For in vivo experiments, brain tissue was collected on the day of the last injection or 1 day after the last vehicle or metformin injection. Mice were anesthetized with isoflurane (Fresenius Kabi, Ontario, Canada) and euthanized by cervical dislocation. The brains were removed, and NPCs were isolated from the SVZ of mice at various ages as previously described (6, 40). Following the microdissection of the SVZ, cells were incubated in an enzyme solution [containing trypsin (1.3 mg/ml), hyaluronidase (0.76 mg/ml), kynurenic acid (0.12 mg/ml); Sigma-Aldrich, Missouri, USA] for 30 min at 37°C and then centrifuged at 1500 revolutions per minute (RPM) for 5 min. Cells were resuspended in a trypsin inhibitor solution (0.67 mg/ml; Worthington Biochemical Corporation, New Jersey, USA) and triturated, and then centrifuged at 1500 RPM for 5 min. Cells were resuspended in SFM, triturated five times, centrifuged at 1500 RPM for 3 min, resuspended in 1 ml of SFM, and then counted on a hemocytometer. Cells were plated at clonal density (10 cells/μl) (41) in the presence of fibroblast growth factor (20 ng/ml, Gibco, New York, USA), epidermal growth factor (20 ng/ml, PeproTech, Quebec, Canada), and heparin (2 μg/ml, Sigma-Aldrich, Missouri, USA) in SFM (neurosphere media) for 7 days in the presence or absence of metformin, and the number of neurospheres was counted. For experiments with early postnatal mice, cells were plated at 5 cells/μl. For passaging, neurospheres were collected, dissociated into a single cell suspension, and replated at 2.5 cells/μl in neurosphere media for 7 days. For coculture experiments, primary YFP cells were plated at 1 cell/μl as a control and cocultured with 9 cells/μl from C57Bl/6 mice of the opposite sex.

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