Brain stereotaxic technology and specific operation methods for rat and mouse experiments

Experimental principle   

Brain stereotaxic technology is widely used for precise localization of brain damage, stimulation, and EEG recording, becoming an essential tool for studying brain structure and function. Brain stereotaxic technology mainly uses a brain stereotaxic device as a positioning instrument, using a three-dimensional coordinate system specified by certain landmarks outside the skull (such as the anterior fontanelle, posterior fontanelle, external auditory canal, eye socket, sagittal suture, etc.) or other reference points to determine the position of certain neural structures under the cortex, in order to conduct targeted stimulation, destruction, drug injection, guided potential and other research under non direct visual exposure. It is an important research method in the fields of neuroanatomy, neurophysiology, neuropharmacology and neurosurgery. Commonly used experimental animals, such as rats, mice, cats and other higher mammals, as well as birds, all have complete external auditory canals that can be located using ear sticks. After determining the extracranial markers, the localization operation can be performed based on the data provided by the brain stereotaxic map. Experimental equipment: Brain stereotaxic device, MC-5 micro manipulator, conventional surgical instruments, drilling needle, gauze, dry cotton ball, alcohol, 0.4% pentobarbital sodium (anesthetic, ready to use), physiological saline, 1ml syringe, 3% hydrogen peroxide, mice.

Procedure

The mouse brain localization system can be roughly divided into two types:

(1) Using the center of the ear line for positioning, first place the tips of the two ear sticks in contact with each other in the middle of the positioning device slide (the readings of the two ear sticks are the same), and tighten the screws. Then remove one ear stick and keep the other ear stick still. Adjust the mobile thruster so that the tip of the verification electrode contacts the center point of the ear rod tip, which is point A (i.e. the center point of the ear line), and record the scale value. Then, move the thruster horizontally above the latch hook and bring the latch hook plane into contact with the tip of the verification electrode. At this point, the zero point of the horizontal section between the center point of the external auditory canal and the upper edge of the incisor plate is determined, denoted as H0. At this point, the animal's anterior and posterior fontanels are basically on the same horizontal plane, with a difference of 0-0.1mm. In addition, it is stipulated that the distance above the center point of the ear line is+, and the distance below is -; To the mouth side is+, to the tail side is -.

(2) Using skull landmarks for localization (commonly the anterior fontanelle), that is, the anterior fontanelle is the 0 point on the tail side of the mouth, the+point from the anterior fontanelle towards the mouth side, and the - point towards the tail side. The other positioning methods are the same as above.

Experimental content 

(1) Animal anesthesia: Mice weighing 20-30g must be slowly anesthetized with 0.4% pentobarbital sodium intraperitoneally after weighing, and the animal's condition must be monitored at all times.

(2) Mouse Head Fixation: Fix the incisors of the mouse to the maxillary fixator of the brain locator, and then push one ear cup into the animal's external passage, so that the animal's head is in the center of the two slides. Push the other earbud into the other side of the external auditory canal. At this point, observe that the scales on the two ear sticks are consistent. Tighten the fixing screws on the two ear sticks, and then press down on the nose ring 2 on the tooth holder and tighten it (the tightness of the nose ring and ear stick should be adjusted appropriately). At this point, pushing the animal's head from all directions will not cause any movement.

(3) Preparation of skin before craniotomy and drilling: Cut off the animal's head hair, disinfect the head skin with 2% iodine and 75% alcohol cotton balls, make a 3cm long skin incision along the sagittal suture, separate the subcutaneous tissue, clean the fascia and muscles on the surface of the skull with hydrogen peroxide and peel them off, push open the periosteum, and expose the anterior fontanelle, herringbone suture, and sagittal suture.

(4) Determine the standard midline: Move the metal positioning needle downwards above the sagittal suture, and then move the positioning needle back and forth to position it at the anterior fontanelle.

(5) Mouse hippocampus positioning: Use a positioning needle to locate a point 2mm behind the anterior fontanelle and 2.5mm next to the sagittal suture, which is the planar position of the hippocampus. Then, use a drilling needle to drill a small circular hole on the skull at this point.

(6) Injecting drugs: The mouse hippocampus is located 2 mm below the circular hole. A 1ml syringe is used to inject the drug and placed on the MC-5 micro manipulator. The instrument is operated to lower the syringe needle 2mm from the mouse brain hole to complete the drug injection into the mouse hippocampus.

(7) Making brain tissue slices: Cut the mouse brain into slices and observe the position of red dye in the mouse brain under a microscope to verify whether the localization of the mouse hippocampus is accurate