Co-culturing of Ventral Hippocampal and Nucleus Accumbens
Transcript
Before tissue collection, pre-incubate 12-millimeter Poly-D-Lysine-laminated coated glass coverslips in the 24-well tissue culture plates with 500 microliters of culture media at 37 degrees Celsius for at least 30 minutes. Then, remove the media before plating the micro-slices. To collect tissue for culture, remove the top of the skull to expose the whole brain of each sacrificed pup.
Under a dissecting microscope, starting at the juncture where the cerebral cortices part from each other caudally, obtain a coronal section containing the posterior cortices, including the ventral hippocampi. Next, dissect the ventral hippocampal tissues containing the CA1-subiculum regions out. After that, transfer the tissues to a 35-millimeter culture dish containing cold culture media.
Subsequently, cut the tissues further into small, thin slices. Plate the slices with a fire-polished Pasteur pipette at the center of the coverslip with 50 microliters of media to facilitate the attachment of the explants. Then, plate the ventral hippocampal micro-slices on the coverslip.
After the explants have settled onto the coverslip, gently add 100 microliters of additional media by the wall so that the level of the media is high enough to completely cover the explants on the periphery, but not those at the center of the coverslip. Then, incubate the explants overnight in 5% CO2 at 37 degrees Celsius before the dispersion of the nucleus accumbens neurons. Now, obtain the nucleus accumbens tissues from embryonic day 18 to postnatal day one wild-type mice.
Cut them into small pieces. Then, transfer them to a 15-milliliter tube. Treat the tissues with two milliliters of 0.25% trypsin for 15 minutes at 37 degrees Celsius. Subsequently, wash the tissues three times with five milliliters of four degrees Celsius washing media, and allow the suspension to rest for five minutes in each wash step.
Then, wash the tissues in five milliliters of four degrees Celsius culture media once. Next, dissociate the cells by gentle trituration in two milliliters of culture media with a lightly fire-polished Pasteur pipette. Afterward, transfer the cell suspension to another 15-milliliter tube, and centrifuge at 2,000 RPM for five minutes.
After five minutes, remove the supernatant, and resuspend the cells in culture media at one milliliter per six pups. Then, disperse the cells by gently pipetting the media several times with a fire-polished Pasteur pipette. At the end, add 0.25 milliliters of the dispersed nucleus accumbens cells to each coverslip with the ventral hippocampal explants. Place the culture plates on damp and sterile gauze pads to mechanically stabilize and maintain humidity in order to facilitate synapse formation. Then, incubate the plates at 5% CO2 at 37 degrees Celsius.
