This procedure starts off with a well perfused monkey head. With the calva removed, the head is then placed into the stereotaxic frame and a series of stereotaxic coronal slices are made with the manipulator arm, the brain is then removed, measured, and cryo protected. Hi, I'm Dr.Mark Burke from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal.
I'm Dr.Shaheen Zur, also from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal. I'm Dr.Mo Petto, director of the Visual Neuroscience Laboratory at the School of Optometry, university of Montreal. Today we will show you a procedure for blocking the non-human primate brain in stereotactic space.
We use this procedure in our laboratory to study various aspects of brain organization in normal development and in response to developmental intrusions. So Let's get started. This protocol begins following euthanasia fixation and decapitation of a vervet monkey.
Prior to brain removal, the stereotaxic frame must be prepared. First one needs to find the coordinates of the Horsley Clark Interoral plane zero or the theoretical midpoint between the ears. To measure this plane, fit the ear bars equally into the apparatus.
Then place a scalpel blade in the stereotaxic manipulator arm and measure the midpoint between the ear bars. Next, prepare the head to go into the stereotaxic frame. Use bone rgo and a scalpel to remove the lower jaw.
At this point, chip away any remaining calva throughout these steps. Be careful not to remove the temporal bones since you will need the ear canals intact to anchor the head in the stereotaxic frame. Using forceps and scissors, carefully cut away the visible dur matter from the exposed brain.
Now the skull is ready to go in the stereotaxic manipulator. To begin stereotaxic blocking the brain, place the head into the frame, adjust the eye palette and ear bars as you would for stereotaxic surgery. Place the stereotaxic manipulator in the predetermined anterior, posterior, or AP axis.
To begin blocking the brain. Position the manipulator to the far left or right side of the brain and lower the blade to make your first cut. The blade will be perpendicular to the midline of the brain cutting in the coronal plane.
Raise the blade to completely clear the brain surface. Then move medially to just overlap your first cut and lower the blade.Again. Continue to make overlapping cuts until you reach the opposite edge of the brain.
This completes the first coronal block. Move the manipulator one centimeter forward or back along the AP axis and make another series of cuts in the coronal plane parallel to your previous ones. Continue this way until you have blocked the entire brain.
Now that you have finished cutting, it is time to remove the brain from the skull. First, take the head out of the frame. Try to secure the brain by lightly coddling it in the palm of your hand to keep the brain from jiggling in the skull and becoming damaged.
Place your pinky finger across the frontal lobes to keep the peel surface of the brain from drying out. Wet a piece of gauze with PBS and drape it over the brain. Hold the head firmly by the skull and chip away the remaining occipital and temporal bones along with the spinal column.
This exposes the base and lateral aspects of the brain. Now remove the remaining dura. Lastly, remove the nasal bone and any remaining frontal bone, which will allow access to the olfactory bulbs.
It is important to remove the frontal bone. Last, because as you remove the base of the skull, the brain moves slightly and the frontal bone's. Jagged edges could damage the frontal lobes.
Remove any remaining dura matter carefully lift the front of the brain, slide the scalpel under the brain and free the brain from the skull. It will stay together like an onion sliced up the stem because the cutting blade does not reach all the way to the ventral side. There are a number of useful measurements you can make before you freeze the brain.
First, a caliper can be used to measure the AP axis of the brain. Next, find its specific density, weigh the brain. Then measure the volume as the displacement of water in a graduated cylinder.
Now you have the density mass divided by volume. Now that you have made measurements on the whole brain, it is time to release the individual blocks of tissue from where they're attached on the brain's ventral side, and prepare them for freezing. Take a tissue slicing blade and extend each cut through the ventral side of the brain releasing the blocks.
A caliper can be used to measure the medial, lateral and anterior posterior AEs. The brain blocks are now ready to be cryo protected and frozen. We've just shown you how to block a non-human primate brain in stereotaxic space.
When doing this procedure, it is important to fully retract the blade before any medial lateral movement is made with a stereotaxic manipulator. Also, when chipping away the bones from the brain, be careful not to damage a cortex with the ERs. So that's it.
Thanks for watching and good luck with your experiments.
