Glutamate

Human DPSC were plated at a density of 5 × 10⁴ cells / well in 100 μL of DPSC cell culture medium in a 96 well plate. Cells were allowed to adhere for 3 h under standard culture conditions. At 3 h post-plating, cells were incubated with various concentrations of Glutamate (Sigma, G1251) for either 15 min to simulate the up-regulation of Glutamate seen during TMS, 24 h or one trial where Glutamate was added for 15 min, followed by a washout step for 45 min with the addition of Glutamate and washout was repeated for a total of six times to replicate the in vivo paradigm of TMS used with each experiment completed in triplicate. Twenty-four hours after cells were initially plated, 10 μL of WST-1 cell proliferation reagent (Scientifix, Cheltenham, VIC, Australia) was added and cell proliferation was read at 450 nm and 690 nm.

Rat hippocampal or cortical neurons were treated with 1 µM dexamethasone (Sigma) for 24 h. The cells were then harvested, extracted and analyzed for expression of CPE-ΔN. FGF2 (R&D) or BDNF (Promega) was added to neurons for 15 min. The cells were then harvested, extracted and analyzed for activation of ERK and AKT. In other experiments, primary neurons were transduced with adenoviral vectors (Type 5 (dE1/E3)), carrying the cDNA of CPE ΔN (custom made by Vector Biolabs) or LacZ (Vector Biolabs) as a negative control, at 50 MOI for 72 h. The neurons were then treated with 100 µM H₂O₂ (Sigma), 40 µM glutamate (Sigma) or 0.4 µM staurosporine (Sigma) for 24 h. In specific experiments, 10 µM LY294002 (Cell Signaling), 5 µM U0126 (Cell Signaling), 1 µM PD166285 (Sigma) or SU5402 (Sigma) were added to the neurons during the 20–24 h treatment with glutamate, staurosporine or H₂O₂. Cell viability, cell cytotoxicity and TUNEL assays were then used to measure cell death after the treatments.

Primary rat hippocampal neurons were prepared from rat feti (Sprague-Dawley, day 18 of gestation; Envigo, Indianapolis, IN, USA) as described previously [5 (link),32 (link),33 (link),34 (link)]. Briefly, neurons (0.3 × 10⁶ cells/35 mm plate) were seeded on poly-l-lysin coated plates and grown in a neurobasal medium supplemented with B-27, glutamine, and antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) for 21 days in vitro (DIV). shRNA lentiviral particle transduction: Primary hippocampal neurons were transduced with control shRNA lentiviral particles or Bcl-xL shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA); copGFP control lentiviral particles or Bcl-xL shRNA-GFP lentiviral particles (Santa Cruz Biotechnology) at DIV 7. Glutamate treatment: Glutamate (Sigma-Aldrich, St. Louis, MO, USA) was freshly prepared in sterile PBS (Thermo Fisher Scientific) and added to the cell culture medium (final concentration: 20 μM). The vehicle control group for the Glutamate experiment was treated with isovolumetric sterile PBS. ABT-263 treatment: ABT-263 was prepared in dimethyl sulfoxide (DMSO) and added to the cell culture medium (final concentration 1 μM). The vehicle control group for ABT-263 was treated with isovolumetric DMSO. The protocol was approved by the Institutional Animal Care Committee (IACUC) of the University of Alabama, Tuscaloosa, AL, USA.