The aim of this procedure is to culture sympathetic neurons so as to be able to manipulate their dendritic growth. This is accomplished by first isolating the superior cervical ganglia from the perinatal rodent pups under sterile conditions. The second step is to dissociate the ganglia to form a single cell suspension that is plated on the prepared substrates.
Inde defined medium. Then after treating the cultures with anti-mitotic to eliminate the non neuronal cells, treat the cultures with BMPs or matrigel to induce dendritic growth. Ultimately, map two immunoreactivity is used to show the extended dendrites of the cultured sympathetic neurons.
The main advantage of this technique over existing methods like primary culture of hippocampal neurons, is that it is possible to manipulate the morphologic state of DYS neurons such that less than 5%of the neurons in culture have dendrites, or more than 95%of the neurons in culture exhibit robust ntic outgrowth To prepare the associated cultures of the SCG from the prenatal rat pups at 19 to 21 embryonic days, first shave the abdomen of a euthanized pregnant rat, clean the skinless 70%ethanol, then remove the uterine horns under sterile conditions and avoid damaging the intestines or other internal organs. Place the uterine horns in a sterile 150 millimeter Petri dish. Next, transfer the Petri dish onto a pan of ice.
For anesthetizing the embryonic pups to euthanize the pup, cut the spinal cord along the midline under its shoulder. This also severs the vena caver, which would reduce bleeding from the carotid artery During the dissection of the SCG place the embryos in sterile leibovitz's L 15 medium supplemented with penicillin and streptomycin. Then transfer the embryos to a 150 millimeter Petri dish, half filled with sil guard and place them on their backs.
Use the sterile 20 gauge needles to pin the thorax and the head gently hyperextending the neck to facilitate the dissection of the SCG under the microscope. Make a midline incision along the neck at the level of the clavicles to reveal the submandibular glands. Remove the glands using fine forceps in order to expose the muscle layer.
Then cut the sternocleidomastoid muscle near the clavicle, gently lifting inci the omohyoid muscle next to the trachea. This muscle is very thin, so avoid cutting too deeply, which would tear the underlying carotid artery and SCG. Once these muscle layers are removed, the carotid artery and the vagus nerve should be clearly visible.
The SCG lies just below the bifurcation of the carotid artery blunt. Dissect the tissue away from both sides of the carotid artery with the fine forceps in order to expose the ganglion. Then use two fine forceps to grasp the carotid artery just above and below the rostral and cordal lens of the SCG respectively.
To remove the section of the carotid artery lying above the SCG as well as the SCG itself, it is likely that the noose ganglion, which lies alongside the SCG at this stage of development, will also be removed at this step. Place the dissected tissue into a sterile 35 millimeter culture dish containing L 15 medium and antibiotics. After that, remove the SCG from all the pups before progressing to the next steps.
Next, gently separate the SCG from the carotid artery and any other tissues. Remove during the dissection. Remove the capsule from the ganglia.
Then transfer the ganglia to another sterile 35 millimeter dish containing L 15 medium. To dissociate the SCG remove L 15 medium. Replace it with collagenase dys basing calcium and magnesium free hanks balance sort solution.
Then incubate it at 37 degrees Celsius for 40 minutes. After that, transfer the SCG to a sterile 15 milliliter conical centrifuge tube. Bring the volume up to 10 milliliters with L 15 supplemented with BSA to a final concentration of one to two milligrams per milliliter.
Centrifuge at 200 times G at room temperature for five minutes. After five minutes, discard the supinate and wash it once more with L 15 supplemented with BSA after the second wash. Remove L 15 medium and rein the ganglia in two milliliters of culture.
Medium tri rate the cells about three to four times using a fire polished bent tip pipette. Make sure the pipette is coated with BSAL 15 before TRI to minimize sticking of cells to the glass. Let the large clumps settle for about one minute, then transfer the cell suspension to a 15 milliliter conical.
Subsequently add more culture media to the remaining SCG and tri rate. After the clumps have settled, transfer the cell suspension from the second tri to that collected after the first tri. Repeat this process several times until the ganglia are mostly dissociated.
Discard any remaining clumps after the fourth tri for the cells dissociated from one liter of rat pups, the total volume of medium in the cell suspension should be brought to eight to 10 milliliters. By adding additional culture medium, gently resuspend the cells using a pipette. Then collect a small aliquot for the determination of cell density.
Adjust the volume of the cell suspension in order to achieve two times the cell density desired in the culture. Next, aliquot 250 microliters of cell suspension per well. For the morphometric studies of dendritic growth, we typically plate cells in 24, well plates at low density, about five times 10 of the four cells per well.
Then place the SCG cultures on the porcelain desiccate plate in the glass desiccate containing sterile water. Inject approximately 120 milliliters of carbon dioxide into the desiccate prior to sealing it shut. After that, place the entire desiccate into an incubator and set it 35.5 degrees Celsius.
In this procedure. Feed the culture by exchanging half of the medium 24 hours after plating and repeat three times a week to eliminate non neuronal cells from the culture. The anti-mitotic agent, RRC as a concentration of one to two micromolar is typically added to the culture medium at this time.
Then add the fresh culture medium with RSC to each well and culture for 48 hours to induce dendritic growth after the non neuronal cells have been eliminated and RSC level has been significantly reduced. Add matrigel or BMP seven to the culture medium on day five. In vitro then feed the culture as described previously shown here as a neuron grown under the control conditions with the lack of dendrites as evidenced by the lack of MAP two immuno positive processes.
In contrast, this neuron, which has been exposed to BMP seven has several tapered map two immuno positive dendrites shown here. A cultured sympathetic neurons are five to seven days in culture prior to treatment with BMP seven and post-treatment with BMP seven at 30 to 50 nanograms per milliliter for two days. Note, the beginning of dendritic growth.
Culture's grown for seven to 14 days in the absence of bmp.Seven. Continue to exhibit only axonal growth while cultures treated with BMP.Seven. At 30 to 50 nanograms per milliliter for seven to 14 days.
Exhibit robust dendritic growth. After watching this video, you should have a good understanding of how to isolate SCG. Prepare dissociated culture of sympathetic neurons for the studies of neuronal morphogenesis and experimentally manipulate dendritic outgrowth.
