Neurobasal a b27 glutamax

Mouse cerebral cortices were extracted from neonatal P0-1 pups and placed into an ice-cold HBSS/FBS solution: Hank’s Balanced Salt Solution (Sigma, St. Louis, MO), 4.2 mM NaHCO₃, 1 mM HEPES, and 20% FBS. The tissue was washed three times with HBSS and then digested at 37°C/15 min with Papain (Worthington Biochemical, Lakewood) in a solution that contained 137 mM NaCl, 5 mM KCl, 7 mM Na₂HPO₄, and 25 mM HEPES (pH 7.2). The tissue was washed 3 times with 20% FBS, three times with HBSS, and three times with growth medium containing Neurobasal A, B27, Glutamax and Penicillin/Streptomycin (Invitrogen, Carlsbad, CA). Tissue was then triturated in growth medium containing 50 units of DNAse I (Invitrogen, Carlsbad, CA). The neurons were pelleted by centrifugation (600 × g for 10 min) and plated at a density of 40,000 cells/cm² on poly-D-lysine-coated (Sigma-Aldrich, St. Louis, MO) 60 mm dishes. Neurons were incubated at 37°C/5% CO₂, and half of the medium was replaced with fresh medium once a week. 0.5 µM dexamethasone (Sigma-Aldrich, St. Louis, MO) or vehicle (EtOH) was applied after 14 days in culture for 7 days.

Striatal neurons from 4-month old male WT mice were isolated as described above and cultured for 7–9 days in Neurobasal A/B27/Glutamax (Invitrogen, Eugene, OR) with 10 ng/mL FGF2 and 10 ng/mL PGDFbb (Invitrogen, Eugene, OR). Then, cells were pretreated for 30 min with either AMD3100 (0.1 μM) or PD98059 (10 μM) and subsequently incubated with gp120_IIIB for 24 hrs. After 24 hrs, a co-treatment of gp120 _IIIB/AMD3100 or gp120 _IIIB/PD98059 was given for an additional 24 hrs (in new media). Finally, cells were fixed and incubated with Bgtx as described below.