Murine neural stem cell (mNSC) sphere culturing

mNSCs were derived from the hippocampus tissue of albino mouse (Charles River, Japan) embryos on embryonic day 15. Cells were cultured in a medial hormone mix (MHM) with 10 μg/ml epidermal (EGF; Sigma aldrich, Japan, Cat-No: SRP3196) and 10 μg/ml basic fibroblast growth factors (bFGF; Sigma aldrich, Japan, Cat-No: F5392) for six days at 37°C and 5% CO2. After six days, the grown NSC spheres were harvested and prepared for differentiation and/or proliferation studies (for methods, see below).

Differentiation study

mNSC spheres were collected and plated on a 30 μg/ml poly-l-lysine (Sigma aldrich, Japan, Cat-No: P1274) and 20 μg/ml laminine (Invitrogen, Japan, Cat-No: 23017–015) coated 8-well glass cover-slip and incubated for another 4 days. Each well had a final concentration of 22.8x104 cells/ml cultured in MHM containing 1% fetal bovine serum (FBS) and without EGF and bFGF. The cells were treated directly after seeding to the cover-slip using different concentration (0 nM, 1 nM, 10 nM, 100 nM) of methylphenidate (MPH; Sigma aldrich, Japan, Cat-No: M2892). On the last day, cells were fixed with 4% paraformaldehyde (Fluka, Japan, Cat-No: 76240) for 20 minutes at room temperature (RT) and stained with different antibodies (see “Immunocytochemistry”).

BrdU incorporation

The proliferation of NSC was identified by in vitro labeling with the thymidine analog 5-bromo-2-desoxyuridine (BrdU; Wako, Japan, Cat-No: 023–15563). NSC spheres were collected and plated on coated 8-well glass cover-slip and incubated for 24 h. Each well had a final concentration of 22.8x104 cells/ml cultured in MHM, containing EGF and bFGF and without FBS. The cells were treated with different concentration of MPH (0 nM, 1 nM, 10 nM, 100 nM). After 24 h, BrdU was added to each well to reach a final concentration of 10 μM and incubated for 4 h at RT. Before BrdU immunostaining, DNA was denatured and the nucleus-membrane was broken by treating cells with 2M HCL for 35 min at RT; afterward, cells were rinsed with 1% phosphate buffer saline (PBS), followed by treatment with sodium borate (pH=8.5) for 10 min at RT for the neutralization of HCL. The prepared cells were stained against BrdU (see “Immunocytochemistry”).

Immunocytochemistry

Fixed and prepared cells were stained for differentiation studies with rabbit monoclonal antibody against glial fibrillary acidic protein (GFAP; 1:200, Sigma aldrich, USA, Cat-No: C4546) and/or with mouse monoclonal antibody against β-tubulin III (Tuj 1; 1:300, Sigma aldrich, USA, Cat-No: T3952) overnight at 4°C. For proliferation studies, cells were stained with rat monoclonal antibody against BrdU (1:100, Accurate Chemical & Scientific, Japan, Cat-No: OBT0030) overnight at 4°C. In both studies, fixed cells were also stained with Hoechst 33258 (1:100; Invitrogen, Cat-No: H3569) for 15 min at RT to visualize the cell nuclei. After overnight incubation, the primary antibodies were visualized with secondary antibodies against rabbit, mouse, or rat conjugated to the following fluorochromes: Alexa Fluor® -488 and Alexa Fluor® -555 (Life Technologies, Japan).

Statistical analysis

The stainings were analyzed for the cell count of astrocytes, immature neurons and proliferated cells in comparison to the total number of cells using the Mann–Whitney (U-Rang) Test in the StatView software program (Stat View 5.0. software, SAS Institute Inc. Cary, NC, USA). A p-value < 0.05 was set as significant. There were at least five independent experiments, and four wells per slide were analyzed. In each case, the total amount of Hoechst stained cells/well were manually counted, and afterward, either BrdU positive, GFAP positive, or Tuj 1 positive cells were counted and the percentage of positive cells to the total amount was calculated. In the proliferation and differentiation studies a comparison of the treated mNSC to the control mNSC (MPH untreated) was done.