Technology of Stereotaxic Instrument and Specific Operation Method for Rat and Mouse Experiments

Experimental Principle   

        Stereotaxic technology is widely used in the precise positioning of brain lesions, stimulation, and electroencephalographic recording, and has become an indispensable tool for studying brain structure and function. The core of stereotaxic technology lies in using a stereotaxic instrument as the positioning device. It utilizes a three-dimensional coordinate system defined by certain extracranial landmarks (such as bregma, lambda, external auditory canal, eye socket, sagittal suture, etc.) or other reference points to determine the location of specific subcortical neural structures. This enables targeted operations (including stimulation, destruction, drug injection, and potential recording) on these structures without direct visual exposure, making it a crucial research method in fields such as neuroanatomy, neurophysiology, neuropharmacology, and neurosurgery. For commonly used experimental animals, such as rats, mice, cats (and other higher mammals) as well as birds, all possess a complete external auditory canal, which allows the use of ear bars for positioning. Once the extracranial landmarks are identified, positioning operations can be performed in accordance with the data provided in the stereotaxic atlas of the brain.
        Experimental Equipment: Stereotaxic instrument, MC-5 micromanipulator, conventional surgical instruments, drill bit, gauze, dry cotton balls, alcohol, 0.4% sodium pentobarbital (anesthetic, prepared freshly before use), normal saline, 1ml syringe, 3% hydrogen peroxide, mice.

Experimental Procedures

There are roughly two systems for mouse brain positioning:

(1) Positioning based on the interaural line center: First, bring the tips of the two ear bars into contact with each other at the middle part of the stereotaxic instrument's slideway (ensuring both ear bars show the same reading), then tighten the screws. Next, remove one ear bar while keeping the other fixed. Adjust the mobile manipulator so that the tip of the calibration electrode touches the center point of the fixed ear bar tip—this point is designated as Point A (i.e., the center of the interaural line), and record its scale value. Then, move the manipulator horizontally to a position above the incisor hook, and bring the plane of the incisor hook into contact with the tip of the calibration electrode. At this point, the horizontal plane zero point (recorded as H0) between the center of the external auditory canal and the upper edge of the incisor plate is determined. At this position, the bregma and lambda of the animal are basically on the same horizontal plane, with a difference of 0-0.1mm. Additionally, the convention is that values above the center of the interaural line are marked as "+", and below as "-"; values toward the rostral (mouth) side are marked as "+", and toward the caudal (tail) side as "-".

(2) Positioning based on skull landmarks (bregma is commonly used): With bregma as the zero point for the rostral-caudal axis, positions rostral to bregma are marked as "+", and caudal to bregma as "-". The other positioning rules are the same as the method described above.

 Experimental Content 

(1) Animal Anesthesia: For mice (weighing 20-30g), after weighing, administer intraperitoneal injection of 0.4% sodium pentobarbital. The injection must be slow, and the animal's condition should be monitored closely at all times.

(2) Mouse Head Fixation: Secure the mouse's incisors to the maxillary fixator of the stereotaxic instrument. Then, insert one ear bar into the animal's external auditory canal, ensuring the animal's head is centered between the two slideways. Insert the other ear bar into the external auditory canal on the opposite side. Check that the scales on both ear bars are consistent, then tighten the fixing screws on the ear bars. Next, press down the nasal ring 2 on the tooth fixator and tighten it (the tightness of the nasal ring and ear bars should be adjusted to an appropriate level—secure enough to prevent movement, but not overly tight). At this point, the animal's head should not move when pressure is applied from any direction.

(3) Skin Preparation before Craniotomy and Drilling: Clip the hair on the animal's head. Disinfect the scalp using 2% iodine tincture and 75% alcohol cotton balls. Make a 3cm-long skin incision along the sagittal suture, dissect the subcutaneous tissue, clean and剥离 (peel off) the fascia and muscles on the skull surface using 3% hydrogen peroxide, and push aside the periosteum to expose the bregma, lambdoid suture, and sagittal suture.

(4) Determination of the Standard Midline: Lower the metal positioning needle to a position above the sagittal suture, then move the positioning needle forward and backward to align it with the bregma.

(5) Mouse Hippocampus Positioning: Use the positioning needle to mark a point 2mm caudal to the bregma and 2.5mm lateral to the sagittal suture—this is the planar position of the hippocampus. Then, use a drill bit to make a small circular hole in the skull at this marked point.

(6) Drug Injection: The mouse hippocampus is located 2mm below this circular hole. Fill a 1ml syringe with the drug and attach it to the MC-5 micromanipulator. Operate the instrument to lower the syringe needle 2mm from the craniotomy hole in the mouse's brain, completing the drug injection into the mouse hippocampus.

(7) Preparation of Brain Tissue Sections: Prepare sections of the mouse brain. Observe the position of the red dye in the mouse brain under a microscope to verify the accuracy of the mouse hippocampus positioning.