Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

This method can help to investigate sex-dependent differences in the neurogenic niches, and to understand their neuroplasticity, changes that underline social interactions in the prairie vole. The assay of neurospheres is an excellent tool to determine the proliferation and differentiation potential of neural stem and progenitor cells. To begin, place a Petri dish on a surface surrounded by ice.

Deposit the brain on the dish and add 20 milliliters of cold wash solution. In the coronal plane, divide the brain into two blocks of tissue using a scalpel, performing the cut at bregma level in the anterior-posterior axis. Extract the subventricular zone, or VZ, tissue from the rostral block, and the dentate gyrus, or DG, tissue from the caudal block.

To dissect the VZ, hold one of the hemispheres with a Dumont forceps, and insert the fine tips of a second forceps under the tissue that lines the caudate-putamen. Open the forceps along the dorsal-ventral axis to separate the tissue, and collect the VZ into a centrifuge tube with two milliliters of cold wash solution. Do not pool the tissue of more than two animals.

Repeat the microdissection in the other hemisphere and store the tube with the tissue on ice. To dissect the DG, use a scalpel to make a coronal cut into the caudal block, to obtain two slices in which the hippocampal formation is observed. Use Dumont forceps to hold one of the slices, and make a horizontal cut between DG and CA1 with another forceps.

Then, make a vertical incision between the DG and CA3, to separate the DG.Repeat the dissection in the other hemisphere of the first slice, then in both hemispheres in the second slice. Collect the four DG pieces of each vole in a centrifuge tube. Place the centrifuge tubes inside the biosafety cabinet and wait approximately 10 minutes for the tissue fragments to precipitate by gravity.

Then, remove the wash solution and add one milliliter of warm enzymatic solution to each tube. Incubate the tubes at 37 degrees for 10 minutes. To disintegrate the tissue fragments, pipette them up and down with a one milliliter tip, but do not pipette more than 30 times.

Repeat the incubation at 37 degrees Celsius, then pipette the tissue again. Add nine milliliters of N-2 medium to each tube to dilute the enzymatic treatment, and centrifuge the tubes at 200 times g for four minutes. Discard the supernatant, wash the cells with 10 milliliters of N-2 medium, and repeat the centrifugation.

Remove the supernatant from each tube, and resuspend the cell pellets of the VZ and DG in two milliliters and one milliliter of the B-27 medium, respectively. Filter each cell suspension with a 40 micrometer cell strainer to remove any non-digested tissue. Culture the filtered cells in an ultra-low attachment 24-well plate, using two wells for the VZ and one well for the DG.Add 20 nanograms per milliliter of fibroblast growth factor 2, and 20 nanograms per milliliter of epidermal growth factor to each well, then incubate the plate at 37 degrees Celsius, 5%carbon dioxide, and high humidity for 48 hours.

After the incubation, remove half of the culture medium and replace it with fresh B-27 medium, supplemented with double concentrations of the growth factors. Repeat this process every third day. On days when it is not necessary to change the culture medium, add growth factors to a final concentration of 1X.

Neurospheres were formed from the neural stem cells isolated from the subventricular zone and dentate gyrus of both female and male adult prairie voles. Although there was debris in the primary culture, only neurospheres were present after the first passage. A higher number of neurospheres were obtained from the female subventricular zone than from the male subventricular zone, or the dentate gyrus of both females and males, suggesting that the number of neurospheres obtained depends on the proliferative zone and the vole sex.

The diameter of the neurospheres was measured on days 8, 11, and 14. It increased progressively for male and female voles in both neuronal regions, but neurospheres derived from the male brains were smaller in comparison to those derived from the female brains. Adhered neurospheres were characterized on day six in the presence of growth factors, or on day 15 without growth factors.

At day six, under undifferentiated conditions, the neurosphere-derived cells expressed Nestin, a marker for neural progenitors. It was also possible to identify doublecortin-positive cells and the proliferation marker Ki-67, which indicate the presence of either neuronal precursors or immature neurons. However, the lack of colocalization of Ki-67 with DCX suggested the presence of post-mitotic neuroblasts.

At day 15, under differentiation conditions, mature neurons and cells with the glial phenotype were found, demonstrating the differentiation potential of the isolated cells. When attempting this protocol, keep in mind that being able to recognize and having previous practice in microdissection is fundamental to isolate only cells from the neurogenic niches. Following this protocol, experiments can be designed to identify mechanisms involved in proliferation, differentiation and survival of neural stem and progenitor cells, processes that are still unknown in the prairie vole.