Ucph 101

DL-TBOA, UCPH-101, WAY 213613, DQP-1105, QNZ-46, TCN-201, (Z)-4-OH-tamoxifen DNQX, MK-801 hydrogen maleate were purchased from Tocris Bioscience; MSO from Sigma-Aldrich. All drugs were diluted in DMSO or ddH2O as required for treatment and corresponding concentration of the dissolving agent used as vehicle controls. For washout, slices were washed 3 times and transferred to fresh medium containing luciferin. SCN slices were kept in standard culture medium (DMEM based with 5% serum and Glutamax supplement 1X (Life Technologies), unless otherwise specified (see main text).

Glutamate transporters activity was evaluated by uptake assays using d-aspartate, a transportable analogue of l-glutamate, which is not metabolized and does not interact with glutamate receptors. At the end of treatment with TNF-α, plates were placed at the surface of a 37°C water bath. Cells were rinsed three times with Krebs buffer (25 mmol/L HEPES, 4.8 mmol/L KCl, 1.2 mmol/L KH₂PO₄, 1.3 mmol/L CaCl₂, 1.2 mmol/L MgSO₄, 6 mmol/L glucose and 140 mmol/L NaCl, pH 7.4). d-[³H]-aspartate (50 nmol/L, specific activity of 11.3 Ci/mmol, Perkin Elmer) was added on the cells in presence or not of the appropriate inhibitors of glutamate transporters. Inhibitors of GLT-1 (WAY-213613, Tocris, Bristol, UK) and GLAST (UCPH-101, Tocris) were used at 100 and 10 µmol/L, respectively. After 6 min, uptake was stopped by three rinses with cold Krebs buffer (NaCl replaced by choline chloride) and cells were lysed with NaOH 0.1 N. Radioactivity was measured using the liquid scintillation solution Microscint 40 and the TopCount NXT Microplate Scintillation and Luminescence Counter (Perkin Elmer). A fraction of the lysate was used for protein determination by Bradford method using BioRad Protein Assay Dye Reagent (BioRad). Glutamate uptake rate was expressed as picomole of d-[³H]-aspartate transported per minute and per milligram of protein.