Materials Needed:

• Stereotaxic instrument
• Anesthesia (e.g., isoflurane or injectable anesthetic)
• Subject 
• Surgical instruments (scalpel, forceps, scissors)
• Sterile saline or PBS
• Antiseptic solution (e.g., iodine or chlorhexidine)
• Dental cement or sutures
• Heating pad
• Analgesics
• Personal protective equipment (PPE)

Procedure:

1. Preparation:

   • Ensure all surgical instruments and the stereotaxic apparatus are sterilized.
   • Prepare the anesthetic setup (e.g., isoflurane vaporizer).

2. Anesthesia:

   • Anesthetize the subject using an appropriate method (e.g., isoflurane inhalation or injectable anesthetics like ketamine/xylazine).
   • Verify the depth of anesthesia by checking the absence of reflexes (e.g., pedal reflex).

3. Positioning:

   • Place the subject on a heating pad to maintain body temperature and continue anesthesia.
   • Move the adapter forward and backward to align the centerline of the left and right ear bars with the mouse’s external auditory canal on the same line. Then, insert the left and right ear bars into the external auditory canal, adjusting the positions of the two ear bars to be symmetrical and at the same height, and tighten the screws to fix the ear bars. Furthermore, adjust the position of the mouse adapter’s incisor clamp up and down so that its position is 3.3±0.4mm below the centerline connecting the left and right ear bars. Ensure the head is stable and level. Optionally, use a nose clamp and bite bar to further stabilize the head.
   • Adjust the positions of the incisor clamp, nose bridge, and ear bars again, pressing firmly against the nose bridge. At this point, the head is fixed, the skull is horizontally leveled (note: this is a preliminary judgment criterion and further assessment is needed), and the nose is inside the anesthesia mask of the adapter, with breathing maintained by gas from the anesthesia machine
   • Check that the subject’s skull is successfully fixed: the nose is in the center, the tail is not slipping, visually confirm the brain is level, and pressing the subject’s head from various directions (top, left, right, and side) does not result in movement.

4. Surgical Site Preparation:

   • Shave the the surgical site (usually the scalp).
   • Clean the area with an antiseptic solution.

5. Incision:

   • Make a midline scalp incision using a scalpel to expose the skull.
   • Retract the skin to clearly expose the bregma and lambda landmarks on the skull.
   • Zero Point Localization: Secure a metal positioning needle in the stereotaxic instrument. Move the metal positioning needle above the sagittal suture, then move it forward and backward until the needle just touches the Bregma. 
   • Secondary Skull Plane Adjustment: Starting from the Bregma, for the first step, move the positioning needle backward along the sagittal line to approximately 2.5mm and 2mm to the left and right, respectively. Observe whether the positioning needle is at the same height from the skull surface at the Bregma and the 2.5mm position along the sagittal line, as well as at the 2mm positions on the left and right. For the second step, continue moving along the sagittal line to approximately 4.2mm and observe whether the positioning needle is at the same height from the skull surface at the Bregma and the 4.2mm position.

6. Locating Stereotaxic Coordinates:

   • Identify the bregma (anterior junction of coronal and sagittal sutures) and lambda (posterior junction) as reference points.
   • Use these landmarks to determine the coordinates for the target brain region (e.g., striatum) according to a stereotaxic atlas.. Select appropriate coordinate points (e.g., AP +0.98mm, ML ±2.0mm, DV -3.0mm).

7. Drilling:

   • Mark the target coordinates on the skull.
   • Drilling: Start moving the positioning needle from the Bregma. Locate a point 0.98mm anterior to the Bregma and 2.0mm lateral to the left or right side of the sagittal suture. Mark this point with a marker pen, which represents the position of the striatum in the X-Y plane.
   • Select an appropriately sized drill bit slightly larger than the injection needle. Hold the skull drill manually or secure it to the stereotaxic instrument and drill at the marked point. Caution: Maintain a gentle touch and avoid exerting excessive force. When the drill bit breaks through, there will be a distinct sense of breakthrough. At this point, withdraw the skull drill.

     After drilling, carefully puncture the dura mater with the fine needle of a medical syringe to avoid damage. If there is bleeding during this process, use a very small medical cotton ball to absorb the blood.

8. Implantation or Injection:

   • Attach the appropriate instrument (e.g., needle, electrode, cannula) to the stereotaxic arm.
   • Lower the instrument slowly and precisely to the desired depth in the brain or spinal cord.
   • Perform the desired procedure (e.g., injection of a substance, implantation of an electrode). For example,  for injections, set the injection parameters (syringe specifications [capacity, piston inner diameter], target injection volume, injection rate), and then start the injection (for example: striatum injection 1ul, rate approximately 0.5ul/min; virus injection 1ul, rate approximately 0.2ul/min). After injection, keep the needle in place for 5-10 minutes to allow for complete diffusion and absorption of the drug, then slowly retract the needle*.

9. Sealing:

   • If necessary, seal the hole in the skull with dental cement or suture the overlying skin.
   • Ensure that the surgical site is clean and free of debris.

10. Recovery:

    • Remove the rodent from the stereotaxic apparatus and place it in a clean, warm recovery area.
    • Monitor the rodent until it fully recovers from anesthesia.
    • Administer post-operative analgesics as required.

11. Post-Operative Care:

    • Continue to monitor the rodent for signs of distress, infection, or pain.
    • Provide any additional care as needed to ensure a smooth recovery.

*Before conducting the formal drug injection, it is necessary to perform a pre-experiment to verify the accuracy of the localization of the target brain area. It is recommended to use tracer dyes such as DiI (detectable after 1 week), fluorescent gold dye (detectable after 2 weeks), or red or black dyes (detectable after 1 week). Then, take samples for frozen sectioning, and compare them with some reference points in the brain atlas to determine the accuracy of brain area localization (selecting larger and clearer reference points, such as the hippocampus, lateral ventricle, and optic chiasm nucleus, etc.).

Note: When sectioning, approach the target nucleus from the outside, and ensure that the angle of brain sections is consistent with the brain atlas.