformaldehyde solution.
3. Decapitate the mouse with tissue dissecting scissors (following institutionally approved protocols). Remove the skin by making an incision along the midline from the neck to the nose and then expose the skull. Clear off residual muscle on the skull with forceps or scissors.
4. Position the tip of one blade of the fine scissors between the foramen magnum and the brain, with the sharp edge facing the bone, at the rostral end of the mid-sagittal suture. Proceed to slide and cut the cranium along the mid-sagittal suture. Employ forceps to remove the skull, revealing the exposed brain.
   NOTE: Exercise caution to avoid causing any damage to the brain during the operation. Before removing the bone chips, carefully peel off any meninges that may be attached to the skull. This precaution is crucial to prevent inadvertent slicing of the brain tissue during the process.
5. Turn on the power of the rotary microtome cryostat (see Table of Materials) and set the chamber temperature to -20 °C. Pour a little OCT compound to cover the surface of the specimen disc evenly. Place it into the cryo-chamber and allow the OCT to solidify.
6. Attach the disc to the specimen head. Bring the blade closer to the block and trim the OCT block to make a flat plane that is parallel to the disc.
7. Cut the brain in a coronal direction away from the injection site in a brain slice mold by a shaving blade. Place the brain tissue containing the injection site with the cut side down on the OCT flat plane.
   1. Pour OCT onto the brain and put the disc into the cryo-chamber, allowing the specimen block to solidify. Pour OCT repeatedly onto the brain until the brain is fully embedded with OCT.
      NOTE: Add the OCT compound layer by layer. Once the previous layer of OCT is frozen, pour the subsequent layer of OCT compound onto the brain.
8. Trim the specimen block until the target region is approaching. Make several sections from the injection site level. Collect the coronal brain slices into a 6-well plate containing room-temperature PBS using a small writing brush kept at -20 °C.
   NOTE: Set the cutting thickness to a larger one to trim the block, and then set the cutting thickness to 40 µm as the blade approaches the injection site. If neural circuit tracers are used, the mice are not sacrificed immediately. Place the mouse in a sterile, postoperative recovery cage over a heating blanket for recovery from anesthesia. Return the mice to the original cage or a new cage once awake.

4. Imaging

1. Wash the OCT with room temperature PBS. Use a brush to pick up brain sections and place them on a slide. Allow sections to air dry at room temperature.
2. Observe the injected region visually or use a microscope when the target nuclei are minuscule. Compare the location of the injected dye with the corresponding region in the brain atlas.
   NOTE: Adjust the values of the x, y, and z axes according to the direction and distance of the injection point deviation. It may be necessary to repeat all steps in this protocol multiple times to determine the coordinates accurately.

Representative Results

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.

Discussion

This article has described a stable strategy to verify the accuracy of stereotaxic brain injections^5,6more 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 process^{11,12,13,14,15}, 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.

Materials

Name	Company	Catalog Number	Comments
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