Preparation of Organotypic Slice Cultures from a Rat Pup's Hippocampus

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Hippocampal Dissection

1. Spray the postnatal day 7 (P7) Sprague Dawley rat pup with 70% ethanol outside of the laminar flow clean bench and quickly decapitate the animal using large sterile surgical scissors inside the laminar flow bench. Let the head drop into an ice-cold dissecting solution in one of the Petri dishes.
2. In the Petri dish, rinse off the blood and quickly transfer the head to sterilized filter paper, ventral side down.
3. Using the scalpel, cut along the dorsal surface in the sagittal plane to expose the underlying skull. Cut through the skin, but not the underlying bone, which is soft and easily penetrable in rats of this age. Set aside this "dirty" scalpel and do not use it on brain tissue.
4. Using the small dissector scissors (angled to the side) and forceps, cut open the skull along the sagittal suture of the skull to bregma, the anatomical point on the skull where the coronal suture is intersected perpendicularly by the sagittal suture. Use forceps to pull skull flaps up and away from the midline of the skull.
5. Place the micro spoon on the underside of the brain, beneath the brain stem, to gently lift the brain out of the skull. Lift the brain to expose the optic nerves and olfactory bulb on the basal surface of the brain. Cut these structures with small scissors to fully detach the brain from the skull. Remove and transfer the intact brain to the other large Petri dish containing an ice-cold dissecting solution.
6. Using the micro spoon, transfer the brain to a small Petri-dish lid containing sterile filter paper. With a sterile Pasteur pipette, rinse the brain with a few drops of dissecting solution to moisten the tissue.
7. Using a "clean" scalpel blade, cut the two hemispheres apart. Transfer the left hemisphere back to a large Petri dish with a micro spoon and place the hemisphere pia side down in an ice-cold dissecting solution for subsequent use.
8. View the medial face of the right hemisphere and identify the edge fornix, a prominent band of white matter along the medial edge of the hippocampus. Using a sterile scalpel, make a sagittal cut through the fornix, but take care because only 0.5 cm of the scalpel tip will be sufficient to cut the fornix.
9. Using two micro-spatulas, remove the first hippocampus from the right hemisphere by placing the right-hand-spatula on the brain stem and lifting the overlying cortex with the left-hand spatula. Gently lift the cortex to reveal the lateral ventricle and medial surface of the hippocampus. A white curved line, the fimbria, should now be visible.
10. Align the curvature of the spatula with the curvature of the fimbria and gently press the spatula under the fimbria. Slide the spatula left and ride along the rostral-caudal axis, and then lift the spatula in the dorsal direction to remove the hippocampus.
11. Transfer the hippocampus to a 2^nd small Petri dish with an ice-cold dissecting solution. Repeat the same procedure on the left hemisphere to remove the left hippocampus.
12. Using a micro spatula, carefully transfer the hippocampi to the tissue chopper stage. Arrange them adjacent and parallel to each other and perpendicular to the axis of the chopper blade. Use a paintbrush to position the tissue and add a few drops of the dissecting solution on top of the hippocampi.
13. Cut the tissue into 400 μm slices without pausing to remove individual slices (usually, they will not adhere to the blade). After the whole hippocampus has been cut, use the paintbrush to gently transfer the sections to a 2^nd small Petri dish with the dissection solution.
14. Under a dissecting microscope, carefully separate the floating slices using a micro-spatula and paintbrush.
15. Remove the pre-prepared culture plate with culture insert from the incubator and place it in a laminar flow hood.
16. Using a fire-polished Pasteur pipette, draw 4-5 slices into the pipette and transfer slices to the apical surface of the culture insert membrane. Next, adjust the positioning with a paintbrush and leave space between individual sections and the border of the culture insert.
17. Using a sterile Pasteur pipette, remove the excess dissecting solution from the apical surface of the membrane.
    NOTE: While removing the solution, avoid drawing tissue sections into the pipette. Alternatively, use a regular pipette (P200 or P1000) with sterilized pipette tips to slowly remove the solution.
18. Place the culture plate with serum-containing culture medium and the hippocampal slices back into the incubator at 35 °C and 5% CO₂.
    NOTE: If the experiment calls for generating cultures from multiple animals, thoroughly clean the laminar flow clean bench between dissections. Return instruments to alcohol solution and replace all Petri dishes, filter papers, and sterile razor blades before the second dissection.

2. Feeding and Maintaining Organotypic Slices

1. Feed cultures in a sterile laminar flow clean bench.
2. Perform the first feeding of the cultured sections two days post-dissection. Aspirate old culture medium using the sterilized glass pipette.
3. Use the sterile 5 ml serological pipette to add 1 ml of fresh, sterile, serum-containing medium to the wells.
4. Gently replace the culture insert and remove any air bubbles that may have formed underneath the membrane surface.
5. After the first feeding, change the medium every other day.