pCREB Experiments

The experiments have been performed on primary cultures of rat cortical neurons. These cells express mGlu5Rs (Koga et al., 2010).

Primary cultures of cerebral cortical neurons were prepared from 1-day-old rats. Cortices free of meninges were dissociated in 0.025% (w/v) trypsin at 37°C followed by mechanical repeated gentle pipetting through wide- and narrow-bore fire-polished Pasteur pipettes in culture medium [Neurobasal medium (Gibco, Grand Island, NY, United States) supplemented with 0.1 mM glutamine (Sigma-Aldrich, Milan, Italy), 10 μg/ml gentamicin (Sigma-Aldrich, Milan, Italy) and 2% B-27^® Supplement (50X), serum free (Gibco, Grand Island, NY, United States)]. Cells were counted and then plated on poly-L-lysine (5 μg/ml)-coated multiwells. Cytosine arabinoside (10 μM; Sigma-Aldrich, Milan, Italy) was added within 24 h of plating to prevent glial cell proliferation. After 8 days of in vitro incubation (days in vitro: DIV), cultures were used for experiments.

pCREB measurement was performed with the pCREB (Ser133) Assay kit (PerkinElmer Italy, Milan). Briefly, cortical neurons were seeded in 96 well tissue culture plates at a density of 4,000 cells/well and incubated at 37°C overnight. Thereafter, the concentration-response curves of glutamate receptor agonists (i.e., glutamate, L-quisqualate, CHPG) were assessed in the absence or in the presence of GET73. Briefly, the cells were incubated for 1 h with the glutamate receptor agonists, lysed with the freshly prepared lysis buffer. When required, GET73 was applied at different concentrations 15 min before the addition of glutamate receptor agonists. The lysates were transferred to a 96-well ½AreaPlate^TM for the assay. The Acceptor Mix was added for 1 h at room temperature and the Donor mix was added to wells under subdue light and incubate for 1 h at room temperature. At the end of this period, raw “AlphaScreen Signal counts” were measured by an Alpha Technology^®-compatible plate reader, using standard AlphaLISA settings (excitation 680 nm, emission 615 nm).