First, the purpose of the experiment
1. Understand the brain stereo positioning technology.
2. Master the use of brain stereotaxic instruments and brain maps.
Second, the experimental principle
Stereotactic technology is widely used in the brain's destruction, stimulation and precise location of EEG recordings, and is an indispensable tool for studying brain structure and function. The stereotactic technique of the brain mainly uses the brain stereotactic device as a positioning instrument, and utilizes the third coordinate system defined by some signs outside the skull (such as anterior or posterior iliac crest, external auditory canal, eyelid, sagittal suture, etc.) or other reference points. To determine the location of certain neural structures under the cortex in order to direct the stimulation, destruction, injection of drugs, guiding potentials, etc. under non-direct vision exposure, in the fields of neuroanatomy, neurophysiology, neuropharmacology and neurosurgery. Important research methods within. Commonly used experimental animals, such as rats, mice, cats and other high mammals as well as birds, have complete external auditory canal and can be positioned with (ear sticks). After the extracranial marker is determined, the positioning operation can be performed according to the data provided by the stereotaxic map of the brain.
Third, experimental equipment
ZS-B/C brain stereo locator 516000 (specification reference website: ), MC-5 micro-operator, conventional surgical instruments, drilling needle, gauze, dry cotton ball, alcohol, 0.4% pentabar Bitot sodium (anesthetic, now available), saline, 1 ml syringe, 3% hydrogen peroxide, mice.
Fourth, the experimental steps
1. Use of brain locator
1.1 Â Calibration instrument
After the locator has been moved or used for a long time, it needs to be verified before use. The key point is to check whether the slides of the electrode moving frame maintain a right angle. It is possible to determine whether the angle formed by each slider is a right angle by a triangular plate; whether the joints and the screws are loose; whether the slider is too loose; checking the parallelism of the arms of the main frame Finally, observe the degree of symmetry on both sides of the fixed head device, and whether the small frame is parallel to the main frame. After checking that the instrument is not faulty, the following checksum operations can be performed:
(1) Loosen the ear column on both sides, slide it back and forth on the main frame, and then install it according to the original specification to see if the ear tips on both sides are completely aligned.
(2) Remove one side ear rod, and install one side electrode moving frame, move each sliding ruler up and down, left and right, so that the metal positioning needle tip mounted on the electrode holder is facing the center of the ear rod tip, and record the respective sliders. Grab the reading, then remove the moving frame and then install it, and measure the tip of the ear stick according to the above method, record the readings of the three sliders, and repeat the operation to take the average. First, place two of the horizontal stereo positioning instruments. The slide is adjusted to the appropriate height according to the requirements of the experiment.
(3) Then use the level to adjust the front, back, left and right levels of the two slides. At this time, the manual micro propeller placed on the slide is adjusted vertically according to the above scale.
1.2 Determine the stereo position of the brain, the mouse brain positioning system is roughly divided into two types:
(1) Using the center of the ear line, first connect the two ear tips to each other in the middle of the locator slide (the two ear sticks read the same) and tighten the wire. Then remove one ear stick and the other ear stick does not move. Adjust the moving pusher so that the tip of the proof electrode touches the center point of the tip of the ear stick, that is, point A (ie, the center point of the ear line), and record the scale value. Then, the pusher is moved horizontally above the incisor hook to bring the incisor hook plane into contact with the check electrode tip. At this time, the horizontal point 0 between the center point of the external auditory canal and the edge of the incisor tooth surface is determined as H0. At this time, the animal's anterior and posterior iliac crests are basically on a horizontal plane with a difference of 0-0.1 mm. In addition, it is specified that the center point of the interaural line is +, and the downward direction is -; the side to the mouth is +, and the side to the tail is -.
(2) Positioning with the skull mark (usually used before and after), that is, 0 points from the front side of the mouth, + from the front to the mouth side, and - to the tail side, the other is the same as the above positioning method (pictured).
2. Â Experimental content
(1) Animal anesthesia: The mice, weighing 20-30 g, must be slowed by intraperitoneal injection with 0.4% pentobarbital sodium after weighing, and the animal state should be paid attention to at any time.
(2) Mouse head fixation: The incisors of the mouse were fixed to the maxillary fixture of the brain locator, and then the ear of one side was pushed into the outer lane of the animal, so that the head of the animal was in the middle of the two slides. Push the other ear to the external auditory canal on the other side. At this time, observe that the scales of the two ear sticks are the same, then tighten the fixing screws on the two ear sticks and tighten them after pressing the nose ring on the tooth holder (the tightness of the nose ring and the ear stick is suitable as well. ), at this time pushing the animal's head from all directions "no movement will occur.
(3) Preparation of skin before craniotomy: cut off the animal's head hair, use 2% iodine and 75% alcohol cotton ball for disinfection of the head skin, make a 3cm long skin incision along the sagittal suture, separate Under the skin, the fascia and muscles on the surface of the skull were cleaned with hydrogen peroxide and peeled off, and the periosteum was pushed open to expose the anterior, herringbone and sagittal suture.
(4) Determine the standard center line: After moving the metal positioning needle down to the sagittal suture, move the positioning needle back and forth to position the positioning needle to the front sill.
(5) Mouse hippocampus positioning: use the positioning needle 2mm behind the anterior iliac crest, 2.5mm next to the sagittal suture, which is the plane position of the hippocampus, then drill a small hole on the skull with a drilling needle at this point. Round hole.
(6) Injecting the drug: The mouse hippocampus is located 2 mm below the round hole, and the 1 ml syringe is inhaled into the drug and placed on the MC-5 micromanipulator. The instrument is operated to make the syringe needle drop 2 mm from the mouse brain hole to complete the drug. Injection into the hippocampus of the mouse brain.
(7) Making brain tissue sections: The mouse brain was sliced, and the position of the red dye in the mouse brain was microscopically observed to verify whether the mouse hippocampus was positioned accurately.
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